EP2235172A1 - Crystal structure of a type iic p-type atpase - Google Patents

Abstract

The present invention relates to a crystal structure of a type IIC P-type ATPase. The invention further describes method for identification of modulators of ATPases as well as uses of such modulators. Based on the provided three dimensional structure of the ATPase, various method, such as computer implemented methods may be used for identification of modulators, such putative modulators may be further analysed using in vitro and in vivo experiments to confirm there functionality. Several modulator interaction regions are described as target of regulation by ATPase modulators.

Description

Crystal structure of a type MC P-type ATPase

All patent and non-patent references cited in the application, or in the present application, are also hereby incorporated by reference in their entirety.

Field of invention

The present invention relates to the three dimensional structure of a type NC P-type ATPases illustrated by the atomic coordinates obtained from crystallization experiments and X-ray diffraction results for the Na+/K+ ATPase from pig renal . The invention further relates to a method for purifying a Na+/K+ ATPase (type NC P-type ATPase) and methods of growing crystals of a Na+/K+ ATPase (type NC P-type ATPase). Based on the three dimensional structure, detailed information regarding specific functionalities of the ATPase is obtained. The invention further relates to methods for identification of modulators, specifically inhibitors of type NC P-type ATPase's. The invention further relates to computer implemented methods for identification of ATPase modulators, based on the structural information obtained from the above described experiments.

Background of invention

The Na⁺, K⁺-ATPase, originally described in 1957¹, is a membrane-bound ion pump belonging to the family of P-type ATPases. By using energy derived from ATP hydrolysis, the Na⁺, K⁺-ATPase generates electrochemical gradients for Na⁺ and K⁺ across the plasma membranes of animal cells, as required for electrical excitability, cellular uptake of ions, nutrients and neurotransmitters, and regulation of cell volume and intracellular pH. The transport is accomplished by enzyme conformational changes between two states, E1 and E2, that selectively bind three Na⁺ and two K⁺ ions, respectively (Fig. 1 a); the ions become transiently 'occluded', that is, inaccessible to the medium on either side of the membrane²³. The pump is sensitive to the membrane potential — the major voltage-dependent steps being associated with the binding and release of one of the three Na⁺ ions⁴⁵.

Na+/K+-ATPases extrudes Na+ from animal cells and takes up K+ from the extracellular solution. The pump maintains the resting potential and are vital for cell survival. Altered expression or functionality of Na+/K+-ATPases have been implicated in a range of diseases such as diabetes, hypertension, cancers and Alzheimer^'s disease.

The type NC P-type ATPases include NaVK⁺- and H⁺-/K⁺-ATPases found in animals which all have a P-subunit in addition to the catalytic α subunit. The alpha-subunit contains the sites for binding of Na⁺, K⁺ or H+, K+ and ATP and is homologous to single-subunit P-type ATPases, like the Ca²⁺-ATPaSe. The beta-subunit is unique to the K⁺-counter-transporting P-type ATPases, Na⁺, K⁺-ATPase and H⁺,K⁺-ATPase; it is required for routeing of the alpha-subunit to the plasma membrane and for occlusion of the K⁺ ions⁶⁷. In addition to the Psubunit, the NaVK⁺ and H+/K+ ATPases may be associated with a small proteolipid, the Ysubunit, which may be important for modulating K⁺ activation of the pump. The γ-subunit is a protein belonging to the FXYD family which may regulate the pumping activity in a tissue- and isoform-specific way⁸⁹.

Although the first crystal structure of a P-type ATPase, the sarco(endo)plasmic reticulum Ca²⁺-ATPaSe (SERCA), appeared in 2000¹⁰ and was followed by structures of this enzyme in several conformations (for example, refs 1 1-13), the present work for the first time reveals the structure of a multi-subunit P-type ATPase, the pig renal Na⁺, K⁺-ATPase alpha-beta-gamma complex, with bound K7Rb⁺ counterions.

The Na+-K+ pump is present in the membrane virtually every human cell and is important for maintain cell potential and regulate cellular volume as described above.

Potent inhibitors of Na+/K+-ATPases are the cardiac glycosides (for example ouabain and digitalis/digoxin), found in both plants and animals. Cardiac glycosides are used in the treatment of congestive heart failure and cardiac arrhythmia and could potentially be used in the treatment of some forms of cancer. Cardiac glycosides bind from the extracellular side of the enzyme to block the ion transport pathway and lock the enzyme a single conformational transition. The inhibitory effect increases the contractions strength of the heart.

The role of the Na+-K+ pump in cardiac contraction is due to the role of Na+ in controlling the intracellular concentration of Ca2+ via the Na+/Ca2+ exchanger, as the resulting lower Na+ concentration gradient will give rise to an increased accumulation of Ca2+ for executing the cardiac contraction. Therefore inhibition of the Na+, K+ pump leads to stronger cardiac contractility.

Pharmacological use of this knowledge is well known exemplified by cardiac glycosides which are used in the treatment of congestive heart failure and cardiac arrhythmia. By inhibiting the Na+/K+ pump an increase in the level of sodium ions in the myocytes is obtained, which subsequently leads to a rise in the level of calcium ions. This improves cardiac output and reduces distention of the heart.

It is envision that the cardiac glycosides stabilize the E2-P transition state of the

Na+/K+ pump (se figure 1A). The proposed mechanism is the following: inhibition of the Na+/K+ pump leads to increased intracellular Na+ levels, which in turn slows down the extrusion of Ca2+ via the Na+/Ca2+ exchange pump. Increased amounts of Ca2+ are then stored in the sarcoplasmic reticulum and released by each action potential.

Cardiac glycosides have been used for many years both as poisons and as drugs and there actions include both beneficial and toxic effects on the heart. Cardiac glycosides are widely used in treatment of congestive heart failure, atrial fibrillation and atrial flutter although their toxicity remains a serious problem. Dosing of the drugs is the major problem as the cardiac glycosides may completely block the Na+/K+ pump as described above resulting in toxic effects.

It is therefore desirable to develop new modulators or inhibitors of the Na+/K+ pump, whose inhibitory activity on the Na+/K+ pump can be controlled e.g. enabling easier dosing of the compounds to avoid toxic effects.

By providing a crystal structure of a Na+, K+ ATPase the applicants have provided new insight into the regulatory mechanism of the Na+, K+ ATPase which is used for identification of potential modulators of the ATPase as described herein.

Because of the high conservation between the Na+, K+ ATPase and the H+, K+ ATPase, in the regulatory region identified herein (se below), the structure of the Na+. K+ ATPase is further useful for identification of modulators of the H+, K+ ATPase. H₊, K+-ATPase

Both pH and potassium levels are regulated within very narrow ranges in mammals. Ion concentrations outside of these levels are life-threatening. The H+, K+-ATPase hydrolyzes ATP to drive exchange of ions along the nephron and the pump functions to conserve potassium and acidify the urine. The H+/K+ ATPase is the proton pump of the stomach and is responsible for the acidification of the stomach contents. The H+/K+ ATPase is found in parietal cells which are highly specialised epithelial cells located in the inner cell lining of the stomach.

This cat-ion exchange molecule is thus important for regulation of salt balance in mammals and is know to be involved in dyspepsia, peptic ulcer disease, gastroesohageal reflux disease and Zollinger-Ellison syndrome which is therefore treatable using inhibitors of the H+, K+ ATPase. Known proton pump inhibitors are mostly benzimidazole derivatives which are given in an inactive form. In an acid environment, the inactive drug is protonated and rearranges into its active form.

Summary of invention

Here we present the X-ray crystal structure at 3.5 A resolution of the pig renal Na⁺, K⁺- ATPase with two rubidium ions bound (as potassium congeners) in an occluded state in the transmembrane part of the alpha-subunit. Several of the residues forming the cavity for rubidium/potassium occlusion in the Na⁺,K⁺-ATPase are homologous to those binding calcium in the Ca²⁺-ATPaSe of sarco(endo)plasmic reticulum. The beta- and gamma-subunits specific to the Na⁺,K⁺-ATPase are associated with transmembrane helices αM7/αM10 and αM9, respectively. The gamma-subunit corresponds to a fragment of the V-type ATPase c subunit. The carboxy terminus of the alpha-subunit is contained within a pocket between transmembrane helices and seems to be a novel regulatory element controlling sodium affinity, possibly influenced by the membrane potential.

The present application relates to a crystal comprising a type NC P-type ATPase, which is preferably a mammalian ATPase, such as an ATPase from pig. The Crystal is in one embodiment characterized by the cubic space group P2₁2₁2i. The type NC P-type ATPase may be a multi subunit ATPase, preferably comprising three subunits, which may be termed alpha, beta and gamma. It is preferred that the crystal effectively diffracts x-rays for the determination of the atomic coordinates of the ATPase to a resolution better than 6 A, such as better than 5 A, preferably better than 4 A. A part from the protein content the crystal may comprise other components such as a phosphate analogue or one or more cation, such as Na+, K+ and/or Rb+.

The applicant has according to the invention described a method of purifying and type NCP-type ATPase which allow crystallization of said enzyme. This is complicated by the requirements for large quantise of protein material which must further be of high quality to enable crystallization.

An aspect of the invention relates to a method for purifying a type NC P-type ATPase comprising the steps of: a. obtaining a composition comprising a P-type NC ATPase, b. solubilising said ATPase using a non-ionic detergent and c. purifying said ATPase.

The ATPase composition may, according to the invention by obtained by any method known in the art including heterogen expression by use of standard technologies of molecular biology. The composition comprising a type NC P-type ATPase is preferably isolated from animal kidney, such as pig kidney. The ATPase is normally comprised by a membrane fraction isolated by isopycnic zonal centrifugation. The protein must be solubilised, which according to the invention is preferably performed using a non-ionic detergent such as Ci₂E₈. Ci₂E₈ is added to a ratio of 0.5-2 such as 1 -1 -5 or such as 1 - 1.25 preferably such as 1 .12 mg per mg membrane protein. To have a more pure composition insoluble material may be removed.

Components from the buffers or specific components to be included in the crystal may be added during purification or during the growth of the crystals. Such components includes one or more cat-ions such as Na+, K+ and Rb+ and substituted amines as N- methyl-D-glycamine (NMDG).

A further aspect of the invention relates to a method of growing a crystal comprising a type NC P-type ATPase according to any of the claims 1 -12, comprising the steps of: a. obtaining a composition comprising a type NC P-type ATPase, b. subjecting said composition to crystallizations environment including PEG 2000mme and c. obtaining crystals comprising a type NC P-type ATPase.

In a further embodiment the crystallizations environment comprise: a. mixing said composition comprising a type NC P-type ATPase with a precipitating solution comprising PEG2000mme, b. growing ATPase crystals by vapour diffusion from hanging drops

The ATPase composition and said precipitating solution are preferably mixed with a β- DDM solution in ratio of 0.5-2 : 0-5-2 : 0.1 -0.5. To optimise the procedure the method may further comprise the steps of: a. isolating an initial precipitate and b. growing these by vapour diffusion from hanging drops.

An aspect of the invention relates to the use of a crystal according to the invention for determination of the three dimensional structure of a type NC P-type ATPase.

Further aspects relates to a computer-readable data storage medium comprising a data storage material encoded with at least a portion of the structure coordinates of the crystal structure described herein as set forth in figure 18.

The crystal structure as disclosed herein is useful for performing in-silico screening methods for identification of potential inhibitors of type Il C P-type ATPases.

An aspect of the invention relates to the use of atomic coordinates as presented in figure 18 or atomic coordinates selected from a three-dimensional structure that deviates form the tree-dimensional structure as presented in figure 18 by a root mean square deviation over protein backbone atoms of not more than 3 A in a method for identifying a potential modulator of a type NCP-type ATPase.

Further methods are described relating to a method of identifying a potential modulator of a type NCP-type ATPase by determining binding interactions between the potential modulator and a set of binding interaction sites in a regulatory binding pocket, the ion transport path way, the CTS cavity or the Na+, K+ binding sites of a type NC P-type ATPase comprising the steps of: a. generating the spatial structure of the regulatory binding pocket or the Na+, K+ binding sites on a computer screen using atomic coordinates as presented in figure 18 or atomic coordinates selected from a three- dimensional structure that deviates from the tree-dimensional structure presented in figure 18 by a root mean square deviation over protein backbone atoms of not more than 3 A. b. generating potential modulators with their spatial structure on the computer screen, and c. selecting potential modulators that can bind to at least 1 amino acid residues of the set of binding interaction sites with out steric interference.

Such methods are preferably performed as computer-assisted method or computer implemented methods. The application further describes a computer-assisted method for identifying potential modulators of a type NC P-type ATPase using a programmed computer comprising a processor, a data storage system, a data input devise and a data output device, comprising the following steps: a. inputting into the programmed computer through said input device data comprising: atomic coordinates of a subset of the atoms of said ATPase, thereby generating a criteria data set; wherein said atomic coordinates atomic coordinates are selected from the tree-dimensional structure presented in figure 18 or atomic coordinates selected from a three-dimensional structure that deviates from the tree- dimensional structure presented in figure 18 by a root mean square deviation over protein backbone atoms of not more than 3 A, b. comparing, using said processor, the criteria data set to a computer data base of low-molecular weight organic chemical structures stored in the data storage system; and c. selecting from said data base, using computer methods, a chemical structure having a portion that is structurally complementary to the criteria data set and being free of steric interference with the ATPase. In an embodiment the invention relates to a method for identifying a potential modulater capable of modulating the Na+, K+ translocating activity of a type NCP-typer ATPase, said method comprising the following steps: a. selecting a potential modulator using atomic coordinates in conjunction with computer modelling, wherein said atomic coordinates are the atomic coordinates presented in figure 18 or wherein the atomic coordinates are selected from a three-dimensional structure that deviates from the tree-dimensional structure presented in figure 18 by a root mean square deviation over protein backbone atoms of not more than 3 A, by docking potential modulators into a set of binding interaction sites in a regulatory binding pocket, the ion transport path way, the CTS cavity or the Na+, K+ binding sites generated by computer modelling and selecting a potential modulator capable of binding to at least one amino acid in said set of binding interaction sites in a regulatory binding pocket, the ion transport path way, the CTS cavity or the Na+, K+ binding sites, b. providing said potential modulator and said ATPase c. contacting the potential modulator with said ATPase and d. detecting modulation of Na+, K+ translocating activity of said ATPase by the potential modulator.

It is preferred that the docking of potential modulator molecules is performed by employing a three-dimensional structure defined by atomic coordinates of the three dimensional structure presented in figure 18 and such that said potential modulates is capable of binding to at least one amino acids in the binding interaction sites in a regulatory binding pocket, the ion transport path way, the CTS cavity or the Na+, K+ binding sites.

An embodiment of the invention relates to a method of identifying a potential modulator capable of modulating the enzymatic activity of a type NC P-type ATPase said method comprising the following steps; a. introducing into a computer, information derived from atomic coordinates defining a conformation of a regulatory binding pocket, the ion transport path way, the CTS cavity or the Na+, K+ binding sites of said ATPase, based on three-dimensional structure determination, whereby a computer program utilizes or displays on the computer screen the structure of said conformation; wherein said atomic coordinates are selected from the tree-dimensional structure as presented in figure 18 or atomic coordinates selected from a three-dimensional structure that deviates from any one of the tree- dimensional structure represented by figure 18 by a root mean square deviation over protein backbone atoms of not more than 3 A; b. generating a three-dimensional representation of the regulatory binding pocket or the Na+, K+ binding sites of said ATPase by said computer program on a computer screen; c. superimposing a model of a potential modulator on the representation of said regulatory binding pocket, the ion transport path way, the CTS cavity or the Na+, K+ binding sites, d. assessing the possibility of bonding and the absence of steric interference of the potential modulator with the regulatory binding pocket, the ion transport path way, the CTS cavity or the Na+, K+ binding sites of the ATPase; e. incorporating said potential compound in an activity assay of said ATPase and f. determining whether said potential compound modulates the enzymatic activity of said ATPase.

It is preferred that the potential modulator can bind to at least 1 , preferably at least 2, or more preferably at least 3 amino acids most preferably at least 5 amino acids in the regulatory binding pocket, the ion transport path way, the CTS cavity t and/or the Na+, K+ binding sites.

As descried herein the crystal structure of the pig ATPase has revealed a regulatory binding pocket. Preferably information derived from the atomic coordinates of at least one of the following amino acid residues of the regulatory binding pocket: ile761 - pro775, M7 Iys833-met852, M8 phe922-thr938, M10 leu990-trp1009 and β thr28-phe42 are used in the methods according to the invention. It is preferred that the potential inhibitor interacts with one ore more of the AA in the regulatory binding pocket. Additionally modulators may interact with the ion transport pathway or the CTS cavity formed. The entry channel is formed by M1 (AA82-98), M2(AA133-146), M3 (AA325- 331 ) and the exit channel by M1 -M2 AA 105-122), M3-M4 (300-318), M5-6 (AA789- 800). The CTS cavity lined by M1 (108-1 1 1 ), M2 (120-124), M4 (304-318); M5 (780- 785) and M6 (795-799) are further residues of importance. Therefore the atomic coordinates of the above mentioned amino acid residues are preferably used according to the invention for selection of interacting modulators.

The crystal structure has further revealed the residues of a potential 3^rd Na+ binding sites, thus the atomic coordinates of at least one of the following amino acid residues of the 3^rd Na₊, K₊ binding site: Tyr 771 (αM5), Thr 807 (αM6), Asp 808 (αM6), GIn 923 (αM8) and GIu 954 is preferably used in the method of the invention.

It is preferred that the structural information used in the methods of the invention are atomic coordinates determined to a resolution of at least 4 A.

The potential modulator may initially be identified in-silico and binding/inhibition confirmed using in vitro or in vivo assays, thus the potential inhibitor is synthesised and modulation of ATPase activity verified. The potential modulator may be such as a non- hydrolyzable peptide analogue, an organic compounds or an inorganic compound.

Libraries of small organic molecules or potential peptide modulators may be screened.

The modulator may be an inhibitor or an activator of said type NCP-type ATPase.

The invention further relates to a method for producing a potential modulator, which comprise the steps of: a. identification of a potential modulator of a type NCP-type ATPase according to the invention and b. producing said identified potential modulator.

The identified modulator may be synthesis using any suitable procedure known to the person skilled in the art. The invention may further relate to the identification of selective peptide inhibitors of a P-type ATPase comprising the following steps: a. identification of a potential modulator of a type NC P-type ATPase according to the invention, b. contacting the potential peptide modulator with said ATPase, c. contacting the potential peptide modulator with a different ATPase, d. detecting inhibition of said ATPase activity of said ATPase by the potential modulator and e. detecting activity of said different ATPase in the presence of said potential modulator.

An aspect of the invention relates to a medicament comprising a modulator of a type I P-type ATPase identified as described herein. The medicament preferably comprises an inhibitor of a Na+, K+ ATPase or an H+, K+ ATPase. The inhibitor is preferably specific for the indicated ATPase.

In a specific embodiment, wherein the inhibitor is an inhibitor of a Na+, K+ ATPase the medicaments are for treatment of congestive heart failure, atrial fibrillation or atrial flutter.

In a specific embodiment, wherein the inhibitor is an inhibitor of an H+, K+ ATPase the medicaments are for treatment of dyspepsia, peptic ulcer disease, gastroesohageal reflux disease or Zollinger-Ellison syndrome.

In a specific embodiment, wherein the inhibitor is an inhibitor of an H+, K+ ATPase the medicaments are for treatment of cancer.

Detailed description of the invention

The term "crystal" refers to an ordered state of matter. Proteins, by their nature are difficult to purify to homogeneity. Even highly purified proteins may be chronically heterogeneous due to modifications, the binding of ligands or a host of other effects.

In addition, proteins are crystallized from generally complex solutions that may include not only the target molecule but also buffers, salts, precipitating agents, water and any number of small binding proteins. It is important to note that protein crystals are composed not only of protein, but also of a large percentage of solvents molecules, in particular water. These may vary from 30 to even 90%. Protein crystals may accumulate greater quantities and a diverse range of impurities which cannot be listed here or anticipated in detail. Frequently, heterogeneous masses serve as nucleation centers and the crystals simply grow around them. The skilled person knows that some crystals diffract better than others. Crystals vary in size from a barely observable 20 micron to 1 or more millimetres. Crystals useful for X- ray analysis are typically single, 0.05 mm or larger, and free of cracks and defects.

The term "coordinate" as use herein, refers to the information of the three dimensional organization of the atoms contributing to a protein structure. The final map containing the atomic coordinates of the constituents of the crystal may be stored on a data carrier; typically the data is stored in PDB format or inx-plor format, both of which are known to the person skilled in the art. However, crystal coordinates may as well be stored in simple tables or text formats. The PDB format is organized according to the instructions and guidelines given by the Research Collaboratory for structural Bioinformatics.

The term "root mean square deviation" (rmsd) is used as a mean of comparing two closely related structures and relates to a deviation in the distance between related atoms of the two structures after structurally minimizing this distance in an alignment. Related proteins with closely related structures will be characterized by relatively low RMSD values whereas larger differences will result in an increase of the RMSD value.

The term "associating with" or "binding" refers to a condition of proximity between chemical entities or compounds, or portions thereof. The association may be non- covalent-wherein the juxtaposition is energetically favoured by hydrogen bonding or van der Waals or electrostatic interactions-or it may be covalent.

The term "binding pocket", as used herein, refers to a region of a molecule or molecular complex that, as a result of its shape, favourably associates with another molecule, molecular complex, chemical entity or compound. As used herein, the pocket comprises at least a deep cavity and, optionally a shallow cavity. As used herein the term "complex" refers to the combination of a molecule or a protein, conservative analogues or truncations thereof associated with a chemical entity.

ATPase crystal An aspect of the invention relates to a crystal which comprises a type NC P-type ATPase.

Depending on the resolution of a crystal structures larger differences information can be obtained from the data. At a resolution of about 5.5 A the overall shape of a molecule, such as helices are seen with strong intensity. At a resolution of about 3.5 A the main chain is visible (usually with some ambiguities). At a resolution of about 3 the side chains are partly resolved and at a resolution of about 2.5 the side chains are well resolved. The atoms are located within about 0.4 A meaning that the lengths of hydrogen bonds calculated from a PDB file (for example, by RasMol) have at least this uncertainty. The limit of protein crystallography is around 1 .5A or a little less, where atoms are located to about ± 0.1 A⁴⁴.

The crystal of the invention preferably effectively diffracts x-rays for the determination of the atomic coordinates of the protein to a resolution better than 6A. More preferably the three dimensional structure determinations can be determined with a resolution of more than 5 A, such as more than 4 A or most preferably about 3.5 A using the crystals according to the invention. Most preferably the crystal effectively diffracts x-rays for the determination of the atomic coordinates of the protein to a resolution of 3.6 A

The space group of crystals according to the invention is preferably P2₁2₁2₁ and the cell dimensions are preferably 69 ± 4 A, 263 ± 4 A, 336 ± 4A. The cell dimensions can according to the application vary depending on the specific ATPase comprised by the crystal an even on the conformation of the ATPase comprised by the crystal.

Type NC P-type ATPases

The function of the Na+, K+ -ATPase is highly conserved in mammals. The type NC P-type ATPases are multi subunit ATPase comprising as described above an α- and a β-subunit and optionally a third subunit (the γ-subunit) which is a member of the FXYD family. The FXYD family proteins are auxiliary subunits of the Na+, K+- ATPase, expressed primarily in tissues that specialize in fluid or solute transport, or that are electrically excitable. These proteins range in size from about 60 to 160 amino acid residues, and share a core homology of 35 amino acid residues in and around a single transmembrane segment. The gene family was named FXYD (pronounced fix-id) in recognition of invariant amino acids in its signature motif.

The following genes for the alpha and beta subunits are know in humans

Alpha: ATP 1A1 , ATP 1A2, ATP 1 A3, ATP1A4 and beta: ATP 1 B1 , ATP 1 B2, ATP 1 B3, ATP1 B4. A plurality of potential gamma subunits is known. The alpha and beta isoforms combine in tissue-specific ways and display variations in for example enzyme kinetics, ion affinities and voltage sensitivity.

In an embodiment the invention relates to a crystal comprising a type Il C ATPase preferably a Na+, K+ -ATPase or a H+, K+ ATPase from a mammalian species. In a preferred embodiment the ATPase of the crystal originates from a pig, most preferably from the outer medulla of a pig kidney.

In a preferred embodiment the invention relates a crystal comprising a type NC P-type which is derived from a mammalian species. In a more preferred embodiment the ATPase is pig kidney Na+, K+ ATPase. Sequences of pig ATPase subunits are known and can be found in suitable databases. The sequence of one alfa, beta and gamma subunit is given here below. The alpha-subunit is identified by SEQ ID NO 1 , the beta- subunit by SEQ ID NO 2 and the optional gamma subunit by SEQ ID NO 3, respectively.

The first 5 amino acids of the alpha-chain of the Na+ ,K+ ATPase are normally not counted because they are regarded a signal sequence, they are shown in italic and underlined. All transmembrane regions have been underlined. TM regions also underlined.

>gi|47523570|ref|NP_999414.1 | Na+/K+ -ATPase alpha 1 subunit [Sus scrofa] (SEQ ID NO 1 )

10 20 30 40 50 60

MGKGVGRDKY EPAAVSEHGD KKKAKKERDM DELKKEVSMD DHKLSLDELH RKYGTDLSRG 70 80 90 100 110 120

LTPARAAEIL ARDGPNALTP PPTTPEWVKF CROLFGGFSM LLWIGAILCF LAYGIOAATE

130 140 150 160 170 180

EEPONDNLYL GVVLSAVVII TGCFSYYOEA KSSKIMESFK NMVPQQALVI RNGEKMSINA

190 200 210 220 230 240 EEVVVGDLVE VKGGDRIPAD LRIISANGCK VDNSSLTGES EPQTRSPDFT NENPLETRNI

250 260 270 280 290 300 AFFSTNCVEG TARGIVVYTG DRTVMGRIAT LASGLEGGQT PIAAEIEHFI HIITGVAVFL

310 320 330 340 350 360 GVSFFILSLI LEYTWLEAVI FLIGIIVANV PEGLLATVTV CLTLTAKRMA RKNCLVKNLE

370 380 390 400 410 420 AVETLGSTST ICSDKTGTLT QNRMTVAHMW SDNQIHEADT TENQSGVSFD KTSATWLALS

430 440 450 460 470 480 RIAGLCNRAV FQANQENLPI LKRAVAGDAS ESALLKCIEL CCGSVKEMRE RYTKIVEIPF 490 500 510 520 530 540

NSTNKYQLSI HKNPNTAEPR HLLVMKGAPE RILDRCSSIL IHGKEQPLDE ELKDAFQNAY

550 560 570 580 590 600 LELGGLGERV LGFCHLFLPD EQFPEGFQFD TDDVNFPLDN LCFVGLISMI DPPRAAVPDA

610 620 630 640 650 660 VGKCRSAGIK VIMVTGDHPI TAKAIAKGVG IISEGNETVE DIAARLNIPV SQVNPRDAKA

670 680 690 700 710 720 CVVHGSDLKD MTSEQLDDIL KYHTEIVFAR TSPQQKLIIV EGCQRQGAIV AVTGDGVNDS

730 740 750 760 770 780 PASKKADIGV AMGIAGSDVS KQAADMILLD DNFASIVTGV EEGRLIFDNL KKSIAYTLTS 790 800 810 820 830 840

NIPEITPFLI FIIANIPLPL GTVTILCIDL GTDMVPAISL AYEOAESDIM KRQPRNPKTD

850 860 870 880 890 900 KLVNEOLISM AYGOIGMIOA LGGFFTYFVI LAENGFLPIH LLGLRVNWDD RWINDVEDSY

910 920 930 940 950 960 GQQWTYEQRK IVEFTCHTPF FVTIVVVQWA DLVICKTRRN SVFQQGMKNK ILIFGLFEET 970 980 990 1000 1010 1020

ALAAFLSYCP GMGVALRMYP LKPTWWFCAF PYSLLIFVYD EVRKLIIRRR PGGWVEKETY

The alpha chain after removal of the signal peptide is identified by SEQ ID NO 16.

>gi|225192|prf||121 1232A ATPase beta,Na/K (SEQ ID NO 2)

The Beta chain has one TM region underlined here below. 10 20 30 40 50 60

MARGKAKEEG SWKKFIWNSE KKEFLGRTGG SWFKILLFYV IFYGCLAGIF IGTIOVMLLT

70 80 90 100 110 120 XSEFKPTYQD RVAPPGLTQI PQSQKTEISF RPNDPQSYES YVVSIVRFLE KYKDLAQKDD

130 140 150 160 170 180 MIFEDCGNVP SELKERGEYN NERGERKVCR FRLEWLGNCS GLNDETYGYK DGKPCVIIKL

190 200 210 220 230 240 NRVLGFKPKP PKNESLETYP VMKYNPYVLP VHCTGKRDED KEKVGTMEYF GLGGYPGFPL 250 260 270 280 290 300 QYYPYYGKLL QPKYLQPLMA VQFTNLTMDT EIRIECKAYG ENIGYSEKDR FQGRFDVKIE

VKS

The Gamma Chain like wise has one transmembrane region.

>gi|62177154|ref|NP_001014427.1 1 FXYD domain containing ion transport regulator 2 [Sus scrota] (SEQ ID NO 3)

MAGLSTDDGGSPKGDVDPFYYDYETVRNGGLIFAALAFIVGLIIILSKRLRCGGKKHRPINEDEL

The invention further encompasses type NC P-type ATPase from different species such human and other animals. Such ATPase from other species can be interpreted as homologues of the pig ATPase identified by SEQ ID NO 1 , 2 and 3. According to the inventions homologues of the pig ATPase identified by SEQ ID NO 1 also covers sequences obtained by modifications of a type NC P-type ATPase from pig or different species. The level of identity is preferably measured by comparison of the sequence with SEQ ID NO1 , 2 and 3 (see below).

A "predetermined sequence" is a defined sequence used as a basis for a sequence comparison; a predetermined sequence may be a subset of a larger sequence, for example, as a segment of a full-length sequence given in a sequence listing.

In further preferred embodiment of the ATPase is a homologue of pig kidney ATPase. Homologues of polypeptides can be determined on the basis of their degree of identity with a predetermined amino acid sequence, said predetermined amino acid sequence for the present invention being SEQ ID NO: 1 , 2 and 3, when the homologue is a fragment, a fragment of the aforementioned amino acid sequences is used from determining their degree of identity (se below).

Accordingly, homologues preferably have at least 75% sequence identity, for example at least 80% sequence identity, such as at least 85 % sequence identity, for example at least 90 % sequence identity, such as at least 91 % sequence identity, for example at least 91% sequence identity, such as at least 92 % sequence identity, for example at least 93 % sequence identity, such as at least 94 % sequence identity, for example at least 95 % sequence identity, such as at least 96 % sequence identity, for example at least 97% sequence identity, such as at least 98 % sequence identity, for example 99% sequence identity with the predetermined sequence.

The percent identity is determined with the algorithms GAP, BESTFIT, or FASTA in the Wisconsin Genetics Software Package Release 7.0, using default gap weights.

The term "sequence identity" means that two polypeptide sequences are identical (i.e., on a residue-by-residue basis) over the window of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residues occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

As the ATPase is a multi subunit ATPase the level of sequence identity may be calculated per subunit. The percentage of sequence identity is preferably above the aforementioned percentage of identify for at least one subunit, more preferably at least two subunits and most preferably all three subunits are at least 85 % identical, preferably more than 90 % of identity with pig ATPase identified by SEQ ID 1 , 2 and 3 respectively.

The invention relates to a crystal comprising any of the above mentioned ATPases or homologues thereof having, such as more than at least 75 % of identity to SEQ ID 1 , 2 and/or 3. In an embodiment the crystal according to the invention comprises a type NC P-type ATPase comprising an α-subunit at least 75 % identical to SEQ ID NO 1.

The level of identity should be calculated over the homologues sequences with may be such as a fragment of SEQ ID NO 1 , 2 and/or 3. The level of identity may be different for different subunits of the ATPase.

The degree of identity may be calculated using suitable available programs, such as the program mentioned herein. The region of homology preferably covers at least 500 AA, such as 600 AA, more preferably 700 AA, most preferably at least 800 AA. According to the invention the ATPase comprised by the crystal is not the necessarily a full-length protein. Truncated versions can readily be prepared by conventional methods of molecular biology (Sambrook and Russell, 2001 ). According to the invention it is preferred that the ATPase of the crystal comprise more than 75 %, more preferred 80 %, and mostly preferred more than 90 % of the full length protein sequences. Particularly the trans-membrane regions should be included, such that the proteins includes 5 or more of the trans-membrane helixes, preferably 7 or more, such as 8 or even more preferred 9 or mostly preferred 10 trans-membrane helixes of the alpha- subunit. In a further preferred embodiment the crystal comprise at least the 10 transmembrane regions of the alpha-subunit, the beta-transmembrane regions and mostly preferred also the gamma transmembrane regions.

The sequences of the transmembrane segments of the alpha, beta and gamma sequences of the Na+, K+ ATPase described herein are denoted, SEQ ID NO 4-13 (TM1 -10 of alpha subunit, SEQ ID NO 14 (TM-β) and SEQ ID NO 15 (TM-γ), respectively and listed here below). As seen from the below the numbering is according to the ATPase after removal of the signal peptide, thus M1 aa80 corresponds to aa85 of the full length alpha chain of SEQ ID NO 1.

M1 aa 80-1 1 1 PEWVKF CRQLFGGFSM LLWIGAILCF LAYGIQ

M2 aa 1 15-141 EEEPQNDNLYL GVVLSAVVII TGCFSY

M3 aa 278-305 AAEIEHFI HIITGVAVFL GVSFFILSLI

M4 aa 310-340 WLEAVI FLIGIIVANV PEGLLATVTV CLTLT M5 aa 763-789 DNL KKSIAYTLTS NIPEITPFLI FIIA

M6 aa 792-818 PLPL GTVTILCIDL GTDMVPAISL AYE

M7 aa 839-868 NEQLISM AYGQIGMIQA LGGFFTYFVI LAE

M8 aa 906-931 IVEFTCHTPF FVTIVVVQWA DLVICK

M9 aa 943-964 KNK I LI FG LFE ETALAAFLS YC M10 aa 980-1005 WFCAF PYSLLIFVYD EVRKLIIRRR

TM Beta aa 33-61 FKILLFYV IFYGCLAGIF IGTIQVMLLTI

TM Gamma a 23-51 YDYETVRNGGLIFAALAFIVGLIIILSKRLR

It is known in the art that fragments of an ATPase or event subunits of an ATPase can be joined by ordinary techniques. Sequence identity is in one embodiment determined by utilising fragments of the alpha subunit (SEQ ID NO 1 ) comprising at least 400 amino acids. Fragments of an ATPase comprising such as most or all of the trans-membrane helixes are preferably used.

A homologue comprising fragments of the alpha-subunit preferably includes least 100, preferably contiguous amino acids of SEQ ID NO 1 and has an amino acid sequence fragments which are at least 80%, such as 85%, for example 90%, such as 95%, for example 99% identical to the amino acid sequence of at least 8 of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:

10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively. More preferable the homologue comprise a sequence with the aforementioned levels of identity to at least 9, further preferably at least 10, and more preferably at least 1 1 and mostly preferred all 12 trans-membrane sequences as identified by SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.

Since two polypeptide sequences may each comprise (1 ) a portion of the complete polypeptide sequence that is similar between the two polypeptides, and (2) a sequence that is divergent between the two polypeptides, sequence comparisons between two (or more) polypeptides are typically performed by comparing sequences of the two polypeptides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window", as used herein, refers to a conceptual segment of at least 20 contiguous peptide positions wherein a polypeptide sequence may be compared to a predetermined sequence of at least 20 contiguous peptides and wherein the portion of the polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the predetermined sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.

Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981 ) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. MoI. Biol. 48: 443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected.

The a preferred embodiment the crystal includes a homologue of a type NC P-type ATPase, such as the pig ATPase, wherein one ore more of the amino acids residues are conserved or substituted by an amino acid residue with similar properties, e.g. the ATPase may comprise conserved amino acid substitutions (see below). Preferably more than 1 , more than 2, more than 5 AA of the above mentioned AA are conserved or represented by a conserved amino acid substitution. Preferably the ATPase homologue comprised by the crystal comprises all the amino acid residues mentioned herein. Alternatively the ATPase may comprise conserved amino acid substitutions for one or more of the mentioned amino acid residues.

Conservative amino acid substitutions refer to the inter-changeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine, a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

Additionally, homologues are also determined based on a predetermined number of conservative amino acid substitutions as defined herein below. Conservative amino acid substitution as used herein relates to the substitution of one amino acid (within a predetermined group of amino acids) for another amino acid (within the same group), wherein the amino acids exhibit similar or substantially similar characteristics. Within the meaning of the term "conservative amino acid substitution" as applied herein, one amino acid may be substituted for another within the groups of amino acids indicated herein below:

i) Amino acids having polar side chains (Asp, GIu, Lys, Arg, His, Asn, GIn, Ser, Thr, Tyr, and Cys,)

ii) Amino acids having non-polar side chains (GIy, Ala, VaI, Leu, lie, Phe, Trp, Pro, and Met)

iii) Amino acids having aliphatic side chains (GIy, Ala VaI, Leu, lie)

iv) Amino acids having cyclic side chains (Phe, Tyr, Trp, His, Pro)

v) Amino acids having aromatic side chains (Phe, Tyr, Trp)

vi) Amino acids having acidic side chains (Asp, GIu)

vii) Amino acids having basic side chains (Lys, Arg, His)

viii) Amino acids having amide side chains (Asn, GIn)

ix) Amino acids having hydroxy side chains (Ser, Thr)

x) Amino acids having sulphor-containing side chains (Cys, Met),

xi) Neutral, weakly hydrophobic amino acids (Pro, Ala, GIy, Ser, Thr)

xii) Hydrophilic, acidic amino acids (GIn, Asn, GIu, Asp), and

xiii) Hydrophobic amino acids (Leu, lie, VaI)

Accordingly, a homologue or a fragment thereof according to the invention may comprise, within the same homologue of the sequence or fragments thereof, or among different variants of the sequence or fragments thereof, at least one substitution, such as a plurality of substitutions introduced independently of one another.

It is clear from the above outline that the same homologue or fragment thereof may comprise more than one conservative amino acid substitution from more than one group of conservative amino acids as defined herein above.

The addition or deletion of at least one amino acid may be an addition or deletion of from preferably 2 to 250 amino acids, such as from 10 to 20 amino acids, for example from 20 to 30 amino acids, such as from 40 to 50 amino acids. However, additions or deletions of more than 50 amino acids, such as additions from 50 to 100 amino acids, addition of 100 to 150 amino acids, addition of 150-250 amino acids, are also comprised within the present invention. The deletion and/or the addition may - independently of one another - be a deletion and/or an addition within a sequence and/or at the end of a sequence.

The polypeptide fragments according to the present invention, including any functional equivalents thereof, may in one embodiment comprise less than 250 amino acid residues, such as less than 240 amino acid residues, for example less than 225 amino acid residues, such as less than 200 amino acid residues, for example less than 180 amino acid residues, such as less than 160 amino acid residues, for example less than 150 amino acid residues, such as less than 140 amino acid residues, for example less than 130 amino acid residues, such as less than 120 amino acid residues, for example less than 1 10 amino acid residues, such as less than 100 amino acid residues, for example less than 90 amino acid residues, such as less than 85 amino acid residues, for example less than 80 amino acid residues, such as less than 75 amino acid residues, for example less than 70 amino acid residues, such as less than 65 amino acid residues, for example less than 60 amino acid residues, such as less than 55 amino acid residues, for example less than 50 amino acid residues.

The homology between amino acid sequences may be calculated using well known scoring matrices such as any one of BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50, BLOSUM 55, BLOSUM 60, BLOSUM 62, BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85, and BLOSUM 90. In addition to conservative substitutions introduced into any position of a preferred predetermined sequence, or a fragment thereof, it may also be desirable to introduce non-conservative substitutions in any one or more positions of such a sequence.

A non-conservative substitution leading to the formation of a functionally equivalent fragment of SEQ ID NO 1 , SEQ ID NO 2 and/or SEQ ID NO 3.would for example i) differ substantially in polarity, for example a residue with a non-polar side chain (Ala, Leu, Pro, Trp, VaI, lie, Leu, Phe or Met) substituted for a residue with a polar side chain such as GIy, Ser, Thr, Cys, Tyr, Asn, or GIn or a charged amino acid such as Asp, GIu, Arg, or Lys, or substituting a charged or a polar residue for a non-polar one; and/or ii) differ substantially in its effect on polypeptide backbone orientation such as substitution of or for Pro or GIy by another residue; and/or iii) differ substantially in electric charge, for example substitution of a negatively charged residue such as GIu or Asp for a positively charged residue such as Lys, His or Arg (and vice versa); and/or iv) differ substantially in steric bulk, for example substitution of a bulky residue such as His, Trp, Phe or Tyr for one having a minor side chain, e.g. Ala, GIy or Ser (and vice versa).

Homologues obtained by substitution of amino acids may in one preferred embodiment be made based upon the hydrophobicity and hydrophilicity values and the relative similarity of the amino acid side-chain substituents, including charge, size, and the like. Exemplary amino acid substitutions which take several of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

In a further embodiment the present invention relates to functional variants comprising substituted amino acids having hydrophilic values or hydropathic indices that are within +/-4.9, for example within +/-4.7, such as within +/-4.5, for example within +/-4.3, such as within +/-4.1 , for example within +/-3.9, such as within +/-3.7, for example within +/- 3.5, such as within +/-3.3, for example within +/- 3.1 , such as within +/- 2.9, for example within +/- 2.7, such as within +/-2.5, for example within +/- 2.3, such as within +/- 2.1 , for example within +/- 2.0, such as within +/- 1.8, for example within +/- 1.6, such as within +/- 1.5, for example within +/- 1.4, such as within +/- 1.3 for example within +/- 1 .2, such as within +/- 1.1 , for example within +/- 1 .0, such as within +/- 0.9, for example within +/- 0.8, such as within +/- 0.7, for example within +/- 0.6, such as within +/- 0.5, for example within +/- 0.4, such as within +/- 0.3, for example within +/- 0.25, such as within +/- 0.2 of the value of the amino acid it has substituted.

The importance of the hydrophilic and hydropathic amino acid indices in conferring interactive biologic function on a protein is well understood in the art (Kyte & Doolittle, 1982 and Hopp, U.S. Pat. No. 4,554,101 , each incorporated herein by reference).

The amino acid hydropathic index values as used herein are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1 .8); glycine (-0.4 ); threonine (-0.7 ); serine (-0.8 ); tryptophan (-0.9); tyrosine (-1.3); proline (-1 .6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5) (Kyte & Doolittle,

1982).

The amino acid hydrophilicity values are: arginine (+3.0); lysine (+3.0); aspartate

(+3.0.+-.1 ); glutamate (+3.0.+-.1 ); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-.1 ); alanine (-0.5); histidine (-0.5); cysteine

(-1 .0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4) (U.S. 4,554,101 ).

ATPase complex

In order to stabilize the protein one or more compounds may be added during purification of the ATPase (see below) enabling formation of an ATPase complex suited for crystallization. This may further enable fixing of the protein in a specific state which is needed to obtain detailed information regarding the functionality of the ATPase.

For various purposes different cat-ions may be included in the crystal. Such cat-ions may be included in the crystal by growing the crystal in the presence of said cat-ions or by submerging the crystals in a solution comprising cat-ions. Heavy atoms are frequently included for initial structure determination.

The crystal structure may further comprise cat-ions such as a cat-ions selected from the group of: Rb₊, H⁺, Mg²⁺, Ho³⁺, K⁺, Pt⁴⁺ and Ta²⁺, preferably Rb₊ or K₊. The cat-ions are preferably included in the crystal by including suitable salt in the buffer during or after purification of the ATPase, such salts include RbF, MgCI, KCI and/or KF.

According to the invention the crystal may comprise one or more compounds for stabilising the protein, such as ATP, ATP analogues. Such analogues may provide stability by fixing the protein in a specific state. In an embodiment the crystal comprises a non-hydrolysable ATP analogue preferably AMPPCP. In a further preferred embodiment the crystal comprises MgF₄ ^2", as a phosphate analogue.

The phosphate analogue MgF₄ ^2" in the structure locks the pump in an occluded state, which is described in the example and demonstrated in Figure 1 , using Rb+ as congener of K+. Thus in a preferred embodiment the ATPase comprised by the crystal is in the E2-P state.

Other compounds, such as compounds such as further buffer components may be included in the crystal such as substituted amines included for stabilisation of the protein. Preferably N-methyl-D-glucamine (NMDG) is used.

Source The protein material subjected to crystallization experiments according to the invention may be obtained from various sources, such as purified from a mammalian organism, preferably from pigs.

Alternatively the ATPase may be produced by recombinant method known by a person skilled in the art. Recombination methods enable expression of proteins at a high level wherefore proteins for crystallization experiment is preferably obtain using recombinant methods. The protein may be expressed in a host different from the organism from where the gene is derived. Heterogen expression is widely used in the art although complications may occur, particular when multi-domain proteins are expressed or where secondary modifications are involved. Expression of a Na+, K+ ATPase may be performed in yeast such as S. cerevisiae or Pichia pastoris which have previously been used to express a two subunit ATPase (J. Biol. Chem., Vol. 278, Issue 46, 46064- 46073, November 14, 2003). By analogy an individual skilled in the art may express three subunits of a type NC ATPase in a heterogen host. Purification

Independent of the source of the ATPase the protein must be purified before crystallization. The purification may be performed by conventional methods known in the art, which may differ dependent on the source of ATPase. Particularly as mentioned above the method of purification may depend on the use of one or more particular tags if the ATPase is heterogeneously expressed.

In a preferred embodiment the ATPase is isolated from pig kidney outer medulla.

Solubilization

ATPases of the invention are transmembrane proteins and thus comprises domains which are membrane integral as well as both intra and- extra cellular domains. Thus both hydrophilic and hydrophobic domains are present which complicates expression and purification of the protein. Detergents are usually required for solubilisation of membrane proteins, but such detergents often interfere with crystallization.

The applicant have success full established a procedure for isolation, purification and crystallization of a type NC P-type ATPase.

The ATPase is isolated from pig kidney outer medulla and the membrane fractions collected by a series of sequential centrifugation steps (se examples). The ATPase according to the invention is solubilised in a suitable detergent, preferably a non-ionic detergent.

Preferred detergents include: Octaethylene glycol monododecyl ether (C12E8) and n- Dodecyl-β-D-maltoside (DDM)

An aspect of the invention relates to a method for purifying a type NC P-type ATPase comprising the steps of: a. obtaining a composition comprising a type NC P-type ATPase, b. solubilising said ATPase using a non-ionic detergent and c. purifying said ATPase.

As mentioned above the ATPase is preferably isolated from pig kidney. The composition comprising the type NC P-type ATPase is according to the invention preferably a membrane fraction isolated by isopycnic zonal centrifugation

In a preferred embodiment the non-ionic detergent is Ci₂E₈ which in order to achieve solubilisation of the ATPase is added to a ratio of 0.5-2 such as 1 -1 -5 or preferably such as or more preferably 1 -1.25 and most preferably such as 1.12 mg per mg membrane protein.

To improve the crystallization process the method of purifying the ATPase may further comprise a step of removing insoluble material.

As described above the different cat-ions may be included in the purification buffers, thus according to the invention the composition comprising a type NC P-type ATPase may further comprise one or more cat-ions, such as one or more cat-ions selected from the group comprising: Na+, K+, Rb+, and Mg2+. The solution may further comprise N- methyl-D-glycamine (NMDG).

Method of growing ATPase crystal

Growing of a crystal comprising a type NC P-type ATPase may according to the invention be performed by any suitable method known in the art, such as vapour diffusions methods and/or hanging drops systems known by the person skilled in the art.

As described above the crystal may contain one or more compounds/cations, such as ATP, ATP analogues and/or cat-ions conveniently added after the purification process and before crystallization is initiated. Alternatively crystals may be submerged in a solution comprising the indication compounds/cat-ions prior to crystallization.

An aspect of the invention relates to a method of growing crystal comprising a type NC P-type ATPase. Such method includes the steps of obtaining an ATPase composition of sufficient quality for growing of a crystal and growing of ATPase crystals. As described herein, both steps can be modulated to optimise the out come.

Initiation of crystal formation can be nucleated by lowering the solubility of the ATPase. According to the invention PEG is included in the crystallizations environment. PEG is preferably selected from the group of PEGs comprising: PEG 100, PEG 200, PEG 400, PEG 600, PEG 800, PEG 1000, PEG 2000, PEG 3000, PEG 4000, PEG 5000, PEG 6000, PEG 7000 and PEG 8000.

An aspect of the invention relates to a method for growing a crystal comprising a type NC P-type ATPase comprising the steps of: a. obtaining a composition comprising a type NC P-type ATPase, b. subjecting said composition to crystallizations environment including PEG 2000mme and c. obtaining a crystal comprising a type NC P-type ATPase.

The crystallization environment may according to the invention be obtained by mixing a composition comprising a type NC P-type ATPase with a precipitating solution comprising PEG2000mme. As mentioned above any suitable method of growing crystals may be used, although vapour diffusion from hanging drops is preferred

In an embodiment the invention relates to a method of growing a crystal comprising a type NC P-type ATPase, comprising the steps of: a. obtaining a composition comprising a type NC P-type ATPase, b. mixing said composition comprising a type NC P-type ATPase with a precipitating solution comprising PEG2000mme, c. growing ATPase crystals by vapour diffusion from hanging drops d. obtaining crystals comprising a type NC P-type ATPase.

To optimize nucleation of crystals the composition comprising a type NC P-type ATPase and the precipitating solution may according to an embodiment be mixed with β-dodecyl maltoside (β-DDM) in solution in a ratio of 0.5-2 : 0-5-2 : 0.1 -0.5, most preferred is a ratio of 1 :1 :0.2. It is further preferred that the concentration of the β-DDM solution is 0.1 -0.35 %.

The precipitating solution used in Example 1 herein comprises 14 % PEG2000mme, 200 mM choline choloride, 4 mM DTT, 4 % Glycerol and 4 % MPD, which is the most preferred precipitating solution according to the invention. The inventors have observed that crystals of improved quality are obtained when the method of growing a crystal further comprising the steps of: a. isolating an initial precipitate and b. growing these by vapour diffusion from hanging drops.

The crystal structure of Na⁺, K⁺-ATPase from pig kidney outer medulla was obtained as described in example 1 and summarized here below.

Na⁺, K⁺-ATPase was isolated from pig kidney outer medulla and purified by mild SDS treatment followed by isopycnic zonal centrifugation⁴⁶. This preparation consists of α1 - and β1 -subunits together with the γ-subunit (γ_A and γ_B), Fig. 1 1 a, b. Crystals were obtained in the presence of 5 mM Rb⁺ by the vapour-diffusion method in hanging drops (Fig. 1 1c and Methods). The structure was determined on the basis of experimental electron-density maps. A low-resolution molecular replacement solution allowed site identification in derivative crystals for heavy-atom-based phasing. Phase extension by density modification and intercrystal averaging produced final experimental maps at 3.5 A resolution, forming the basis for model building (Fig. 2d). The final model yields an R-factor of 27.7% and a free R-factor of 31.2% (Table 1 ). The magnesium fluoride complex at the catalytic site was modelled as the tetrahedral MgF₄ ^2", as in the corresponding conformation of SERCA determined at 2.3 A resolution 12

The data for the model is summarized in table 1 .

Table 1 Data collection, phasing and refinement statistics*

^*For a full account of the data collection and structure determination see Methods. fValues in parentheses here and below refer to the high-resolution shell as indicated. ^Phasing power is the root mean squared (r.m.s.) value of F_h divided by the r.m.s. lack-of- closure, as given by SHARP22. lsomorphous and anomalous differences, respectively ^§R_free is the R-factor calculated for a randomly picked subset with approximately 1 ,000 reflections excluded from the refinement throughout fractions of residues in 'most favourable', 'allowed', 'generously allowed' and 'disallowed' regions of the Ramachandran plot after refinement.

Table 1 Data collection, phasing and refinement statistics* Those of skill in the art will understand that a set of structure coordinates for a protein or protein complex or a portion thereof, is a relative set of points that define a shape in three dimensions. Thus, it is possible that an entirely different set of coordinates could define a similar or identical shape. The variations in coordinates may be generated by mathematical manipulations of the structure coordinates. For example, the structure coordinates set forth in figure 1 1 could be manipulated by crystallographic permutations of the structure coordinates, fractionalization or matrix operations to sets of the structure coordinates or any combination of the above.

Coordinates stored on machine readable storage medium

In a further aspect the invention provide a computer-readable data storage medium comprising a data storage material encoded with the structure coordinates, or at least a portion of the structure coordinates set forth in figure 18. Examples of such computer readable data storage media are well known to those skilled in the art and include, for example CD-ROM and diskette ("floppy disks"). Thus, in accordance with the present invention, the structure coordinates of an ATPase, and portions thereof can be stored in a machine-readable storage medium. Such data may be used for a variety of purposes, such as drug discovery and X-ray crystallographic analysis of protein crystal.

The storage medium may be local to a computer as described above, or the storage medium may be located in a net-worked storage medium including the internet, to which remote accessibility is possible.

Use of crystal Provided that crystals of sufficient quality have been obtained, the crystals may according to the invention be used for X-ray diffraction experiments.

An aspect of the invention relates to the use of type NC P-type ATPase crystals for determination of the three dimensional structure of said ATPase.

Before data collection crystals may be treated by standard methods known in the art. Which include preparation of samples for heavy atom derivatization by dusting a dry powder of Ta₆Br₁₂ or Orange Pt directly to the drop until the crystal appears light green or faint orange. Crystals are according to the invention preferably dehydrated by conventional methods such as using cryo-prolectants such as sucrose, glycol and salt etc. Dehydration may be performed by increasing the concentration of the precipitating agent, such as PEG2000mme.

The crystals are mounted in nylon loops and flashed cooled in liquid. Excess mother liquor of the crystallisation mixture can be removed prior to flash cooling by gently touching a glass cover slip with the edge of the loop,

Data collection and data processing can be performed by any suitable systems know by the person skilled in the art. Data may be collected at 100 K on the end stations X06SA and X10SA at the Swiss Light Source SLS in Willingen. Processing may be performed using XDS⁴⁶. Data processing is further described in the examples.

Method using information derived from a three dimensional structure of an ATPase

Three dimensional structures provide information regarding the spatial localization of the peptide backbone and the side chains of the amino acid residues of the protein complex. Such information can not be derived from the primary amino acid sequence or from the knowledge of the secondary structure of the protein. The level of order of the crystal determines the level of details that can be obtained. The quality of a three dimensional structure is evaluated by the resolution obtained, which is an expression for the minimum spacing observed in diffraction. As mentioned above the application relates to crystals of high quality e.g. crystals with a resolution of less than 6 A preferably less than 4 A, most preferably around 3.6 A or less, which is required to have a sufficiently detailed model for selecting potential binding molecules e.g. modulators such as inhibitors of Na+, K+ or H+, K+ ATPase activity.

In order to employ virtual screening (by database docking programs such as Dock, FlexX, Gold) detailed structural information of the molecule is necessary.

The over all three dimensional structure of the ATPase is described in details in Example 1 , and summarized here below. The structure includes two potassium/rubidium binding sites and a regulatory binding pocket capable of accommodating the C-terminal regulatory peptide (se below). It further suggests the position of three Na+ binding sites

The α- subunit

Two cat-ion binding sites (herein denoted 1 and 2) are found between the transmembrane helices αM4, αM5 and αM6. The side chains of residues GIu 327 (αM4), Ser 775, Asn 776, GIu 779 (αM5) and Asp 804 (αM6) are sufficiently close to the Rb⁺ ions of the crystal to donate ligands for binding either directly or through an intervening water molecule. Asp 808 (αM6) is somewhat further away, but could be indirectly involved, and the same holds for GIn 923 (αM8). Asp 804 seems to donate a side-chain oxygen ligand to each Rb⁺ ion. GIu 327 is associated exclusively with K7Rb⁺ site 2 and may control the extracellular gate of the occlusion cavity, possibly guided by contact with Leu 97 of αM1 (ref. 17) (Fig. 3d). The residues GIu 327, Asn 776, GIu 779, Asp 804, Asp 808, and GIn 923 of the Na⁺, K⁺-ATPase correspond to residues in

SERCA which provide oxygen ligands for Ca²⁺ binding in the E1 form of SERCA^{10 23 24}, and they are therefore candidates for liganding residues in two of the three Na⁺ sites in the E1 form of the Na⁺,K⁺-ATPase.

The β-subunit.

The β-subunit is part of the regulatory binding pocket (se below). Particularly Tyr 39, Phe 42 and Tyr 43 in βM which are within interaction distance with αM7 residues around GIy 848 are likely involved in regulation of Na+ binding and transport.

The v-subunit.

The transmembrane region of the gamma subunit is clearly close to αM9 yet located on the outside of αM9 and not in the groove between αM9 and αM2, where it has been placed in modelling studies that are based on the Ca²⁺-ATPaSe structure. Several αM9 residues are within interaction distance of γlvl, including Phe 949, GIu 953, Leu 957 and Phe 960. The part of the γ-chain showing the most intimate interaction with αM9 around GIu 953 contains GIy 41.

C-terminal of αM10

The αM10 helix ends with three arginines (1003-1005) followed by the PGG motif and an extension of eight residues relative to the C terminus of the Ca²⁺-ATPaSe (SERCAI a isoform). The small α-helix formed by the first part of this extension is accommodated between αM, αM7 and αM10, and the two C-terminal tyrosine residues are recognized by a binding pocket between αM7, αM8 and αM5. Tyr 1016 seems to interact with Lys 766 of αM5 and Arg 933 in the loop connecting αM8 and αM9. Ser 936 (also in loop) is located within interaction distance of Arg 1003).

A regulatory peptide in the C-terminal (a ligand binding molecule) comprising the switch helix consisting of the amino acid residues val1010-tyr1016 (VEKETYY) was further identified. The truncated enzyme (ΔKETYY) exhibited an extraordinary 26-fold reduction of the Na⁺ affinity, yet the affinity for activating K⁺ was like wild-type.

The Na⁺-selective effect of the ΔKETYY truncation mimics the effects observed previously for mutation of Tyr 771 (αM5)³⁹ and Thr 807 (αM6)¹⁸. Together with GIu 954 (αM9) these residues have been suggested to make up a third Na⁺-binding site (Na⁺ sites 1 and 2 probably being formed by almost the same coordinating side chains as the two K7Rb⁺ binding sites)^40"42. We find these residues to cluster and to be lined by Asp 808 (αM6), bridging to KVRb⁺ site 2. In addition, GIn 923 (αM8) is found in the same cluster and could be involved with the third Na⁺ site in the E1 form⁴² (Rg. 6a and Fig. 7).

In light of the sensitivity of the Na⁺,K⁺-pump activity to the membrane potential⁴⁵, it is notable that Arg 1003, Arg 1004 and Arg 1005 at the end of αM10, together with Arg 933, Arg 934 and Arg 998, make the area around the C terminus in the membrane edge region highly electropositive.

Regulatory binding pocket and ligand binding molecule

Based on the three dimensional structure disclosed herein a binding pocket and a ligand binding molecule was identified

The binding pocket is formed by the transmembrane helices 5, 7, 8, 10 and β.

Thus the amino acid residues present in the α peptides including of Ne 761 - Ser 778 (M5), Lys 833- Met 852 (M7), Phe916 -Thr932 (M8), Leu 990- Trp 1009 (M10) and the amino acid residues present in the β peptide Thr 28 - Phe 42 are relevant for formation of the binding pocket of the Na⁺, K⁺ ATPase. The binding pocket may alternatively include Asn839-Gln854 (M7) and Ser31 -Cys45 (beta-subunit). This is further supported by the identification of point mutations in the area which affects the ion transport across the membrane as shown in figure 13. Amino acid residues (in the human alpha 2 isoform) R937, S940, D999, R1002Q, Y1020 and Y1021 all interferes with ion transport possibly via different mechanisms, whereof D999, R1002Q lies within the above defined binding pocket.

The following amino acid interactions are considered important for formation of the binding pocket: a possible salt bridge between the carboxy-terminal of Tyr 1016 and the primary amino group on Lys 766, a possible interaction between the side chains of Tyr 1015-1016 and Arg 933, a possible interaction between side chain of GIn 841 and Asn 839 with Tyr 1015-1016 and a possible interaction between side chain of Arg1003 and Ser936. It is further noted that Ser 936 may be phosphorylated.

Interactions between drug and the binding pocket would preferably be of electrostatic nature.

Entry and exit channel

An entry and exit channel or ion transport pathway is defined by the trans-membrane regions. The entry channel formed by M1 (AA82-98), M2(AA133-146), M3 (AA325-331 ) and the exit channel by M1 -M2 AA 105-122), M3-M4 (300-318), M5-6 (AA789-800). These amino acids are thus considered important for ion transport across the membrane.

In the regard Phe90 (M1 ), Glu282 (M3) and His286 (M3) are expected to be highly involved in ion transport shown by the direct contact to the positively charged metal cluster Ta₆Br₁₂ ²⁺. The localisation of two TaBr clusters within this region including possible interactions with the above mentioned amino acid residues are shown in figure 12. The effect of Ta₆Br₁₂ ²⁺ on Na+, K+ ATPase activity is shown in figure 15 clearly demonstrating the importance of this region.

CTS cavity

Cardiac glycosides or cariotonic steroids are well known inhibitors of the Na+, K+ ATPase. These compounds bind in CTS cavity of the molecule lined by residues of M1 (108-1 1 1 ), M2 (120-124), M4 (304-318); M5 (780-785) and M6 (795-799). Figure 13 shows ouabain in a large cavity of Na+, K+ in the E2 form, e.g. the form where the ATPase is accessible to extracellular K+. The cavity at the top of the trans-membrane region can thus accommodate CTS molecules such as ouabain including their bulky glucosylations. Within this cavity ouabain has close contact to Q1 1 1 , N122, Y308 and E312 and possibly F783, L793 and T797 (see figure 13C). Figure 14 displays the binding of bufalin in the cavity, within close proximity of Q1 1 1 , N122, E312, 1315, F783 and T797.

Na⁺, K⁺ binding sites As mentioned above the cat-ion binding sites (herein denoted 1 st and 2nd) are found between the transmembrane helices αM4 (lie 321 - Leu330), αM5 (asp763-pro782) and αM6 (Thr799 - Val810).

The side chains of residues GIu 327 (αM4), Ser 775, Asn 776, GIu 779 (αM5), Asp 804 (αM6), Asp 808 (αM6) and GIn 923 (αM8) could be indirectly involved as ligands of

Rb/K+. GIu 327 of αM4 and Leu 97 of αM1 may further be involved coordinating K7Rb⁺ in site 2 and to control the extracellular gate of the occlusion cavity, respectively.

Based on the structure the residue Asp 804, Asn 776, GIu 327, Ser 775, Gu779 and Gln923 re interpreted as being specifically important for cat-ion binding and may contribute to selectivity of the pump.

The β subunit may further contribute to cat-ion binding and transport as Tyr 39, Phe 42 and Tyr 43 in βM are within interaction distance with αM7 residues around GIy 848.

Third Na+ binding site

Tyr 771 (αM5), Thr 807, Gln923 (αM8) and Asp808 (αM6) together with GIu 954 (αM9) is suggested to make up the third Na⁺-binding site while the 1 ^st and 2^nd Na⁺ binding site is probably formed by almost the same coordinating side chains as the two K7Rb⁺ binding sites (described above). AA AspδOδ (αM6) may bridge to the 2^nd KVRb⁺ site 2 and GIn 923 (αM8) may further be part of the third Na⁺ site.

Identification of modulators

According to the invention various strategies can be followed to identify and generate modulators of Na+, K+ ATPases based on the structural information described herein. Modulators according to the invention may stimulate or inhibit the overall function of the ATPase, alternatively, a modulator may specifically address either the Na+ translocating or the K+ translocating function of the ATPase.

Potential modulators are molecules that can bind to the regulatory binding pocket, the ion transport pathway, the CTS cavity and/or the Na+, K+ binding sites which can be identified trough virtual screening of chemical databases. Virtual screening are performed with different database docking programs ( for instance Dock, FlexX, Gold, Flo, Fred, Glide, LigFit, MOE or MVP, but not limited to these) and used with different scoring functions (e.g. Warren et. al., 2005; Jain, 2006; Seifert et al., 2007). The scoring functions may include, but are not limited to force-field scoring functions (affinities estimated by summing Van der Waals and electrostatic interactions of all atoms in the complex between the type NC P-type ATPase and the ligand), empirical scoring functions (counting the number of various interactions, for instance number of hydrogen bonds, hydrophobic-hydrophobic contacts and hydrophilic-hydrophobic contacts, between the type NC P-type ATPase and the ligand), and knowledge based scoring functions (with basis on statistical findings of intermolecular contacts involving certain types of atoms or functional groups). Scoring functions involving terms from any of the two of the mentioned scoring functions may also be combined into a single function used in database virtual screening of chemical libraries.

Identified potential modulators are confirmed by in vitro and in vivo experiments before further developments. The binding of modulators may further be confirmed by x-ray experiments. Even when modulating activity is confirmed further drug development may be required before a compound suitable as a drug is identified.

As seen from the above and the examples the three dimensional structure described herein has identified a binding pocket for the regulatory C-terminal and specified the amino acid residues involved in Na+, K+ binding and transport. Based on this knowledge potential modulators of a type NC P-type ATPase can be identified. It is preferred that the structure used is the atomic coordinates presented in Figure 18, but a structure that deviates from the tree-dimensional structures as presented in Figure 18 by a root mean square deviation over protein backbone atoms of not more than 3 A may like wise used. It is preferred that the deviate is less than 2 A, more preferably less than 1 A. Such methods are preferable performed using computers, whereby the atomic coordinates are introduced into the computer, allowing generation of a model on the computer screen which allows visual selection of binding molecules.

Methods of selecting or identifying potential modulators Preferably, potential modulators are selected by their potential of binding to the regulatory binding pocket, the ion transport pathway, the CTS cavity and/or the Na+, K+ binding sites. The pocket and the binding sites are described above. Compounds which bind to at least one of these regions can be expected to interfere with the function of the ATPase and is thus a potential modulator of the ATPase. When selecting a potential modulator by computer modelling, the 3D structure of the ATPase is loaded from a data storage device into a computer memory and may be displayed (generated) on a computer screen using a suitable computer program. Preferably, only a subset of interest of the coordinates of the whole structure of the ATPase is loaded in the computer memory or displayed on the computer screen. This subset of interest may comprise the coordinates of the regulatory binding pocket or the Na+, K+ binding sites. This subset may be called a criteria data set; this subset of atoms may be used for designing a modulator.

An aspect of the invention relates to a method of identifying a potential modulator of a type NC P-type ATPase by determining binding interactions between the potential modulator and a set of binding interaction sites in a regulatory binding pocket or the Na+, K+ binding sites of a type NC P-type ATPase comprising the steps of: a. generating the spatial structure of the regulatory binding pocket, the ion transport pathway, the CTS cavity and/ or the Na+, K+ binding sites on a computer screen using atomic coordinates as presented in figure 18 or atomic coordinates selected from a three-dimensional structure that deviates from the tree-dimensional structure presented in figure 18 by a root mean square deviation over protein backbone atoms of not more than 3 A. b. generating potential modulators with their spatial structure on the computer screen, and c. selecting potential modulators that can bind to at least 1 amino acid residues of the set of binding inter action sites with out steric interference. An alternative wording for binding interaction sites set of may be a criteria data set.

In an a further aspect the potential modulators are identified using a computer, wherein the computer comprise programs and processor capable of utilizing the three dimensional structure information for selecting potential inhibitors bases on a criteria data set which defines target regions of the ATPase. Data bases of potential inhibitors, such as data bases of low molecular weight organic and/or inorganic chemical structures can be stored in the computer, e.g. in a storage system and used by the processor of the computer to identify potential inhibitors which in a region are structurally complementary to the criteria data set and being free of steric interference with the ATPase. Modulators being, in a region, complementary to the criteria data set, can be interpreted as inhibitors capable of accommodating a three-dimensional cavity defined by the criteria data set with out interfering with the structure of the target. Complementary indicates that the ATPase and the modulator interact with each other in an energy favourable way minimizing the availability of polar and charged residues (see below). The storage medium may be local to the computer as described above, or the storage medium may be remote such as a net-worked storage medium including the internet.

The low molecular weight organic chemical structures may include structures such as lipids, nucleic acids, peptides, proteins, antibodies and saccharides.

A computer-assisted method for identifying potential modulators of a type NC P-type ATPase using a programmed computer comprising a processor, a data storage system, a data input devise and a data output device, comprising the following steps: a. inputting into the programmed computer through said input device data comprising: atomic coordinates of a subset of the atoms of said ATPase, thereby generating a criteria data set; wherein said atomic coordinates atomic coordinates are selected from the tree-dimensional structure presented in figure 18 or atomic coordinates selected from a three-dimensional structure that deviates from the tree- dimensional structure presented in figure 18 by a root mean square deviation over protein backbone atoms of not more than 3 A, b. comparing, using said processor, the criteria data set to a computer data base of low-molecular weight organic chemical structures stored in the data storage system; and c. selecting from said data base, using computer methods, a chemical structure having a portion that is structurally complementary to the criteria data set and being free of steric interference with the ATPase.

The set of binding interaction sites or the criteria data set may according to the invention comprise at least some of the residues forming the regulatory binding pocket, the ion transport pathway, the CTS cavity and/or the Na+, K+ binding sites.

According to the invention the criteria data set may preferably comprise at least one or more of the amino acid residues forming the regulatory binding pocket between the transmembrane helixes 5, 7, 8, 10 and β, preferably including Ne 761 - Ser 775 (M5), Lys 833- Met 852 (M7), Phe916-Thr932(M8), Leu 990- Tyr 1016 (M10) and Thr 28 - Phe 42 (β peptide) more preferably at least some of the following amino acid residues:

The criteria data set may in addition or as an alternative in an embodiment include at least one or more of the following AA residues which with out being bound by the theory are considered important for formation of the binding pocket: Tyr 1016, Lys 766, Tyr 1015, Tyr 1016, Arg 933, GIn 841 , Asn 839, Tyr 1015-1016, Arg 1003 and Ser936.

Most preferably amino acids at least some of: Lys 766 (M5) GIn 841 and Asn 839(M7), Ser936 (M8), Arg933 and Trp1009-Tyr1016 (M10) are used.

According to the invention the criteria data set may as an alternative or in addition comprise at least some of the amino acid residues αM1 , αM2, αM3, αM4, αM5 and αM6 forming the ion transport pathway including the entry channel formed by αM1 (AA82- 98), αM2(AA133-146), αM3 (AA325-331 ) and the exit channel by αM1 -αM2 (AA 105- 122), αM3-M4 (300-318), αM5-6 (AA789-800). The criteria data set may in addition or as an alternative in an embodiment include at least one or more of the following AA residues which with out being bound by the theory are considered important for formation of the ion transport pathway: Phe90 (M1 ), Glu282(M3) and His286 (M3) According to the invention the criteria data set may as an alternative or in addition comprise at least some of the amino acid residues forming the CTS cavity formed by the trans-membrane helixes of αM1 , αM2, αM4, αM5 and αM6, preferably at least one or more of the following amino acid residues: αM1 (108-1 1 1 ), αM2 (120-124), αM4 (304-318); αM5 (780-785) and αM6 (795-799).

More preferred the criteria data may comprise at least one or more of the following amino acid residues: Q1 1 1 , N122, Y308, 1315, E312, F783, L793 and T797.

According to the invention the criteria data set may as an alternative or in addition comprise at least some of the amino acid residues forming the cat-ion binding sites formed between the transmembrane helices αM4, αM5 and αM6, preferably at least one or more of the following amino acid residues; ile 321 -Leu330 (αM4), Thr772- Thr781 (αM5) and Thr799-Val810 (αM6).

More preferred the criteria data may comprise at least one or more of the following amino acid residues: GIu 327 (αM4), Ser 775, Asn 776, GIu 779 (αM5), Asp 804 (αM6), Asp 808 (αM6), GIy 848 (αM7), GIn 923 (αM8), Leu 97 (αM1 ), Tyr 771 (αM5), Thr 807 (αM6), GIu 954 (αM9), Asp808 (αM6) and GIn 923 (αM8).

In the methods described herein the one or more amino acid residues comprised by be at least one, preferably at least 2 or 3, more preferably at least 5 or mostly preferred at least at least 6, 7 or 8 AA selected from the identified groups. In further embodiments the data criteria set may comprise more that 10 amin acids residues.

A potential inhibitor may then be designed de novo in conjunction with computer modelling. Models of chemical structures or molecule fragments may be generated on a computer screen using information derived from known low-molecular weight organic chemical structures stored in a computer data base or are built using the general knowledge of an organic chemist regarding bonding types, conformations etc. Suitable computer programs may aid in this process in order to build chemical structures of realistic geometries. Chemical structures or molecule fragments may be selected and/or used to construct a potential inhibitor such that favourable interactions to said subset or criteria data set become possible. The more favourable interactions become possible, the stronger the potential inhibitor will bind to the ATPase. Preferably, favourable interactions to at least one amino acid residues should become possible. Such favourable interactions may occur with any atom of the amino acid residue e.g. atoms of the peptide back-bone or/and atoms of the side chains.

Favourable interactions are any non-covalent attractive forces which may exist between chemical structures such as hydrophobic or van-der-Waals interactions and polar interactions such as hydrogen bonding, salt-bridges etc. Unfavourable interactions such as hydrophobic-hydrophilic interactions should be avoided but may be accepted if they are weaker than the sum of the attractive forces. Steric interference such as clashes or overlaps of portions of the inhibitor being selected or constructed with protein moieties will prevent binding unless resolvable by conformational changes. The binding strength of a potential inhibitor thus created may be assessed by comparing favourable and unfavourable interactions on the computer screen or by using computational methods implemented in commercial computer programs.

Conformational freedom of the potential inhibitor and amino acid side chains of the ATPase should be taken into account. Accessible conformations of a potential inhibitor may be determined using known rules of molecular geometry, notably torsion angles, or computationally using computer programs having implemented procedures of molecular mechanics and/or dynamics or quantum mechanics or combinations thereof.

A potential inhibitor is at least partially complementary to at least a portion of the active site of the ATPase in terms of shape and in terms of hydrophilic or hydrophobic properties.

Databases of chemical structures (e. g. Cambridge structural database or from Chemical Abstracts Service; for a review see: Rusinko (1993) Chem. Des. Auto. News 8,44-47) may be used to varying extents. In a totally automatic embodiment, all structures in a data base may be compared to the active site or to the binding pockets of the ATPase for complementarity and lack of steric interference computationally using the processor of the computer and a suitable computer program. In this case, computer modelling which comprises manual user interaction at a computer screen may not be necessary. Alternatively, molecular fragments may be selected from a data base and assembled or constructed on a computer screen e. g. manually. Also, the ratio of automation to manual interaction by a person skilled in the art in the process of selecting may vary a lot. As computer programs for drug design and docking of molecules to each other become better, the need for manual interaction decreases.

A preferred approach of selecting or identifying potential inhibitors of type NC P-type ATPases makes use of the structure of the pig Na+, K+ ATPase of this invention.

Analogously to the principles of drug design and computer modelling outlined above, chemical structures or fragments thereof may be selected or constructed based on non-covalent interactions with the potential inhibitor with the regulatory binding pocket or the Na+, K+ binding or the H+, K+ binding sites of an type NC P-type ATPase.

Potential inhibitors may be selected or designed such that they interfere with binding of and organic compound bound by the ATPase, such as ATP or an ATP analogues such as MgF₄ ^2" present in the crystal structure or alternatively any cat-ions associated with the ATPase such as in the structure (see section relating to the ATPase crystal). Such inhibitors may prevent binding of ATP or ATP analogues or cat-ions to the ATPase.

Programs usable for computer modelling include Quanta (Molecular Simulations, Inc.) and Sibyl (Tripos Associates). Other useful programs are Autodock (Scripps Research Institute, La JoIIa, described in Goodsell and Olsen (1990) Proteins: Structure, Function and Genetics, 8, 195-201 ), Dock (University of California, San Francisco, described in: Kuntz et al. (1982) J. MoI. Biol. 161 ,269-288.

The present invention in an aspect relates to a method for identifying a potential modulator capable of modulating the Na+, K+ translocating activity of a type NC P-typer ATPase, said method comprising the following steps: a. selecting a potential modulator using atomic coordinates in conjunction with computer modelling, wherein said atomic coordinates are the atomic coordinates presented in Figure 18 or wherein the atomic coordinates are selected from a three-dimensional structure that deviates from the tree- dimensional structure presented in figure 18 by a root mean square deviation over protein backbone atoms of not more than 3 A, by docking potential modulators into a set of binding interaction sites in a regulatory binding pocket, the ion transport pathway, the CTS cavity and/or the Na+, K+ binding sites generated by computer modelling and selecting a potential modulator capable of binding to at least one amino acid in said set of binding interaction sites in a regulatory binding pocket or the Na+, K+ binding sites, b. providing said potential modulator and said ATPase c. contacting the potential modulator with said ATPase and d. detecting modulation of Na+, K+ translocating activity of said ATPase by the potential modulator.

In a preferred embodiment docking of potential modulator molecules is performed by employing a three-dimensional structure defined by atomic coordinates of the three dimensional structure presented in Figure 18 and such that said potential modulator is capable of binding to at least one amino acid in the regulatory binding pocket or the Na+, K+ binding sites.

Suitable binding amino acid residues are defined above in relation to the criteria data set.

Any of the above described regions of ATPase, e.g. the regulatory binding pocket, the ion transport pathway, the CTS cavity and/or any of the Na+, K+ binding sites may be a target for modulator binding. Based on the homology to the Ca+ pump highest specificity is expected when the 3^rd Na+ binding site is target. Thus one or more of these regions may be use for indentifying potential modulator molecules. The representation of any one of these regions can be superimposed with models of potentials molecules to indentify a potential molecule that bind at least 1 amino acid in any one of said regions. The evaluation may be performed by manual visualisation or by suitable programs capable of selecting binding molecules based on the representation and the structure of the potential inhibitors.

A more potent modulator may according to the invention be identified if further interactions are present, such as at least 2, more preferably at least 3, even further preferred at least 4 or mostly preferred at least 5 interactions of the potential modulator with the binding amino acid residues are present.

In a preferred embodiment the docking of potential modulator molecules is performed by employing a three-dimensional structure defined by atomic coordinates of the three dimensional structure presented in figure 18 and such that said potential modulates is capable of binding to at least three amino acids in the binding interaction sites in a regulatory binding pocket, the ion transport pathway, the CTS cavity or the Na+, K+ binding sites.

A further method of the invention relates to a method of identifying a potential modulator capable of modulating the enzymatic activity of a type NC P-type ATPase said method comprising the following steps; a. introducing into a computer, information derived from atomic coordinates defining a conformation of a regulatory binding pocket, the ion transport pathway, the CTS cavity and/or the Na+, K+ binding sites of said ATPase, based on three-dimensional structure determination, whereby a computer program utilizes or displays on the computer screen the structure of said conformation; wherein said atomic coordinates are selected from the tree-dimensional structure as presented in figure 18 or atomic coordinates selected from a three-dimensional structure that deviates from any one of the tree- dimensional structure represented by figure 18 by a root mean square deviation over protein backbone atoms of not more than 3 A; b. generating a three-dimensional representation of the regulatory binding pocket or the Na+, K+ binding sites of said ATPase by said computer program on a computer screen; c. superimposing a model of a potential modulator on the representation of said regulatory binding pocket or the Na+, K+ binding sites, d. assessing the possibility of bonding and the absence of steric interference of the potential modulator with the regulatory binding pocket or the Na+, K+ binding sites of the ATPase; e. incorporating said potential compound in an activity assay of said ATPase and f. determining whether said potential compound modulates the enzymatic activity of said ATPase.

As described above the regulatory binding pocket is formed between the transmembrane helixes 5, 7, 8, 10 and β. Thus information derived from the atomic coordinates of at least one of these residues: M 5 ile761 -pro775, M7 Iys833-met852, M8 phe922-thr938, M10 Ieu99θ-trp1009 and β thr28-phe42, are preferably used. The ion transport pathway include the entry channel formed by αM1 (AA82-98), αM2(AA133-146), αM3 (AA325-331 ) and the exit channel formed by αM1 -αM2 (AA 105-122), αM3-M4 (300-318), αM5-6 (AA789-800). Thus information derived from the atomic coordinates of at least one of these residues are preferably used.

Furthermore as the CTS cavity is formed by the trans-membrane helixes of αM1 , αM2, αM4, αM5 and αM6, preferably the atomic coordinates for at least one or more of the following amino acid residues αM1 (108-1 1 1 ), αM2 (120-124), αM4 (304-318); αM5 (780-785) and αM6 (795-799) are used, most preferably one or more of the following amino acid residues: Q1 1 1 , N122, Y308, 1315, E312, F783, L793 and T797.

The cat-ion binding sites formed between the transmembrane helices αM4, αM5 and αM6. Thus information derived from the atomic coordinates of at least one of the following amino acid residues; Ne 321 -Leu330 (αM4), Thr772- Thr781 (αM5) and Thr799-Val810 (αM6) are used and more preferred at least some of the following amino acid residues: GIu 327 (αM4), Ser 775, Asn 776, GIu 779 (αM5), Asp 804 (αM6), Asp 808 (αM6), GIy 848 (αM7), GIn 923 (αM8), Leu 97 (αM1 ), Tyr 771 (αM5), Thr 807 (αM6), GIu 954 (αM9), AspδOδ (αM6) and GIn 923 (αM8) are used according to the invention.

The 3^rd Na+ binding site, lined by amino acid residues: Tyr 771 (αM5), Thr 807 (αM6), Asp 808 (αM6), GIn 923 (αM8) and GIu 954, thus information derived from the atomic coordinates of at least one of these residues may preferably be used according to the invention.

It is preferred that the atomic coordinates employed in the methods according to the invention is determined to a resolution of at least 4 A.

Potential the potential modulator selected according to the invention preferably interacts with to at least 1 , more preferably at least 2, or further preferred as at least 3 amino acids or mostly preferred at least 4 amino acids in the regulatory binding pocket, the ion transport pathway, the CTS cavity and/or the Na+, K+ binding sites. Based on the information obtained from the three dimensional structure, a potential modulator may be a type of molecule which mimicking the short switch helix. Potential possible modulators are chemically synthesised organic compounds, non hydrolyzable peptide analogues, and inorganic compounds.

This knowledge may be used in the method by applying compounds from a preferred group of molecules in the in silica docking experiments. The method according to any of the above claims X-Y, wherein the potential modulator is a non-hydrolyzable peptide analogues, a organic compounds or an inorganic compound.

As mentioned above the effect of a modulator may be both as an inhibitor, an activator or a regulator.

The invention further relates to a method for producing a potential modulator of a type NC P-type ATPase comprising the steps of: a. identification of a potential modulator of a type NC P-type ATPase according to the invention and b. producing said identified potential modulator.

The invention further relates to a method for identifying a selective peptide inhibitor of type NC P-type ATPase comprising the following steps: a. identification of a potential modulator of a type NC P-type ATPase according to the invention, b. contacting the potential peptide modulator with said ATPase, c. contacting the potential peptide modulator with a different ATPase, d. detecting inhibition of ATPase activity of said ATPase by the potential modulator and e. detecting activity of said different ATPase in the presence of said potential modulator.

Specific modulators

As mention in the background section, some features of the Na+, K+ ATPase may based on sequence similarity be conserved in the H+, K+ ATPase, the methods described herein by apply for identification of modulators of the H+, K+ ATPase as well as the Na+, K+ ATPase. By using the knowledge of the various ATPase different parameters may be used to select for modulators that are specific for either of the ATPases.

The specificity may following be tested in vivo or in vitro assays as described in relation to verification of potential inhibitors.

Methods for verification of inhibitors

The activity of identified modulators may be verified by established methods. In vitro verification may be demonstrated by binding, inhibition/activation of ATP hydrolytic activity, inhibition Na+, K+, inhibition of auto-phosphorylation and/or inhibition conformational transitions. In vitro verification may be shown by administration of potential inhibitors to cell cultures such as COS cells. In vivo experiments may be performed on mice. The binding is further confirmed by X-ray studies. Such methods are known in the art and an example is described in Example 2 and 3.

An internal modulator of the Na+, K+ ATPase is demonstrated herein as the C-terminal deletion mutants drastically reduced the Na+ affinity, thus the C-terminal peptide is a modulator of the Na+, K+ ATPase, which specifically interferes with the affinity for Na+.

The potential inhibitors can be synthesized according to the methods of organic chemistry. Preferably, compounds from a database have been selected without remodelling, and their synthesis may already be known.

In any event, the synthetic effort needed to find an inhibitor is greatly reduced by the achievements of this invention due to the pre-selection of promising inhibitors by the above methods. Binding of a potential modulator may be determined after contacting the potential inhibitor with the ATPase. This may be done crystallographically by soaking a crystal of the ATPase with the potential inhibitor or by co-crystallisation and determining the crystal structure of the complex. Preferably, binding may be measured in solution according to methods known in the art. More preferably, inhibition of the catalytic activity of the ATPase by the inhibitor is determined e. g. using the assays described in the examples section. Method of treatment

Use of type NC P-tvpe modulators

The type NC P-type ATPase are involved in regulation of a plurality of specialized functions responsible for maintaining cell potential in a plurality of cell types. As described herein modulators of specific ATPase can be identified according to the invention and can be used accordingly. Some examples are provided here below which are not to be interpreted as limiting for the invention.

Depending on the specific situation the modulator may be an activator or an inhibitor.

Both, Na+, K+ ATPases and H+, K+ ATPases belong to the group type NC P-type ATPases. By incorporating knowledge of the differences of the two subtype of ATPase modulators specific for either of the ATPases can be identified, particularly specificity can be determined or confirmed in an assay for verification of the modulator as described above.

Na+, K+ ATPase modulators

As described in the background section the Na+, K+ ATPase is fundamental for cell viability and cardiac glycosides function by blocking of Na+, K+ ATPase activity.

A modulator identified according to the invention may have functionality in treatment of various diseases which are alleviated by modulation of Na+, K+ ATPase activity. By parallel to the use of cardiac glycosides, inhibitors of Na+, K+ ATPase may be used for treatment of congestive heart failure, atrial fibrillation and atrial flutter, and other indications which benefits from strengthened contractions of the heart.

In further embodiments inhibitors of the Na+, K+ ATPase are useful in the treatment of proliferative diseases and/or disorder, including neoplastic cell growth and proliferation, whether malignant or benign. Proliferative diseases and/or disorders include, but are not limited to, premalignant or precancerous lesions, abnormal cell growths, benign tumours and malignant tumours also termed cancer.

Malignant tumours are distinguished from benign tumours by there ability to spread and to invade and/or destroy the normal tissue. Benign tumours expand when growing and push surrounding tissue which may lead to damage of normal tissue, whereas malignant tumours invades the normal structures and thereby damage the healthy tissue. Tumours, tumour tissue and tumour cells may be benign or malignant, whereas Cancers are per definition always malignant.

Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.

A tumour or tumour tissue may also comprise "tumour-associated non-tumour cells", e.g., vascular cells which form blood vessels to supply the tumour or tumour tissue. Non-tumour cells may be induced to replicate and develop by tumour cells, for example, the induction of angiogenesis in a tumour or tumour tissue.

Proliferative diseases and/or disorders (including benign or malignant neoplasm's) may according to the invention have different location, such as in the: prostate, colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital tract.

Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.

Further applications of N+,K+ modulators particular inhibitors are in the treatment of skin disorders, such as for inhibition of tissue fibrosis and stimulation of wound closure.

Further applications of N+,K+ modulators particular inhibitors are in the treatment of migrane.

H+, K+ ATPase modulators

Based on the homology of the Na+, K+ ATPase with the H+, K+ ATPase, modulators identified according to the invention may be used for the treatment of diseases which are ameliorated by modulation of the salt balance in mammalian cell. Diseases which may be treated using a H+, K+ ATPase modulators, preferably an inhibitor identified according to the invention, include but is not limited to dyspepsia, peptic ulcer disease, gastroesohageal reflux disease and Zollinger-Ellison syndrome.

The invention in an aspect relates to a method of treatment of a disease which is associated with the function of a type NC P-type ATPase comprising administrating to a subject in need thereof a therapeutically effective does of a type NC P-type ATPase modulator identified according to the invention.

The method applies for treatment of congestive heart failure, atrial fibrillation or atrial flutter by administration of Na+, K+ ATPase activators.

In different embodiment the method may be used for treatment of to dyspepsia, peptic ulcer disease, gastroesohageal reflux disease or Zollinger-Ellison syndrome by administration of H+, K+ ATPase inhibitor.

Medicament

Pharmaceutical compositions or medicaments containing a compound of the present invention may be prepared by conventional techniques, e.g. as described in Remington: The Science and Practice of Pharmacy 1995, edited by E. W. Martin, Mack Publishing Company, 19th edition, Easton, Pa. The compositions may appear in conventional forms, for example capsules, tablets, aerosols, solutions, suspensions or topical applications.

An aspect of the invention relates to a medicament comprising a modulator of a type NC P-type ATPase identified according to the invention. In a preferred embodiment the modulator is an inhibitor of the pig renal Na+,K+ ATPase. In a further preferred embodiment the modulator is an inhibitor of a human Na+, K+ ATPase

In an embodiment the medicament is for the treatment of heart failure, atrial fibrillation or atrial flutter.

In a preferred embodiment the medicament is for treatment of a proliferative disease or disorder, such as benign and malignant tumours. The medicament is preferably for treatment of a cancer selected from the group of: carcinoma, lymphoma, blastoma, sarcoma and leukemia or lymphoid malignancies.

In a further embodiment the medicament is for treatment of skin disorders.

In a even further embodiment the medicament is for treatment of migrane.

In an embodiment the medicament is for the treatment of dyspepsia, peptic ulcer disease, gastroesohageal reflux disease or Zollinger-Ellison syndrome

Administration forms

The main routes of drug delivery, in the treatment method are intravenous, oral, and topical. Other drug-administration methods, such as subcutaneous injection or via inhalation, which are effective to deliver the drug to a target site or to introduce the drug into the bloodstream, are also contemplated.

The mucosal membrane to which the pharmaceutical preparation of the invention is administered may be any mucosal membrane of the mammal to which the biologically active substance is to be given, e.g. in the nose, vagina, eye, mouth, genital tract, lungs, gastrointestinal tract, or rectum, preferably the mucosa of the nose, mouth or vagina.

Compounds of the invention may be administered parenterally, that is by intravenous, intramuscular, subcutaneous intranasal, intrarectal, intravaginal or intraperitoneal administration. The subcutaneous and intramuscular forms of parenteral administration are generally preferred. Appropriate dosage forms for such administration may be prepared by conventional techniques. The compounds may also be administered by inhalation, which is by intranasal and oral inhalation administration. Appropriate dosage forms for such administration, such as an aerosol formulation or a metered dose inhaler, may be prepared by conventional techniques.

The compounds according to the invention may be administered with at least one other compound. The compounds may be administered simultaneously, either as separate formulations or combined in a unit dosage form, or administered sequentially. Formulations

Whilst it is possible for the compounds or salts of the present invention to be administered as the raw chemical, it is preferred to present them in the form of a pharmaceutical formulation. Accordingly, the present invention further provides a pharmaceutical formulation, for medicinal application, which comprises a compound of the present invention or a pharmaceutically acceptable salt thereof, as herein defined, and a pharmaceutically acceptable carrier therefore.

The compounds of the present invention may be formulated in a wide variety of oral administration dosage forms. The pharmaceutical compositions and dosage forms may comprise the compounds of the invention or its pharmaceutically acceptable salt or a crystal form thereof as the active component. The pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, wetting agents, tablet disintegrating agents, or an encapsulating material.

Preferably, the composition will be about 0.5% to 75% by weight of a compound or compounds of the invention, with the remainder consisting of suitable pharmaceutical excipients. For oral administration, such excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like.

In powders, the carrier is a finely divided solid which is a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired. Powders and tablets preferably contain from one to about seventy percent of the active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term "preparation" is intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be as solid forms suitable for oral administration.

Drops according to the present invention may comprise sterile or non-sterile aqueous or oil solutions or suspensions, and may be prepared by dissolving the active ingredient in a suitable aqueous solution, optionally including a bactericidal and/or fungicidal agent and/or any other suitable preservative, and optionally including a surface active agent. The resulting solution may then be clarified by filtration, transferred to a suitable container which is then sealed and sterilized by autoclaving or maintaining at 98-100 C for half an hour. Alternatively, the solution may be sterilized by filtration and transferred to the container aseptically. Examples of bactericidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01 %). Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.

Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavours, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

Other forms suitable for oral administration include liquid form preparations including emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions, toothpaste, gel dentrifrice, chewing gum, or solid form preparations which are intended to be converted shortly before use to liquid form preparations. Emulsions may be prepared in solutions in aqueous propylene glycol solutions or may contain emulsifying agents such as lecithin, sorbitan monooleate, or acacia. Aqueous solutions can be prepared by dissolving the active component in water and adding suitable colorants, flavours, stabilizing and thickening agents. Aqueous suspensions can be prepared by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well known suspending agents. Solid form preparations include solutions, suspensions, and emulsions, and may contain, in addition to the active component, colorants, flavours, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

The compounds of the present invention may be formulated for parenteral administration (e.g., by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol. Examples of oily or nonaqueous carriers, diluents, solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate), and may contain formulatory agents such as preserving, wetting, emulsifying or suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water.

Oils useful in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils useful in such formulations include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides; (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-. beta.-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.

The parenteral formulations typically will contain from about 0.5 to about 25% by weight of the active ingredient in solution. Preservatives and buffers may be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

The compounds of the invention can also be delivered topically. Regions for topical administration include the skin surface and also mucous membrane tissues of the vagina, rectum, nose, mouth, and throat. Compositions for topical administration via the skin and mucous membranes should not give rise to signs of irritation, such as swelling or redness.

The topical composition may include a pharmaceutically acceptable carrier adapted for topical administration. Thus, the composition may take the form of a suspension, solution, ointment, lotion, sexual lubricant, cream, foam, aerosol, spray, suppository, implant, inhalant, tablet, capsule, dry powder, syrup, balm or lozenge, for example. Methods for preparing such compositions are well known in the pharmaceutical industry.

The compounds of the present invention may be formulated for topical administration to the epidermis as ointments, creams or lotions, or as a transdermal patch. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also containing one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or colouring agents. Formulations suitable for topical administration in the mouth include lozenges comprising active agents in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing the active ingredient in finely-divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with the aid of suitable machinery, with a greasy or non-greasy base. The base may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives or a fatty acid such as steric or oleic acid together with an alcohol such as propylene glycol or a macrogel. The formulation may incorporate any suitable surface active agent such as an anionic, cationic or non-ionic surfactant such as a sorbitan ester or a polyoxyethylene derivative thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.

Lotions according to the present invention include those suitable for application to the skin or eye. An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those for the preparation of drops. Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturizer such as glycerol or an oil such as castor oil or arachis oil.

Transdermal Delivery

The pharmaceutical agent-chemical modifier complexes described herein can be administered transdermally. Transdermal administration typically involves the delivery of a pharmaceutical agent for percutaneous passage of the drug into the systemic circulation of the patient. The skin sites include anatomic regions for transdermally administering the drug and include the forearm, abdomen, chest, back, buttock, mastoidal area, and the like.

Transdermal delivery is accomplished by exposing a source of the complex to a patient's skin for an extended period of time. Transdermal patches have the added advantage of providing controlled delivery of a pharmaceutical agent-chemical modifier complex to the body. See Transdermal Drug Delivery: Developmental Issues and Research Initiatives, Hadgraft and Guy (eds.), Marcel Dekker, Inc., (1989); Controlled Drug Delivery: Fundamentals and Applications, Robinson and Lee (eds.), Marcel Dekker Inc., (1987); and Transdermal Delivery of Drugs, VoIs. 1 -3, Kydonieus and Berner (eds.), CRC Press, (1987). Such dosage forms can be made by dissolving, dispersing, or otherwise incorporating the pharmaceutical agent-chemical modifier complex in a proper medium, such as an elastomeric matrix material. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.

Passive Transdermal Drug Delivery

A variety of types of transdermal patches will find use in the methods described herein.

For example, a simple adhesive patch can be prepared from a backing material and an acrylate adhesive. The pharmaceutical agent-chemical modifier complex and any enhancer are formulated into the adhesive casting solution and allowed to mix thoroughly. The solution is cast directly onto the backing material and the casting solvent is evaporated in an oven, leaving an adhesive film. The release liner can be attached to complete the system.

Alternatively, a polyurethane matrix patch can be employed to deliver the pharmaceutical agent-chemical modifier complex. The layers of this patch comprise a backing, a polyurethane drug/enhancer matrix, a membrane, an adhesive, and a release liner. The polyurethane matrix is prepared using a room temperature curing polyurethane prepolymer. Addition of water, alcohol, and complex to the prepolymer results in the formation of a tacky firm elastomer that can be directly cast only the backing material.

A further embodiment of this invention will utilize a hydrogel matrix patch. Typically, the hydrogel matrix will comprise alcohol, water, drug, and several hydrophilic polymers.

This hydrogel matrix can be incorporated into a transdermal patch between the backing and the adhesive layer.

The liquid reservoir patch will also find use in the methods described herein. This patch comprises an impermeable or semipermeable, heat sealable backing material, a heat sealable membrane, an acrylate based pressure sensitive skin adhesive, and a siliconized release liner. The backing is heat sealed to the membrane to form a reservoir which can then be filled with a solution of the complex, enhancers, gelling agent, and other excipients.

Foam matrix patches are similar in design and components to the liquid reservoir system, except that the gelled pharmaceutical agent-chemical modifier solution is constrained in a thin foam layer, typically a polyurethane. This foam layer is situated between the backing and the membrane which have been heat sealed at the periphery of the patch.

For passive delivery systems, the rate of release is typically controlled by a membrane placed between the reservoir and the skin, by diffusion from a monolithic device, or by the skin itself serving as a rate-controlling barrier in the delivery system. See U.S. Pat. Nos. 4,816,258; 4,927,408; 4,904,475; 4,588,580, 4,788,062; and the like. The rate of drug delivery will be dependent, in part, upon the nature of the membrane. For example, the rate of drug delivery across membranes within the body is generally higher than across dermal barriers. The rate at which the complex is delivered from the device to the membrane is most advantageously controlled by the use of rate-limiting membranes which are placed between the reservoir and the skin. Assuming that the skin is sufficiently permeable to the complex (i.e., absorption through the skin is greater than the rate of passage through the membrane), the membrane will serve to control the dosage rate experienced by the patient.

Suitable permeable membrane materials may be selected based on the desired degree of permeability, the nature of the complex, and the mechanical considerations related to constructing the device. Exemplary permeable membrane materials include a wide variety of natural and synthetic polymers, such as polydimethylsiloxanes (silicone rubbers), ethylenevinylacetate copolymer (EVA), polyurethanes, polyurethane- polyether copolymers, polyethylenes, polyamides, polyvinylchlorides (PVC), polypropylenes, polycarbonates, polytetrafluoroethylenes (PTFE), cellulosic materials, e.g., cellulose triacetate and cellulose nitrate/acetate, and hydrogels, e.g., 2- hydroxyethylmethacrylate (HEMA). Other items may be contained in the device, such as other conventional components of therapeutic products, depending upon the desired device characteristics. For example, the compositions according to this invention may also include one or more preservatives or bacteriostatic agents, e.g., methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkonium chlorides, and the like. These pharmaceutical compositions also can contain other active ingredients such as antimicrobial agents, particularly antibiotics, anesthetics, analgesics, and antipruritic agents.

The compounds of the present invention may be formulated for administration as suppositories. A low melting wax, such as a mixture of fatty acid glycerides or cocoa butter is first melted and the active component is dispersed homogeneously, for example, by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and to solidify.

The active compound may be formulated into a suppository comprising, for example, about 0.5% to about 50% of a compound of the invention, disposed in a polyethylene glycol (PEG) carrier (e.g., PEG 1000 [96%] and PEG 4000 [4%].

The compounds of the present invention may be formulated for vaginal administration. Pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

The compounds of the present invention may be formulated for nasal administration. The solutions or suspensions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray. The formulations may be provided in a single or multidose form. In the latter case of a dropper or pipette this may be achieved by the patient administering an appropriate, predetermined volume of the solution or suspension. In the case of a spray this may be achieved for example by means of a metering atomizing spray pump.

The compounds of the present invention may be formulated for aerosol administration, particularly to the respiratory tract and including intranasal administration. The compound will generally have a small particle size for example of the order of 5 microns or less. Such a particle size may be obtained by means known in the art, for example by micronization. The active ingredient is provided in a pressurized pack with a suitable propellant such as a chlorofluorocarbon (CFC) for example dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide or other suitable gas. The aerosol may conveniently also contain a surfactant such as lecithin. The dose of drug may be controlled by a metered valve. Alternatively the active ingredients may be provided in a form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidine (PVP). The powder carrier will form a gel in the nasal cavity. The powder composition may be presented in unit dose form for example in capsules or cartridges of e.g., gelatin or blister packs from which the powder may be administered by means of an inhaler.

When desired, formulations can be prepared with enteric coatings adapted for sustained or controlled release administration of the active ingredient.

The pharmaceutical preparations are preferably in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

Pharmaceutically acceptable salts Pharmaceutically acceptable salts of the instant compounds, where they can be prepared, are also intended to be covered by this invention. These salts will be ones which are acceptable in their application to a pharmaceutical use. By that it is meant that the salt will retain the biological activity of the parent compound and the salt will not have untoward or deleterious effects in its application and use in treating diseases.

Pharmaceutically acceptable salts are prepared in a standard manner. If the parent compound is a base it is treated with an excess of an organic or inorganic acid in a suitable solvent. If the parent compound is an acid, it is treated with an inorganic or organic base in a suitable solvent. The compounds of the invention may be administered in the form of an alkali metal or earth alkali metal salt thereof, concurrently, simultaneously, or together with a pharmaceutically acceptable carrier or diluent, especially and preferably in the form of a pharmaceutical composition thereof, whether by oral, rectal, or parenteral (including subcutaneous) route, in an effective amount.

Examples of pharmaceutically acceptable acid addition salts for use in the present inventive pharmaceutical composition include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, p-toluenesulphonic acids, and arylsulphonic, for example.

Detailed description of the drawings

Figure 1 Functional characterization of Na⁺,K⁺-ATPase used for crystallization, a,

The reaction cycle of the Na⁺,K⁺-ATPase²³ showing in red the form crystallized, b, Demonstration of ⁸⁶Rb⁺ occlusion in the MgF₄ ^2"-bound form of the pig renal Na⁺, K⁺- ATPase from outer medulla. The dissociation of ⁸⁶Rb⁺ from membranous enzyme pre- incubated with ⁸⁶Rb⁺ in the absence (lowest panel) or presence (middle panel) of MgF₄ ^2", and from d₂E₈-solubilized enzyme preincubated in the presence of MgF₄ ^2" (uppermost panel), was followed at 25 °C. Error bars, lllll n = 3.

Figure 2 Crystal packing and electron-density map. a, Crystal lattice viewed down the a axis. The unit cell is indicated, b, c, Asymmetric unit viewed down the c and a axis, respectively. The α-, β- and γ-subunits are coloured blue, wheat and red, respectively, d, Experimental electron-density map of the αβy complex calculated at 3.5 A and contoured at 1.Oσ. The α- and γ-subunits are shown in cyan mesh (backbone indicated in yellow and red, respectively) and the extracellular part of the β-subunit as wheat mesh.

Figure 3 Architecture of the Na⁺,K⁺-ATPase alpha-beta-gamma complex and the K⁺/Rb⁺ sites. The cytoplasmic side is up in all panels, a, The α-, β- and γ-subunits are coloured blue, wheat and red, respectively. Helices are represented by cylinders and β- strands by arrows. The D-ectodomain is shown by surface representation of the experimental electron density. The transmembrane segments of the α-subunit are numbered (yellow) starting with the most N-terminal. The small C-terminal helix (S, for switch) is light red. Mg²⁺, MgF₄ ^2' and Rb⁺ ions are grey, orange and pink, respectively. b, The red mesh (anomalous difference Fourier map) and the green mesh (omit F₀-F₀ electron density map) show the positions of Rb⁺ and K⁺ ions, respectively. Oxygen- containing side chains within and close to the coordination sphere are shown, c, Structural alignment of the Na⁺,K⁺-ATPase (blue) with SERCA (yellow; PDB 1WPG) in the E2 MgF₄ ² forms. Yellow and magenta spheres represent water molecules in SERCA and K7Rb⁺ ions in Na⁺,K⁺-ATPase, respectively, d, Interaction between GIu 327 (αM4) and Leu 97 (αM1 )¹⁷. The cyan mesh indicates the electron density map (2F₀-Fc) of αM1 contoured at ^"I .Oσ.

Figure 4 Interactions between alpha and beta and between alpha and gamma transmembrane helices, a, βM (wheat colour) shown in stick representation. The electron-density map (2F_O-F_C) shown as cyan mesh was calculated at 3.5 A and contoured at 1 .Oσ. PL, phospholipid head group modelled as phosphatidylcholine, b, γM represented by sticks and coloured according to the sequence alignment shown in c. The experimental electron density shown as cyan mesh is contoured at 1 .Oσ. Y23 indicates the start of the visible part of the γ-subunit. In a and b the transmembrane helices of the alpha-subunit are shown in blue with yellow numbering, c, The V-type ATPase rotor ring c subunit and sequence alignment with underlined signature sequence of the FXYD family. Structural elements showing sequence homology with the gamma-subunit are coloured in light gray. The sequences are identified by SEQ ID NO 17 and 18.

Figure 5 Structural comparison of the Na⁺, K⁺ ATPase with the Ca²⁺-ATPaSe. a,

The Na⁺, K⁺ ATPase (blue) and the Ca²⁺-ATPaSe (yellow) were structurally aligned using as fix points the highly similar A domain and P domain. In this view the A domain is not apparent. The arrow indicates a protrusion of the P domain unique to the Na⁺, K⁺ ATPase. b, Top view of the aligned transmembrane regions seen from the cytoplasmic surface. Note the triangular pocket between βM, αM7 and αM10 that accommodates the small helix of the C terminus of the α-subunit. Figure 6 The C-terminal switch, a, Side view of the transmembrane domain with the C-terminal switch region shown in the upper right part. In addition, residues of possible importance for interaction between alpha and gamma subunits and between alpha- and beta-subunits (SYGQ) and the third Na⁺ site, as well as the modelled phosphatidylcholine head group (PL), are indicated, b, Top view from the cytoplasmic side of the C-terminal intrusion into the transmembrane region. Possible direct contacts between the tyrosine residues and Lys 766 and Arg 933 are represented by dashed lines, c, Functional analysis of the truncated enzyme DKETYY (open symbols) and the wild type (closed symbols). The apparent affinities for Na⁺, K⁺, ATP and vanadate are indicated as K_{0 5} (the concentration giving half-maximum effect) values in the relevant panels. Error bars, s.e.m.; n = 3-6). The inhibition seen at high K⁺ concentrations for ΔKETYY but not for the wild type, in the upper right panel, is a consequence of the reduced Na⁺ affinity, allowing K⁺ to compete efficiently with Na⁺ at the sites of the E1 form¹, e, Cartoon of the proposed functional elements of the C-terminal switch. The red double arrow indicates a change in membrane potential. The pull/push exerted by the switch on M5 may affect the affinity of the third electrogenic Na⁺ site. The positive charges of the three arginines of αM10 at the membrane surface suggested to sense the membrane potential are indicated in blue. The interaction between the C-terminal tyrosine residue and M5 is indicated by lines.

Figure 7. The third Na+ site. The central part of Fig. 6a shown enlarged with the two bound Rb+ ions (pink) and labelling of residues proposed to be involved with the third Na+ site in the E1 conformation. The cytoplasmic side is up. GIn 923 may also be involved in K+/Rb+ binding in E2 conformation.

Figure 8. Experimental density maps of the cytosolic part of the β-subunit. The cytosolic part of the β-subunit. a, Experimental density maps contoured at 1 .Oσ (α- subunit cyan mesh, β-subunit wheat coloured mesh) and at 0.5σ (yellow). The lower contoured density belonging to the N-terminal cytosolic part of the β-subunit indicates that it has direction around the cytosolic part of the α-subunit toward αM1 . b, same as a viewed along an axis perpendicular to that in a.

Figure 9. Homology of the β-ectodomain to the interleukin receptor.

The β-ectodomain shows weak homology to the interleukin receptor, a, The residues 200 to 292 of the type-1 interleukin receptor⁵⁶ docked into the electron density map corresponding to the extracellular part of the β-subunit. b, Same as a viewed along an axis perpendicular to that in a. c, A Blast search was performed against the PDB database, using the primary structure of the Na⁺,K⁺-ATPase β-subunit isoform 2 from Danio rerio. A weak hit was found, with low sequence homology to a human type-1 interleukin receptor. The Blast result is shown with the domain used for docking in green. The alignment includes the sequences identified by SEQ ID NO 19 and 20.

Figure 10. C-terminal sequence variation Alignment of the Na⁺,K⁺-ATPase α-subunit C-terminal sequences of pig isoform 1 (NKA1 ) (SEQ ID NO 21 ), human isoforms 1 -4 (NKA1 -4) (SEQ ID NO 22-25),, and the two human H⁺, K⁺- AT Pases (AT12A and AT4A) (SEQ ID NO 26 and 27). The isoform divergent region is shown boxed. Various colours are used to distinguish hydrophobic residues (FVIWCM), positive residues, negative residues, polar residues, tyrosine, glycine, histidine, and proline.

Figure 11. Documentation of the presence of γ_a- and γ_b-subunits and crystal dimensions, a, b, Tricine-SDS polyacrylamide gel electrophoresis revealing the presence of γ_a- and γ_b-subunits in the membranous enzyme and in crystals redissolved subsequent to washing by centrifugation. These splice variants deviate only in the N- terminal 6 to 7 amino acid residues, a, Amido black stained gel showing the protein components present in the purified membranous enzyme used for solubilization and crystallization and in the redissolved crystals. Five to six weeks aged crystals were used. The α-subunit of the redissolved crystals migrates as a mixture of monomer and higher oligomers, whereas the membranous enzyme migrates mainly as monomer, b, Western blot, using a γ-specific polyclonal antibody, of a gel run in parallel with the same material as in a. For the redissolved crystals staining is seen at migration positions corresponding to both γ- and α-subunits, the latter indicating very close SDS- resistant association with the α-subunit. c, A crystal of the Na⁺,K⁺-ATPase, the dimensions have been labelled next to the edges.

Figure 12 Binding of Tantalum bromide clusters in the Na+, K+ ATPase.

The figure shows that the Tantalum Bromide clusters bind both in the entry and exit cannels in close proximity of His286 and Glu282 of M3 and Phe90 in M1 .

Figure 13 Ouabain binding in the E2P model of the pig renal Na⁺, K⁺- ATPase. A. View of the transmembrane region of the pig renal Na⁺,K⁺-ATPase in the E2P state from the extracellular side of the membrane. The cation binding site is visualized down the open cation exit pathway. B. Docking model of ouabain within the extracellular cavity on the surface of the Na⁺,K⁺-ATPase E2P homology model. The residues described as important for CTS binding are shown on the surface of the protein. C. Same as B but with cartoons and sticks representations to visualize the interactions of residue side chains with the ouabain molecule. Amino acids N122(M2), E312 (M4), 1315 (M4), F783 (M5) and T797 (M6) are located close to the ouabain molecule.

Figure 14 Binding of bufalin in the Na+, K+ ATPase.

Visualisation of the electron density around the bufalin binding site in the structure of the Na⁺,K⁺-ATPase:bufalin complex in the E2P state at 4.1 A resolution. The preliminary map obtained after molecular replacement and one round of bulk solvent correction shows an additional density in the cavity formed at the extracellular side of the pump. Residues involved in cardiac glycosides binding by several mutagenesis studies - such as Gln1 1 1 and Asn122 in TM1 and TM2, Glu312 and Ne315 in TM4, Phe783 and Thr797 in the TM5-TM6 loop - are lying close to this additional density, therefore providing an interaction network to stabilise the bufalin molecule in its putative binding pocket.

Figure 15

Ta₆Bn₂ inhibits the activity of Na,K-ATPase. ATP hydrolytic activity of the Na,K-ATPase was measured at 40 mM NaCI and 10 mM KCI in the absence (1 ) or in the presence of 20μM Ta₆Br₁₂ (± StDev).

Figure 16 Structure of the sodium pump.

The transmembrane region through which three sodium ions are transported out and two potassium ions in per ATP molecule is indicated by the arrows. On the right is a close up with the C-terminus represented as sticks. Note the two tyrosines in the pocket.

Figure 17

Activity of N+, K+ ATPase mutants.

A) show the normal profile of Na+ transport of the N+, K+ ATPase in the presence of absence of K+ as a function of the membrane potential. Mutations of specific amino acid residues as described in Example 8, introduces ion flow in the opposite direction (C and E) as seen by the altered profile. The KW mutant displays high activity also at low potential (D), whereas the S940E shows a profile close to the wild type (B). (F). The charge moved at the off-set of pulses from -200 mV to 6OmV going to -50 mV sigmoidally fitted and normalized. The curves for the C-terminal mutants are all shifted to the left, suggesting impaired sodium occlusion. The D999H mutant displays an intermediate phenotype when compared to wild type and the R1002Q and YYAA mutants.

As seen in figure 17C, the YYAA mutant display ion transport in the opposite directions, which with out being bound by the theory is believed to be caused by flow of Na+ or H+ ions across the membrane through the ion channel of the ATPase. The KW mutant which is inspired by the sequence of ATPases in certain bacteria and worms, is capable of maintaining the K+ dependent ion transport even at a very low membrane potential (17D). Both D999 and R1002Q are severely inhibited as seen in figure 17F, showing inhibition of Na+ release.

Figure 18. Atomic coordinates

Data including atomic coordinates for the crystal structure of pig Na+, K+ ATPase. The figure lists the atomic structure coordinates for the pig Na+, K+ ATPase as derived by X-ray diffraction from co-crystals of that complex. The data includes information of two complexes (the asymmetric unit) which consists of peptides denoted A, B, G and C, D H respectively, which was used to interpret the data. The structure includes data relating to amino acids 19-1016 of the alpha-subunit (A and C), 28-73 of the beta- subunit (B and D and 23-51 of the gamma (G and H) subunit.

Examples

Example 1

Rb⁺-occl uded enzyme with bound magnesium fluoride

As a congener of K⁺, Rb⁺ is specifically recognized by the Na⁺,K⁺-ATPase and transported into the cell. In the E2 state Rb⁺ is occluded^{23 14}, as indicated by a very slow dissociation of ⁸⁶Rb⁺ in the absence of ATP (lower panel of Fig. 1 b). A Rb⁺- occluded enzyme ([Rb₂]E2 MgF₄ ^2") can also be formed in the presence of the phosphate analogue MgF₄ ^2" (Fig. 1 b, middle panel). In the absence of MgF₄ ², the de- occlusion of ⁸⁶Rb⁺ is markedly accelerated by ATP ², whereas this effect is abolished by binding of the phosphate analogue. The MgF₄ ²-bound enzyme is expected to represent the [K₂]E2 P, product state following dephosphorylation¹⁵ (Fig. 1 a). Hence, in the reaction sequence leading from the non-occluded K₂E2P state to the [K₂]E2 state the ions become occluded before the final Pj dissociation step, probably in relation to the hydrolysis of the covalent bond¹³. Following solubilization with the detergent Ci₂E₈ the occluded [Rb₂]E2 MgF₄ ² enzyme remained stable (Fig. 1 b, upper panel), and this form was used for crystallization.

Overall structure

Figure 2 and Fig. 3a present the crystal lattice and the overall architecture of the α-β-γ- complex. The crystal lattice consists of layers of membrane-spanning regions stacked on each other (Fig. 2a). Between the membrane layers, the molecules are in contact through interactions between the cytoplasmic domains of molecules that are oriented head-to-head. The extracellular parts containing the glycosylated regions of the β- subunit do not contribute to the interlayer contact, but point into large solvent-filled channels of the crystal. The α-chain adopts a topology similar to that of the Ca²⁺- ATPase with three characteristic cytoplasmic domains, the actuator (A), nucleotide- binding (N) and phosphorylation (P) domains, which together with all ten transmembrane segments, αM1-αM10, are well resolved in the electron-density maps (Fig. 2d). The model consists of α-subunit residues 19 to 1016 (complete C terminus, 998 residues), γ-subunit residues 28 to 73 (46 residues, only the transmembrane segment), and a tentative assignment of γ-subunit residues 23 to 51 (29 residues, only the transmembrane segment). The asymmetric unit of the crystal lattice consists of two α-β-γ units with limited contact between the A domains (Fig. 2b, c). There is no contact between the membrane parts of the α-subunits. The only membrane domain interaction occurs between β-subunits that are oppositely oriented relative to the membrane plane: an interaction that does not exist in the native membrane.

The occluded Rb⁺/K⁺-binding sites of the α-subunit

The anomalous scattering properties of rubidium allowed the accurate identification of two Rb⁺ sites in the α -subunit by an anomalous difference Fourier map (Fig. 3b, and Table 1 ). With the enzyme having K⁺ bound instead of Rb⁺, it was possible under identical crystallization conditions to obtain crystals diffracting to 4 A (Table 1 ). Two density peaks exceeding a 4σ level in the annealed omit map indicate the positions of the bound K⁺ ions, which overlap with the Rb⁺ sites (Fig. 3b). This confirms the expectation that K⁺ and Rb⁺ occupy similar sites. Notably, the RbVK⁺ ions are the first counter-ions directly visualized in a P-type ATPase structure. The two sites, here denoted 1 and 2, are found between the transmembrane helices αM4, αM5 and αM6. The Rb⁺ ions are located in a common binding cavity, only ~4 A apart with site 1 slightly closer to the cytoplasmic side of the membrane than site 2. No open pathways leading to the bound ions are apparent, in accordance with an occluded state. The side chains of residues GIu 327 (αM4), Ser 775, Asn 776, GIu 779 (αM5) and Asp 804 (αM6) are sufficiently close to the Rb⁺ ions to donate ligands for binding (Fig. 3b), either directly or through an intervening water molecule. Asp 808 (αM6) is somewhat further away, but could be indirectly involved, and the same holds for GIn 923 (αM8) (see later and Fig. 7]). Asp 804 seems to donate a side-chain oxygen ligand to each Rb⁺ ion (Fig. 3b). GIu 327 is associated exclusively with K7Rb⁺ site 2 and may control the extracellular gate of the occlusion cavity^{16 17}, possibly guided by contact with Leu 97 of αM1 (ref. 17) (Fig. 3d). Most of these residues have been assigned a role in K⁺ interaction by mutagenesis^16"21. Identical residues are found at the corresponding positions in SERCA, except Asp 804 and GIn 923, which are replaced by asparagine and glutamate, respectively. In the E2P state of SERCA, GIu 309 (homologous to GIu 327) is exposed to the lumen for Ca²⁺ release²². The structure of the ion-binding cavity of SERCA in the E2 conformation that is supposed to accommodate the counter- transported protons seems rather similar to that of the K7Rb⁺ cavity described here (Fig. 3c). A higher resolution would, however, be required to reveal subtle differences between the positions of the side chains in the Na⁺,K⁺-ATPase and the Ca²⁺-ATPaSe. Notably, the water molecules identified in the higher resolution E2 MgF₄ ² structure of SERCA¹² do not overlap directly with the Rb⁺ ions observed in Na⁺,K⁺-ATPase (Fig. 3c).

The residues corresponding to GIu 327, Asn 776, GIu 779, Asp 804, Asp 808, and GIn 923 of the Na⁺,K⁺-ATPase all provide oxygen ligands for Ca²⁺ binding in the E1 form of SERCA¹⁰'²³'²⁴, and they are therefore candidates for liganding residues in two of the three Na⁺ sites in the E1 form of the Na⁺,K⁺-ATPase. If these residues do indeed bind the transported Na⁺ ions, then a considerable overlap would seem to exist between the residues that coordinate K⁺ in the E2 form and Na⁺ in the E1 form, supporting the consecutive transport model² in which Na⁺ is released on the extracellular side in exchange for K⁺ being transported to the cytoplasm through the same occlusion cavity and, in more general terms, the alternating access model²⁵.

The β-subunit The transmembrane helix of the β-subunit (βM) is clearly visible in the electron-density map (Fig. 2d). It traverses the membrane with a strong tilt of approximately 45° (Fig. 3a) and makes direct contact with αM7 and αM10 (Figs 3a and 4a). β-M is closest to αM7, and approaches αM10 only near the extracellular end, in agreement with the finding that the β-subunit together with αM7 remains anchored in the membrane when αM8-M10 is released on heat denaturation²⁶. Tyr 39, Phe 42 and Tyr 43 in βM are within interaction distance with αM7 residues around GIy 848, and the conserved glycines in the repeated GXXXG motif of βM (ref. 6) are exposed on the other side of βM (Fig. 4a). A dumb-bell-shaped density present between βM and αM7 may correspond to a phospholipid head group (Fig. 4a, "PL").

The cytosolic amino-terminal part of the β-subunit cannot be modelled, but at low contour-level the density indicates that it continues around the α-subunit (Fig. 8). The first 10-15 residues of the β-ectodomain have been tentatively traced and could come into contact with the αM7-αM8 loop around the SYGQ motif that is found to be crucial for αβ assembly²⁷ (Fig. 6a). Except for this part, it was not possible to build the β- ectodomain, although we find indications of an interleukin-receptor homology (Fig. 9). However, in agreement with electron microscopy data²⁸'²⁹, our density map provides a clear indication that the β-subunit completely covers the extracellular αM5-αM6 and αM7-αM8 loops as a lid (Figs 2d, 3a), which may relate to the essential role of the β- subunit in K⁺ occlusion⁷. We interpret the disorder of the β-ectodomain as a direct consequence of its inherent flexibility in the absence of stabilizing crystal contacts.

The γ-subunit

The transmembrane segment of the γ-subunit (yM) is seen in the electron-density map as a stretch of approximately 30 amino acids with mostly α-helical structure (Fig. 4b). We noticed that the γ-subunit shares a sequence motif with the rotor ring c-subunit of V-type ATPase from Enterococcus hirae³⁰ (Fig. 4c), possibly indicative of a common origin of these subunits. We used this analogy as a first resort in an assignment of DM, aided by the recent nuclear magnetic resonance model of the FXYD1 (ref. 31 ). The density maps further indicate that the extracellular part of the γ-subunit, containing the conserved FXYD motif, moves in between the α- and β-subunits where it may contact the β-subunit (Figs 2d and 4b). γlvl is clearly close to αM9 (Figs 3a and 4b), yet located on the outside of αM9 and not in the groove between αM9 and αM2, where it has been placed in modelling studies that are based on the Ca²⁺-ATPase structure⁸³². Several αM9 residues are within interaction distance of γlvl, including Phe 949, GIu 953,

Leu 957 and Phe 960, in accordance with a mutagenesis study³². The part of the y- chain showing the most intimate interaction with αM9 around GIu 953 contains GIy 41 , which has been found mutated to arginine in familial dominant renal hypomagnesaemia³³ (Fig. 6a).

Unique features of the α-subunit

As in the Ca²⁺-ATPaSe, the αM4 and αM6 helices of the Na⁺,K⁺-ATPase are unwound in the middle, thereby making space for the ions (Fig. 3b), and DM1 shows a characteristic -90° kink near the cytoplasmic surface of the membrane, where it comes into contact with αM3 (Fig. 3d). This contact point may function as a pivot for movement of αM1 in connection with ion binding³⁴. The plant plasma membrane H⁺-ATPase adopts a similar bent structure in M1 (ref. 35), suggesting that it constitutes a general structural motif of P-type ATPases. Significant differences from SERCA are, on the other hand, seen in αM7, which is unwound at GIy 848, resulting in a kink, and at the cytoplasmic end of αM10 (Figs 4a and 5). These differences may be a consequence of the interaction with the β-subunit. Moreover, the C terminus may be influential (Fig. 5b, see further below).

The N domain is smaller than that of SERCA, which has insertions in surface loops, but is otherwise rather similar²⁸'³⁶³⁷. We find the N domain rather loosely associated with the rest of the molecule, rotated away from its interface with the A domain (Fig. 5a).

The architectures of the A domain and P domain are also very similar in the two pumps, the MgF₄ ² in the catalytic site being coordinated by conserved residues from both of these domains. The C-terminal part of the P domain of the Na⁺,K⁺-ATPase contains a 20-residues-long outward-protruding insertion²⁸, which is seen to adopt the form of two small helices connected by a loop, as a possible target for interaction with regulatory proteins (Fig. 5a). The C-terminal extension is crucial for Na⁺ binding

The αM10 helix ends with three arginines (1003-1005) followed by the PGG motif and an extension of eight residues relative to the C terminus of the Ca²⁺-ATPase (SERCAI a isoform). The small α-helix formed by the first part of this extension is accommodated between αM, αM7 and αM10, and the two C-terminal tyrosine residues are recognized by a binding pocket between αM7, αM8 and αM5 (Figs 5b, 6a and 6b). The insertion of Tyr 1015 and Tyr 1016 in this pocket is made possible by the kink of αM7 at GIy 848. Tyr 1016 seems to interact with Lys 766 of αM5 and Arg 933 in the loop connecting αM8 and αM9. This loop also contains Ser 936, a controversial phosphorylation site proposed to be responsible for some of the cAMP-dependent kinase (PKA)-mediated effects on the Na⁺,K⁺-ATPase^{9 38}. Ser 936 is located within interaction distance of Arg 1003 (Fig. 6b). The unexpected features of the C terminus prompted us to study its functional importance by deletion of the five most C-terminal residues (Fig. 6c). The truncated enzyme (ΔKETYY) exhibited an extraordinary 26-fold reduction of the Na+ affinity, yet the affinity for activating K+ was like wild-type (Fig. 6c, upper panels). This is a direct effect of the truncation on the Na+-binding E1 conformation, and not caused by displacement of the E1-E2 conformational equilibrium toward E2. In fact, the conformational equilibrium of ΔKETYY seems to be slightly displaced in the opposite direction towards E1 , because the apparent affinities for ATP (binding preferentially to E1 ) and vanadate (binding only to E2) were found slightly enhanced and reduced, respectively (Fig. 6c, lower panels). The conspicuous and highly Na⁺-selective effect of the DKETYY truncation is reminiscent of the effects observed previously for mutation of Tyr 771 (αM5)³⁹ and Thr 807 (αM6)¹⁸. Together with GIu 954 (αM9) these residues have been suggested to make up a third Na⁺- binding site (Na⁺ sites 1 and 2 probably being formed by almost the same coordinating side chains as the two K7Rb⁺ binding sites)^40"42. We find these residues to cluster and to be lined by Asp 808 (αM6), bridging to KVRb⁺ site 2. In addition, GIn 923 (αM8) is found in the same cluster and could be involved with the third Na⁺ site in the E1 form⁴² (Fig. 6a and Fig. 7). We propose that the direct contact of the C-terminal tail with αM5 and the loop between αM8 and αM9 serves to optimize Na⁺ binding at the third site.

In light of the sensitivity of the Na⁺,K⁺-pump activity to the membrane potential⁴⁵, it is notable that Arg 1003, Arg 1004 and Arg 1005 at the end of αM10, together with Arg 933, Arg 934 and Arg 998, make the area around the C terminus in the membrane edge region highly electropositive (Fig. 6a, b). In various types of voltage-dependent ion channels arginine clusters act as voltage sensors that move in response to membrane depolarization^{43 44}, and in the Na⁺,K⁺-ATPase the arginine cluster associated with the C terminus could function similarly as a control point for a voltage- sensitive switch that alters the relations of the C terminus in its binding pocket during depolarization/repolarization, with consequences for the Na⁺ affinity (Fig. 6d). The proposal of a direct structural and functional relation between the C terminus and the third Na⁺ site is in accordance with the high voltage-sensitivity of the binding and release of one of the three Na⁺ ions⁴⁵. Interestingly, the human α1-α4 isoforms show a compelling pattern of differentiation in the 1003-1005 region (Fig. 10), which may contribute to defining the differential sensitivity of the isoforms to variation in the membrane potential⁴⁵.

Conclusion

The present results provide clear structural evidence for the existence of a state in which the two counter-transported RbVK⁺ ions are occluded, as originally proposed on the basis of kinetic measurements²³. The structural resemblance of the Na⁺,K⁺-ATPase α-subunit to the Ca²⁺-ATPaSe is surprisingly high, even in the cation-binding pocket, thus raising the fundamental issue of how the specific cation selectivity is determined? Our results define a canonical set of cation-binding residues with only two conservative amino acid differences between the Na⁺,K⁺-ATPase and the Ca²⁺-ATPaSe. We find it likely that subtle differences in the positions of the side chains and water molecules also contribute to define the cation selectivity. A unique aspect of the Na⁺,K⁺-ATPase is the non-canonical third Na⁺ site, the location of which is hinted at by the present observations, even though our structure is the RbVK⁺ occluded enzyme. The C terminus of the Na⁺,K⁺-ATPase α-subunit has a previously unknown strategic location, allowing it to affect Na⁺ binding and participate in Na⁺,K⁺-ATPase regulation.

METHODS

Enzyme preparation and biochemical studies Following zonal centcentrifugation⁴⁶ membrane fractions with particularly high specific Na⁺, K⁺-ATPase activity were selected for crystallization experiments. The membranes were incubated in 5 mM RbF (or 20 mM KCI and 5 mM KF for crystallization of K⁺- bound enzyme), 5 mM MgCI₂, 100 mM Λ/-methyl-D-glucamine (NMDG), 40 mM MOPS, pH 7.0. For solubilization of the enzyme, the non-ionic detergent octaethyleneglycol mono-n-dodecylether (Ci₂E₈)¹⁴ was added at a ratio of 1.12 mg Ci₂E₈ per mg membrane protein. Before crystallization, the insoluble material (20-25% of total protein) was removed by ultracentrifugation.

⁸⁶Rb⁺ occlusion in the membranous and soluble enzyme preparations was measured according to the previously described principles¹⁴. Enzyme preincubated with 0.1 mM ⁸⁶Rb⁺ in the presence or absence of magnesium fluoride (2-5 mM) was mixed with a dissociation solution containing an 83-fold excess of non-radioactive Rb⁺ with and without 3 mM ATP at 25 °C. The amount of ⁸⁶Rb⁺ remaining bound to the enzyme was determined at the indicated time intervals by subjecting aliquots of the samples to rapid ionic exchange chromatography, following cooling to 2 °C.

Deletion of the five most C-terminal residues KETYY was carried out by PCR of complementary DNA encoding the rat α1 isoform of the Na⁺,K⁺-ATPase followed by expression in COS cells, and the previously described assays for phosphorylation and ATPase activity¹⁸'⁴⁷ were used for the functional characterization (Fig. 6c). The Na⁺ dependence of phosphorylation (Fig. 6c, upper left panel) was determined in the presence of 2 μM [γ-³²P]ATP in the absence of K⁺. The K⁺ dependence of Na⁺, K⁺- ATPase activity (Fig. 6c, upper right panel) was determined in the presence of 40 mM Na⁺ and 3 mM ATP. The ATP and vanadate dependencies of the Na⁺,K⁺-ATPase activity (Fig. 6c, lower left and right panels, respectively) were determined in the presence of 130 mM Na⁺ and 20 mM K⁺. The ΔKETYY mutant exhibited a maximal catalytic turnover rate of 9720 ± 490 min^"1 (mean ± s.e.m., n = 6) versus 8470 ± 170 min^"1 (ΓΪ = 1 1 ) for the wild type, determined at 37 ⁰C, pH 7.4, in the presence of 3 mM MgATP and saturating Na⁺ and K⁺ concentrations of 130 mM and 20 mM K⁺, respectively.

Crystallization

Crystals were grown by vapour diffusion from hanging drop at 19 °C. Protein solution was mixed with precipitating solution (14% PEG2000mme, 200 mM choline chloride, 4 mM DTT, 4% Glycerol, 4% MPD) in a 1 :1 ratio by adding 4 μl protein, 4 μl precipitating solution, and 0.8 μl 0.1-0.35% β-DDM. The initial precipitate formed was spun down before 2 μl hanging drops were dispensed. The very thin and fragile crystals (Fig. 1 1c) appeared after 3-4 days and grew to their maximum size (0.6 x 0.2 x 0.05 mm³) within a month. The crystals were mounted in Litholoops (Molecular Dimensions) from the mother liquor. Before flash-cooling in liquid nitrogen excess mother liquor was dipped away by gently touching a glass cover slip with the edge of the loop. For heavy-atom derivatization, dry powder of Ta₆Bn₂ ²⁺ or Orange-Pt was dusted directly to the drop until the crystals appeared light green or faint orange, respectively.

Structure determination and analysis

All data sets were collected at 100 K on the end stations X06SA and X10SA at the Swiss Light Source (SLS) in Villingen. The diffraction data were processed and scaled with XDS⁴⁸. The crystal form exhibits P2₁2₁2₁ space-group symmetry with unit cell dimensions a = 68.93 A, b = 261 .5 A and c = 333.8 A. The asymmetric unit contains 2 αβγ complexes. Initial phases were obtained by molecular replacement at 6 A resolution using the program PHASER⁴⁹ and a search model derived from Ca²⁺- ATPase in the E2-AIF₄ form (PDB code 1XP5)¹³. Heavy-atom sites were then identified by difference Fourier maps using the molecular replacement phases and MIRAS (multiple isomorphous replacement with anomalous scattering) phases obtained at 6 A resolution with SHARP/autoSHARP 2.0 ⁵⁰). The MIRAS phases were refined and further extended using DMMULTI⁵¹ and RESOLVE⁵² to 3.5 A resolution, exploiting solvent flattening (75% solvent), two-fold non-crystallographic symmetry-averaging and two-fold inter-crystal averaging, using two data sets exhibiting a low degree of isomorphism. The final experimental map was of sufficient quality to trace the entire model. Model building was performed using O⁵³ and model refinement was performed with CNS1 .2⁵⁴. For Fig. 3c, a data set obtained for Na⁺,K⁺-ATPase with bound K⁺ was used to perform a simulated annealing omit map, calculated at 4 A resolution, using model phases, omitting both Rb⁺ ions and residues in a radius of 4.5 A. All structural representations in this paper were prepared with Pymol (http://www.pymol.org). The coloured figures may be found in Morth et all 2007 (ref. 58) The structure PDB ID: 3B8E

Example 2 Determination of Na⁺, K⁺-ATPase activity

The rate of ATP hydrolysis catalyzed by the isolated membranes is determined at 37 °C by measurement of the amount of Pi liberated over a period of 10 min using the Baginski method (Baginski, E. S., Foa, P. P., and Zak, B. (1967) CHn. Chem. 13, 326- 332). The hydrolysis of ATP is terminated by dilution of 0.5 ml of the reaction mixture in 1 .0 ml of ice-cold 0.5 M HCI, 4 mM ammonium heptamolybdate, 170 mM ascorbic acid. Subsequently, 1.5 ml of 150 mM sodium-m-arsenite, 70 mM sodium citrate, and 0.35 mM CH3COOH are added, and the mixture kept at 37 °C for 10 min to complete the reaction. Measurements of absorbance at 850 nm are related to a standard of known Pi concentration.

To determine the K+ dependence of Na+, K+-ATPase activity measurement of Na+, K+-ATPase activity is performed at 37 °C in a medium containing 30 mM histidine buffer (pH 7.4), 40 mM NaCI, 3 mM ATP, 3 mM MgCI2, 1 mM EGTA, 10 μM ouabain, and various concentrations of K+. Modulators are added in varying concentrations.

Example 3

Determination of H⁺, K⁺-ATPase activity

The same protocol is used as described for Na⁺,K⁺-ATPase activity except that Na+ is absent and the pH is 6.5.

Example 4

Binding of Tantalum bromide clusters to Na+, K+ ATPase.

The tantalum bromide cluster is added to the ATPase and crystals were grown as described above. The clusters were added as a salt (Ta₆Br^]²⁺ x 2 Br ^" ) to the crystals in the drop and equilibrated overnight. The data was collected at the wave length 1 .08 A. This wave length is remote from the absorption edge of Tantalum to reduce the potential danger of radiation damage during data collection. The binding sites identified by the calculation of an anomalous difference Fourier map, using experimentally derived phases. The binding site is shown in figure 12.

Example 5

Oaubain binding in the extracellular site of Na+, K+ ATPase.

To build the E2P homology model of the Na⁺,K⁺-ATPase, the structure of the [Rb]₂E2-

MgF₄ ^2' form of the pig renal Na⁺, K⁺ ATPase is combined with the observed conformation of the Ca²⁺-ATPaSe in the open E2-BeF₃ form (57). The invariant regions between these two E2 states were initially deduced by comparison of the Ca²⁺-ATPaSe structure in the E2-BeF₃ ^' and in the [H₂.₃]E2-AIF₄ ^' forms using the program ESCET (58). Of particular importance, the region consisting of the transmembrane helixs M6 to M10 was found to be significantly invariant. The E2-BeF₃ ^' structure of Ca²⁺-ATPaSe (representing the genuine E2-P state) was then superimposed with the [Rb]₂E2-MgF₄ ^2' form of the sodium pump on the TM6 to TM10 invariant region. The transmembrane helixs TM1 -TM2 and TM3-TM4 and the A domain from the Na⁺, K⁺ -ATPase were then superimosed on the Ca²⁺-ATPaSe E2-BeF₃ ^' structure to impose the E2-P state of the Na⁺, K⁺ ATPase (assuming a conserved mechanism of lumenal/extracellular opening in the E2-P state).

Example 6

Binding of bufalin to Na+, K+ ATPase

Bufalin was co-crystallised with the Na+,K+-ATPase in the E2P state. Crystals were cooled and tested and data processed as described above. The data was collected at the wave lenght 1.0 A. The structure was solved by molecular replacement. The electron density map was improved by a round of bulk solvent correction using Phenix. An additional density present in the map was identified as the possible binding site for bufalin. The results are shown in figure 14.

Example 7

Tantalum Bromide inhibition of Na+, K+ ATPase.

ATP hydrolytic activity of the Na,K-ATPase was measured at 40 mM NaCI and 10 mM

KCI in the absence (1 ) or in the presence (2) of 20 μM Ta₆Bn₂ (± StDev). Assay conditions are 30 mM Histidin, pH: 7,4, 20 mM KCI, 4 mM MgCI2, 3 mM ATP and 40 mM NaCI. Production of Pi was measured by standard calorimetric tests.

Example 8

Voltage dependency of the Na+, K+ ATPase and mutants thereof

Generation of cRNAs encoding the Na⁺, K⁺ -ATPase subunits

Plasmids encoding human alphal , alpha2, alpha3 and betai of the Na⁺,K⁺-ATPase were purchased from Origene (www.origene.com). Mutations Q1 18R and N129D (human alphal numbering) were introduced into all of the alpha isoforms to reduce their ouabain sensitivity (Price and Lingrel 1988, Biochemistry, vol. 27, pp. 8400- 8408.). The sequences encoding Na⁺,K⁺-ATPase subunits were subcloned into the pXOON vector (Jespersen 2002, Biotechniques, vol. 32, pp. 536-8, 540) using Notl (alphal ) or EcoR1/Notl (alpha2 and alpha3). Mutations were introduced by PCR. All constructs were verified by sequencing. From Nhel digested plasmids, cRNAs were transcribed with the mMESSAGE ULTRA kit (Ambion). Mutant YYAA has the two amino acid substitutions Y1020A and Y1021A. Further mutants analysed are R937P, S940E, D999H, R1002Q, and YYKW having the two amino acid substitutions Y1020K and Y1021W. The amino acid numbering refers to the full length human alpha chain very close to SEQ ID NO 1 , and thus Y1020 and Y1021 is equal to Y1015 and Y1016 as numbered in relation to the crystal structure. Like wise R937, S940, D999 and R1002 corresponds to amino acid residues 932, 935, 994 and 997.

Oocytes from Xenopus laevis were isolated and defollicated. Stage V-Vl oocytes were coinjected with 1 ng of betai and 10 ng of one of the alpha subunit cRNAs. After 1 -3 days at 19°C, oocytes were loaded with sodium by incubation for at least two hours in a potassium free solution with 95 mM sodium, 90 mM sulfamic acid, 5 mM Hepes, 10 mM TEACI and 0.1 mM EGTA, pH 7.6. Electrophysiological measurements were performed using the two-electrode voltage-clamp technique with an OC-725C voltage- clamp apparatus (Warner Instrument Corp) in a buffer with 1 15 mM Na, 1 10 mM sulfamic acid, 1 mM MgCI2, 0.5 mM CaCI2, 5 mM BaCI2, 10 mM Hepes, 1 microM ouabain, pH 7.4. Current-voltage curves were determined from the currents activated by replacing 15 mM Na with 15 mM K and running a series of 200 ms voltage steps every 20 mV between -200 and 60 mV. Background was determined from similar voltage steps in potassium-free solution containing 10 mM ouabain.

Charge-movement was determined from similar voltage steps in potassium-free solutions with or wihtout 10 mM ouabain. Data were recorded and analyzed using pClamp 9.2 (Axon Instruments) or Igor (Wavemetrics).

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Claims

Claims

I . A crystal comprising a type NC P-type ATPase.

2. The crystal according to claim 1 , characterized by the cubic space group P2₁2₁2i.

3. The crystal according to claim 1 or 2, wherein the type NC P-type ATPase comprising an α-subunit at least 75 % identical to SEQ ID NO 1.

4. The crystal according to any of the previous claims, wherein the type NC P-type ATPase is a multi subunit APase.

5. The crystal according to any of the previous claims, wherein the type NC P-type ATPase is a mammalian ATPase.

6. The crystal according to any of the previous claims, wherein the type NC P-type ATPase is pig renal Na⁺, K⁺ ATPase.

7. The crystal according to any of the previous claims, which effectively diffracts x- rays for the determination of the atomic coordinates of the ATPase to a resolution better than 6 A, such as better than 5 A, preferably better than 4 A.

8. The cystal according to any of the previous claims wherein the crystal comprise a phosphate analog.

9. The cystal according to claim 8 wherein the phosphate analog is MgF₄ ^2'.

10. The crystal according to any of the previous claims, wherein the crystal comprise one or more cat-ions.

I 1. The crystal according to claim 10, wherein the one or more cat-ions is selected from the group comprising: Na+, K+ and Rb.

12. The crystal according to any of the precious claims, wherein the crystal further comprises two ⁸⁶Rb⁺ ions.

13. A method for purifying a type NC P-type ATPase comprising the steps of: a. obtaining a composition comprising a P-type NC ATPase, b. solubilising said ATPase using a non-ionic detergent and c. purifying said ATPase.

14. The method according to claim 13, wherein the composition comprising a type NC P-type ATPase is isolated from pig kidney.

15. The method according to any of the claims 13-14, wherein the composition comprising the type NC P-type ATPase is a membrane fraction.

16. The method according to claim 15, wherein the membrane fraction is isolated by isopycnic zonal centrifugation

17. The method according to any of the claims 13-16, wherein the non-ionic detergent

18. The method according to claim 17, wherein Ci₂E₈ is added to a ratio of 0.5-2 such as 1 -1 -5 or preferably such as or more preferably 1 -1 .25 and most preferably such as 1.12 mg per mg membrane protein.

19. The method according to any of the claims 13-18, comprising a step of removing insoluble material.

20. The method according to any of the claims 13-19, wherein the composition comprising a type NC P-type ATPase may further comprise one or more cat-ions..

21. The method according to claim 20, wherein the one or more cat-ions are selected from the group comprising: Na+, K+, Mg2+ and Rb+.

22. The method according to claim 20 wherein the solution further comprises N-methyl- D-glycamine (NMDG).

23. A method of growing a crystal comprising a type NC P-type ATPase according to any of the claims 1 -12, comprising the steps of: a. obtaining a composition comprising a type NC P-type ATPase, b. subjecting said composition to a crystallizations environment including PEG 2000mme and c. obtaining crystals comprising a type NC P-type ATPase.

24. A method of growing a crystal comprising a type NC P-type ATPase according to any of the claims 1 -12, comprising the steps of: a. obtaining a composition comprising a type NC P-type ATPase, b. mixing said composition comprising a type NC P-type ATPase with a precipitating solution comprising PEG2000mme, c. growing ATPase crystals by vapour diffusion from hanging drops d. obtaining crystals comprising a type NC P-type ATPase.

25. The method according to claim 24, wherein said composition comprising a type NC P-type ATPase and said precipitating solution are mixed with a β-DDM solution in ratio of 0.5-2 : 0.5-2 : 0.1 -0.5.

26. The method according to claim 25 wherein the ratio is 1 :1 :0.2.

27. The method according to claim 25, wherein the concentration of the β-DDM solution is 0.1 -0.35 %.

28. The method according to any of the claims 23-27, further comprising the steps of: a. isolating an initial precipitate and b. growing these by vapour diffusion from hanging drops.

29. Use of a crystal according to any one of claims 1 -12 for determination of the three dimensional structure of a type NC P-type ATPase.

30. A computer-readable data storage medium comprising a data storage material encoded with at least a portion of the structure coordinates set forth in figure 18.

31 . Use of atomic coordinates as presented in figure 18 or atomic coordinates selected from a three-dimensional structure that deviates form the tree-dimensional structure as presented in figure 18 by a root mean square deviation over protein backbone atoms of not more than 3 A in a method for identifying a potential modulator of a type NC P-type ATPase.

32. A method of identifying a potential modulator of a type NC P-type ATPase by determining binding interactions between the potential modulator and a set of binding interaction sites in a regulatory binding pocket or the Na+, K+ binding sites of a type NC P-type ATPase comprising the steps of: a. generating the spatial structure of the regulatory binding pocket, the ion transport pathway, the CTS cavity and/or the Na+, K+ binding sites on a computer screen using atomic coordinates as presented in figure 18 or atomic coordinates selected from a three-dimensional structure that deviates from the tree-dimensional structure presented in figure 18 by a root mean square deviation over protein backbone atoms of not more than 3 A. b. generating potential modulators with their spatial structure on the computer screen, and c. selecting potential modulators that can bind to at least 1 amino acid residue of the set of binding interaction sites without steric interference.

33. A computer-assisted method for identifying potential modulators of a type NC P- type ATPase using a programmed computer comprising a processor, a data storage system, a data input devise and a data output device, comprising the following steps: a. inputing into the programmed computer through said input device data comprising: atomic coordinates of a subset of the atoms of said ATPase, thereby generating a criteria data set; wherein said atomic coordinates atomic coordinates are selected from the tree-dimensional structure presented in figure 18 or atomic coordinates selected from a three-dimensional structure that deviates from the tree- dimensional structure presented in figure 18 by a root mean square deviation over protein backbone atoms of not more than 3 A, b. comparing, using said processor, the criteria data set to a computer data base of low-molecular weight organic chemical structures stored in the data storage system; and c. selecting from said data base, using computer methods, a chemical structure having a portion that is structurally complementary to the criteria data set and being free of steric interference with the ATPase.

34. A method for identifying a potential modulater capable of modulating the Na+, K+ translocating activity of a type NC P-type ATPase, said method comprising the following steps: a. selecting a potential modulator using atomic coordinates in conjunction with computer modelling, wherein said atomic coordinates are the atomic coordinates presented in figure 18 or wherein the atomic coordinates are selected from a three-dimensional structure that deviates from the tree- dimensional structure presented in figure 18 by a root mean square deviation over protein backbone atoms of not more than 3 A, by docking potential modulators into a set of binding interaction sites in a regulatory binding pocket, the ion transport pathway, the CTS cavity and/or the Na+, K+ binding sites generated by computer modelling and selecting a potential modulator capable of binding to at least one amino acid in said set of binding interaction sites in a regulatory binding pocket, the ion transport pathway, the CTS cavity and/or the Na+, K+ binding sites, b. providing said potential modulator and said ATPase c. contacting the potential modulator with said ATPase and d. detecting modulation of Na+, K+ translocating activity of said ATPase by the potential modulator.

35. The method according to claim 33, wherein docking of potential modulator molecules is performed by employing a three-dimensional structure defined by atomic coordinates of the three dimensional structure presented in figure 18 and such that said potential modulator is capable of binding to at least one amino acids in the binding interaction sites in a regulatory binding pocket, the ion transport pathway, the CTS cavity and/or the Na+, K+ binding sites.

36. A method of identifying a potential modulator capable of modulating the enzymatic activity of a type NC P-type ATPase said method comprising the following steps; a. introducing into a computer, information derived from atomic coordinates defining a conformation of a regulatory binding pocket, the ion transport pathway, the CTS cavity and/or the Na+, K+ binding sites of said ATPase, based on three-dimensional structure determination, whereby a computer program utilizes or displays on the computer screen the structure of said conformation; wherein said atomic coordinates are selected from the tree-dimensional structure as presented in figure 18 or atomic coordinates selected from a three-dimensional structure that deviates from any one of the tree- dimensional structure represented by figure 18 by a root mean square deviation over protein backbone atoms of not more than 3 A; b. generating a three-dimensional representation of the regulatory binding pocket, the ion transport pathway, the CTS cavity and/or the Na+, K+ binding sites of said ATPase by said computer program on a computer screen; c. superimposing a model of a potential modulator on the representation of said regulatory binding pocket, the ion transport pathway, the CTS cavity and/or the Na+, K+ binding sites, d. assessing the possibility of bonding and the absence of steric interference of the potential modulator with the regulatory binding pocket, the ion transport pathway, the CTS cavity and/or the Na+, K+ binding sites of the ATPase; e. incorporating said potential compound in an activity assay of said ATPase and f. determining whether said potential compound modulates the enzymatic activity of said ATPase.

37. The method according to any of the above claims 33-36, wherein information derived from the atomic coordinates of at least one of the following amino acid residues of the regulatory binding pocket: Ne 761 - Ser 775 (M5), Lys 833- Met 852 (M7), Phe916-Thr932 (M8), Leu 990- Tyr 1016 (M10) and Thr 28 - Phe 42 (β peptide) are used.

38. The method according to any of the above claims 37, wherein information derived from the atomic coordinates of at least one of the following amino acid residues of the regulatory binding pocket: Tyr 1016, Lys 766, Tyr 1015, Tyr 1016, Arg 933, GIn 841 , Asn 839, Tyr 1015-1016, Arg 1003 and Ser936 are used.

39. The method according to any of the above claims 33-36, wherein information derived from the atomic coordinates of at least one of the following amino acid residues of the ion transport pathway: αM1 (AA82-98), αM2(AA133-146), αM3 (AA325- 331 ) αM1 -αM2 (AA 105-122), αM3-M4 (300-318), αM5-6 (AA789-800) are. used.

40. The method according to any of the above claims 33-36, wherein information derived from the atomic coordinates of at least one of the following amino acid residues of the CTS cavity: αM1 (108-1 1 1 ), αM2 (120-124), αM4 (304-318); αM5 (780-785) and αM6 (795-799) are used.

41 . The method according to any of the above claims 33-36, wherein information derived from the atomic coordinates of at least one of the following amino acid residues of the CTS cavity: Q1 1 1 , N122, Y308, 1315, E312, F783, L793 and T797.

42. The method according to any of the above claims 33-36, wherein information derived from the atomic coordinates of at least one of the following amino acid residues of the cation binding sites; Ne 321 -Leu330 (αM4), Thr772- Thr781 (αM5) and Thr799-Val810 (αM6) are used.

43. The method according to any of the above claims 33-4042, wherein information derived from the atomic coordinates of at least one of the following amino acid residues: GIu 327 (αM4), Ser 775, Asn 776, GIu 779 (αM5), Asp 804 (αM6), Asp 808 (αM6), GIy 848 (αM7), GIn 923 (αM8), Leu 97 (αM1 ), Tyr 771 (αM5), Thr 807 (αM6), GIu 954 (αM9), Asp808 (αM6) and GIn 923 (αM8) are used.

44. The method according to any of the above claims 33-42, wherein information derived from the atomic coordinates of at least one of the following amino acid residues of the 3^rd Na+, K+ binding site: Tyr 771 (αM5), Thr 807 (αM6), Asp 808 (αM6), GIn 923 (αM8) and GIu 954, are used.

45. The method according to any of the above claims 37-44, wherein the data criteria set or binding interaction set comprise at least 2 or 3 amino acid residues selected from the identified groups.

46. The method according of any of the above claims 33-45, wherein the atomic coordinates are determined to a resolution of at least 4 A.

47. The method according of any of the above claims 33-46, wherein the potential modulator interacts with at least one amino acids in the regulatory binding pocket or the Na+, K+ binding sites.

48. The method according to any of the above claims 33-47, wherein the potential modulator is a non-hydrolyzable peptide analogues, a organic compounds or an inorganic compound.

49. The method according of any of the above claims 33-47, wherein a library of small organic molecules are screened.

50. The method according of any of the above claims 33-47, wherein a library of potential peptide modulators are screened.

51 . The method according to any of the above claims 33-50, wherein the potential modulator is a potential inhibitor.

52. The method according to any of the above claims 33-50, wherein the potential modulator is a potential activator.

53. The method according to any of the above claims 33-50, wherein the potential modulator is a potential regulator.

54. A method for identifying a selective peptide inhibitor of a type NCP-type ATPase comprising the following steps: a. identification of a potential modulator of a type NCP-type ATPase according to any of the claims , b. contacting the potential peptide modulator with said ATPase, c. contacting the potential peptide modulator with a different ATPase, d. detecting inhibition of ATPase activity of said ATPase by the potential modulator and e. detecting activity of said different ATPase in the presence of said potential modulator.

55. A method for producing a potential modulator of a type NCP-type ATPase comprising the steps of: a. identification of a potential modulator of a type NCP-type ATPase according to any of the previous claims 33-50 and b. producing said identified potential modulator.

56. A medicament comprising a modulator of a type I P-type ATPase identified according to any of the claims 33-54.

57. The medicament according to claim 56, wherein the modulator is an inhibitor of a Na+, K+ ATPase.

58. The medicament according to claim 56, wherein the modulator is an inhibitor of a H₊, K₊ ATPase.

59. The medicament according to claim 56 or 57, wherein the medicament is for treatment of congestive heart failure, atrial fibrillation or atrial flutter.

60. The medicament according to claim 57 or 58, wherein the medicament is for treatment of dyspepsia, peptic ulcer disease, gastroesohageal reflux disease or Zollinger-Ellison syndrome.

61. The medicament according to claim 57 or 58, wherein the medicament is for treatment of cancer.