WO2001010906A9 - Crystallization and structure determination of staphylococcus aureus elongation factor p - Google Patents
Crystallization and structure determination of staphylococcus aureus elongation factor pInfo
- Publication number
- WO2001010906A9 WO2001010906A9 PCT/US2000/021528 US0021528W WO0110906A9 WO 2001010906 A9 WO2001010906 A9 WO 2001010906A9 US 0021528 W US0021528 W US 0021528W WO 0110906 A9 WO0110906 A9 WO 0110906A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- aureus
- molecule
- molecular complex
- binding surface
- structure coordinates
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
- C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Definitions
- This invention relates to the crystallization and structure determination of Staphylococcus aureus elongation factor P (S. aureus EF-P).
- Translation is fundamental to the biochemical and cellular process of all cells; therefore, it is not surprising that many antibacterial agents target this process.
- Preparation for translation begins with the binding of the mRNA to the ribosome placing the first codon, AUG, in position for interaction with the fMet-tRNA.
- Translation is initiated with the binding of the fMet-tRNA to the 30S subunit in the P site.
- the second tRNA is transported to the ribosome via the GTP dependent elongation factor-TU which situates the tRNA in the A site enabling the first peptide bond to be synthesized.
- the newly free tRNA localizes to the E site while the tRNA containing the growing amino acid chain moves to the P site vacating the A site for the next aminoacyl-tRNA.
- This translocation step is catalyzed by the GTP dependent elongation factor G.
- the present invention provides a method of crystallizing an S. aureus EF-P molecule or molecular complex that includes preparing purified S. aureus EF-P at a concentration of about 1 mg/ml to about 50 mg/ml; and crystallizing S. aureus EF-P from a solution including about 0 wt. % to about 50 wt. % polyethylene glycol and 0 to about 20 wt. % DMSO, and buffered to a pH of about 3.5 to about 5.5.
- the present invention provides crystalline forms of S. aureus EF-P.
- a crystal of S. aureus EF-P is provided having the orthorhombic space group symmetry P2,2,2 ! .
- the present invention provides a scalable three dimensional configuration of points wherein at least a portion of the points are derived from structure coordinates of a least a portion of an S. aureus EF-P molecule or molecular complex listed in Figure 4, preferably comprising amino acids Val 29, Lys30, Pro31, Gly32, Lys 33, Gly 34, Ser 35, and Ala 36.
- at least a portion of the points are derived from S. aureus EF-P structure coordinates representing the locations of at least the backbone atoms of amino acids defining an S. aureus EF-P or EF-P-like binding surface, the binding surface comprising amino acids selected from the surface residues listed in Table 1.
- At least a portion of points are derived from S. aureus EF-P structure coordinates representing the backbone atoms of amino acids within 4 A, preferably within 7 A, more preferably within 10 A, and most preferably within 15 A of Lys 33, as shown in Table 2.
- the present invention provides a scalable three dimensional configuration of points with at least a portion of the points derived from structure coordinates of at least a portion of a molecule or a molecular complex that is structurally homologous to an S. aureus EF-P molecule or molecular complex, On a molecular scale, with points derived from a molecule or molecular complex preferably have a root mean square deviation of less than about 1.9 A from the structure coordinates.
- the present invention provides a molecule or molecular complex that includes at least a portion of an S. aureus EF-P binding surface.
- the binding surface comprises amino acids selected from the surface residues listed in Table 1.
- the binding surface is further defined by a set of points having a root mean square deviation of less than about 1.9 A from points representing the backbone atoms of amino acids Val 29, Lys30, Pro31, Gly32, Lys 33, Gly 34, Ser 35, and Ala 36 as represented by the structure coordinates listed in Figure 4.
- the binding surface is further defined by a set of points having a root mean square deviation of less than about 1.9 A from points representing the backbone atoms of the amino acids that are within 4 A of Lys33, preferably within 7 A of Lys33, more preferably within 10 A of Lys33, and most preferably within 15 A of Lys33, as shown in Table 2 and represented by the structure coordinates listed in Figure 4. TABLE 1 Identified Surface Residues for S. aureus EF-P
- the present invention provides molecules or molecular complexes that are structurally homologous to an S. aureus EF-P molecule or molecular complex.
- the present invention provides a machine readable storage medium including the structure coordinates of all or a portion of anS. aureus EF-P molecule, molecular complex, a structurally homologous molecule or complex, including structurally equivalent structures, as defined herein, particularly a binding surface thereof, or a similarly shaped homologous binding surface.
- a storage medium encoded with these data is capable of displaying on a computer screen, or similar viewing device, a three-dimensional graphical representation of a molecule or molecular complex which comprises a binding surface or a similarly shaped homologous binding surface.
- the present invention provides a method for identifying inhibitors, ligands, and the like for an S. aureus EF-P molecule by providing the coordinates of a molecule of S. aureus EF-P to a computerized modeling system; identifying chemical entities that are expected to bind to or interfere with the molecule (e.g., screening a small molecule library); and, optionally, procuring or synthesizing then assaying the compounds or analogues derived therefrom for bioactivity.
- the present invention provides methods for designing inhibitors, ligands, and the like by providing the coordinates of a molecule of S.
- aureus EF-P to a computerized modeling system; designing a chemical entity that is likely to bind to or interfere with the molecule; and optionally, synthesizing the chemical entity and assaying the chemical entity for bioactivity.
- the present invention provides inhibitors and ligands designed or identified by the above method.
- a composition is provided that includes an inhibitor or ligand designed or identified by the above method.
- the composition is a pharmaceutical composition.
- the present invention provides a method involving molecular replacement to obtain structural information about a molecule or molecular complex of unknown structure.
- the method includes crystallizing the molecule or molecular complex, generating an x-ray diffraction pattern from the crystallized molecule or molecular complex, and applying at least a portion of the EF-P structure coordinates set forth in Fig. 4 to the x-ray diffraction pattern to generate a three-dimensional electron density map of at least a portion of the molecule or molecular complex.
- the present invention provides a method for homology modeling an S. aureus EF-P homolog.
- Staphylococcus aureus elongation factor P S. aureus EF-P
- IPTG Isopropylthio- ⁇ -D-galactoside
- DTT Dithiothreitol
- DMSO Dimethyl sulfoxide
- MAD Multiple anomalous dispersion
- PEG Polyethylene glycol
- Arg Arginine
- Figure 1 shows a crystal of S. aureus elongation factor P grown in 0.1 M sodium acetate pH 5.4, 1% PEG 4000.
- the width of the crystal is approximately 0.350 mm.
- Figure 3 shows electron density maps for EF-P for residues 73- 77 for a) multiple anomalous dispersion map after solvent flattening calculated to 2.3 A and b) the final 2F 0 -F C map calculated to 1.9 A resolution.
- Figure 4 lists the atomic structure coordinates for S. aureus EF-P as derived by x-ray diffraction from a crystal of that complex. The following abbreviations are used in Figure 1 :
- Atom refers to the element whose coordinates are measured.
- the second column defines the number of the atom in the structure.
- the letters in the third column define the element.
- the fourth and fifth columns define the amino acid and the number of the amino acid in the structure, respectively.
- Occ is an occupancy factor that refers to the fraction of the molecules in which each atom occupies the position specified by the coordinates.
- a value of " 1 " indicates that each atom has the same conformation, i.e., the same position, in all molecules of the crystal.
- FIG. 5 shows structure and sequence of S. aureus EF-P by a) ribbon representation of the X-ray crystal structure of S. aureus EF-P, and b) the amino acid sequence of recombinant EF-P (SEQ ID NO:l, including a His 6 tag) with the beginning of each domain indicated by an arrow. Disordered residues are underlined.
- Figure 6 shows a secondary structure diagram for S. aureus EF-
- Figure 7 shows conserved secondary and tertiary structure between domains 2 and 3 of EF-P.
- Two views (a and b) of a superposition of domain 2 and domain 3 from S. aureus elongation factor P are shown.
- Figure 8 shows two stereoviews (a and b) of EF-P.
- Figure 9 shows a surface representation of S. aureus EF-P. Alternative views (a and b) of the surface charge density (180° apart) of elongation factor P are shown.
- Figure 10 shows a hypothetical superposition of EF-P and tRNA Bln from E. coli (tRNA from M. A. Rould et al., Science 246: 1135-42 (1989), PDB access code lgtr).
- a) ⁇ F-P is oriented with domain 3 at the anticodon stem and domain 1 at the acceptor stem
- Figure 11 shows a structural comparison and sequence alignment of ⁇ F-P homologs. The three solved structures from a) S.
- Figure 12 shows a superposition of S. aureus ⁇ F-P (dark) and eIF5 A from Methanococcus jannaschii (light), a) Alignment of domain 1
- Figure 14 shows certain residues of interest in S. aureus ⁇ F-P.
- Lys33 is the proposed site for post-translational modification based on the hypusine modification found in ⁇ F-P homologs in eukaryotic systems.
- Figure 15 lists the structure factors and multiple anomalous dispersion phases for the crystal structure of S. aureus EF-P (SEQ ID NO:l).
- INDE refers to the indices h, k, and 1 (columns 2, 3, and 4 respectively) of the lattice planes.
- FOBS refers to the structure factors of the observed reflections.
- SIGMA is the standard deviation for the observations.
- PHAS refers to the phase used for the observations.
- FOM refers to the figure of merit.
- crystals comprising S. aureus EF-P which are suitable for x-ray crystallographic analysis.
- the three-dimensional structure of S. aureus EF-P was solved using high resolution x-ray crystallography.
- the crystal has orthorhombic space group symmetry P2 , 2 , 2 , .
- the crystallized enzyme is a monomer and has one molecule in the asymmetric unit.
- Purified S. aureus EF-P at a concentration of about 1 mg/ml to about 50 mg/ml may be crystallized, for example, using the hanging drop procedure from a solution including about 0 wt. % to about 50 wt. % polyethylene glycol (PEG, preferably having a number average molecular weight between about 200 and about 20,000), 0 to about 20 wt. % DMSO, and buffered to a pH of about 3.5 to about 5.5.
- PEG polyethylene glycol
- a buffer having a pK j of between 2.5 and 6.5 is preferred.
- the buffer includes about 10 mM to about 300 mM sodium acetate. Variation in buffer and buffer pH as well as other additives such as PEG is apparent to those skilled in the art and may result in similar crystals.
- Crystals of recombinant S. aureus EF-P were obtained from a crystallization screening solution that contained 100 mM sodium acetate at pH 4.6 and 4% PEG 4000.
- the recombinant S. aureus EF-P used for crystallization contains a six-residue polyhistidine tag at the C-terminus in order to facilitate purification of the recombinant protein. Refinement of the conditions resulted in ideal crystal growth occurring at pH 5.2-5.4. Since there was no homologous structure available for molecular replacement, selenomethionine inco ⁇ orated EF-P was prepared by the downregulation of methionine (T.E. Benson et al., Nat. Struct.
- structure coordinates refers to Cartesian coordinates derived from mathematical equations related to the patterns obtained on diffraction of a monochromatic beam of x-rays by the atoms (scattering centers) of an S. aureus EF-P complex in crystal form.
- the diffraction data are used to calculate an electron density map of the repeating unit of the crystal.
- the electron density maps are then used to establish the positions of the individual atoms of the S. aureus EF-P protein or protein/ligand complex.
- Slight variations in structure coordinates can be generated by mathematically manipulating S. aureus EF-P structure coordinates.
- the structure coordinates set forth in Figure 4 could be manipulated by crystallographic permutations of the structure coordinates, fractionalization of the structure coordinates, integer additions or subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above.
- modifications in the crystal structure due to mutations, additions, substitutions, and/or deletions of amino acids, or other changes in any of the components that make up the crystal could also yield variations in structure coordinates.
- Such slight variations in the individual coordinates will have little effect on overall shape. If such variations are within an acceptable standard error as compared to the original coordinates, the resulting three-dimensional shape is considered to be structurally equivalent. Structural equivalence is described in more detail below.
- the phrase "associating with” refers to a condition of proximity between a chemical entity, or portions thereof, and an S. aureus EF-P molecule or portions thereof.
- the association may be non- covalent, wherein the juxtaposition is energetically favored by hydrogen bonding, van der Waals forces, or electrostatic interactions, or it may be covalent.
- a ligand that bound to or interfered with a binding surface of S. aureus EF-P would also be expected to bind to or interfere with another binding surface whose structure coordinates define a shape that falls within the acceptable error.
- Elongation factor P is primarily comprised of ⁇ strands organized into three distinct domains ( Figures 5 and 6).
- Domain 1 contains four antiparallel ⁇ strands and a single turn of ⁇ helix. Domains two and three are both five stranded antiparallel ⁇ barrels similar to the putative oligonucleotide- oligosaccharide binding fold (A.G. Murzin, EMBO. J. ; 12:861-67 (1993)).
- Domain 2 also contains a single turn of a 3 10 helix between strands ⁇ 7 and ⁇ 8. Superposition of the C ⁇ from domains 2 and 3 resulted in an r.m.s. deviation of 1.55 A ( Figure 7).
- RNA binds RNA
- IF1 M. Sette et al., EMBO. J.; 16:1436-43 (1997)
- CspA W. Jiang et al., J. Biol. Chem.; 272:196-202 (1997); H. Schindelin et al., Proc. Natl. Acad. Sci. U.S.A.; 91:5119-23 (1994)
- EF-Tu P. Nissen et al., Science; 270:1464-72 (1995)
- domains 2 and 3 of EF-P may play a role in interacting with RNA - probably either tRNA or rRNA.
- Other structures that utilize this putative oligonucleotide- oligosaccharide binding fold show specific interactions with their respective ligands with the loops between strands 1 and 2, strand 3 and the ⁇ helix, or strands 4 and 5 (A.G. Murzin, EMBO. J.; 12:861-67 (1993)). Identification of these residues within the two ⁇ barrels for EF-P reveals potential sites for interaction with RNA (Figure 8). Based on evidence from related structures, residues for S.
- aureus ER-P that could be involved in oligonucleotide binding include residues 77-80, 99-105, and 117-120 from domain 2 and residues 149- 150, 164-169, and 177-181 from domain 3. These residues correspond to the loop between strand 1 and strand 2, the loop from strand 3 through helix 1, and the loop between strand 4 and strand 5 in a model beta barrel oligonucleotide binding fold as described in A.G. Murzin, EMBO. J.; 12:861-67 (1993).
- EF-P EF-P polarity of surface charges as shown in a surface representation ( Figure 9).
- Figure 9 Such polarity in a protein which most likely interacts with RNA suggests that the positively charged face of the protein ( Figure 9a) would interact with the negatively charged oligonucleotide.
- Figure 10 this surface representation reveals that EF-P resembles the shape and dimensionality of tRNA ( Figure 10) (M.A. Rould et al., Science 246:1135- 42 (1989)). This similarity in shape to tRNA may provide a hypothesis for the mechanism of action of EF-P.
- S. aureus, M. jannaschii, and P. aerophilum possess a highly conserved motif within domain 1 - xKxGKGxA - which has been identified in yeast and human as a site for post-translational modification of the second lysine to N ⁇ -(4-aminobutyl)lysine (also called deoxyhypusine).
- This modification has been shown to be essential for cell viability in yeast (J. Schnier et al., Mol. Cell. Biol; 11:3105-14 (1991)) and occurs only with eIF5A by the enzyme deoxyhypusine synthase (D.I. Liao et al., Structure; 6:23-32 (1998)).
- Applicants' invention has provided, for the first time, information about the shape and structure of a putative oligonucleotide binding surface of S. aureus EF-P.
- Binding surfaces are of significant utility in fields such as drug discovery.
- the association of natural ligands or substrates with the binding surfaces of their corresponding receptors or enzymes is the basis of many biological mechanisms of action.
- many drugs exert their biological effects through association with the binding surfaces of receptors and enzymes. Such associations may occur with all or any parts of the binding surface.
- An understanding of such associations helps lead to the design of drugs having more favorable associations with their target, and thus improved biological effects. Therefore, this information is valuable in designing potential inhibitors of S. aureus EF-P-like binding surfaces, as discussed in more detail below.
- a “molecular complex” means a protein in colvalent or non- covalent association with a chemical entity.
- binding surface refers to a region of a molecule or molecular complex, that, as a result of its shape, favorably associates with another chemical entity.
- the amino acid constituents of an S. aureus EF-P oligonucleotide binding surface as defined herein, as well as selected constituent atoms thereof, are positioned in three dimensions in accordance with the structure coordinates listed in Figure 4.
- the structure coordinates defining the binding surface of S. aureus EF-P include structure coordinates of substantially all atoms in the constituent amino acids; in another aspect, the structure coordinates of the binding surface include structure coordinates of just the backbone atoms of the constituent atoms.
- a specific chemical entity may bind to any of the amino acid surface residues of S. aureus EF-P as listed in Table 1.
- the surface residues that comprise the binding surface include amino acid K33.
- the surface residues that comprise the binding surface include residues whose backbone atoms are situated within 4 A, preferably within 7 A, more preferably within 10 A, and most preferably within 15 A of K33 as listed in Table 2.
- the term "S. aureus EF-P-like binding surface" refers to a portion of a molecule or molecular complex whose shape is sufficiently similar to at least a portion of a binding surface of S. aureus EF-P as to be expected to bind common or structurally related ligands.
- a structurally equivalent binding surface is defined by a root mean square deviation from the structure coordinates of the backbone atoms of the amino acids that make up the binding surfaces in S. aureus EF-P (as set forth in Figure 4) of at most about 1.9 A. How this calculation is obtained is described below.
- the invention thus provides molecules or molecular complexes comprising an S. aureus EF-P oligonucleotide binding surface or S. aureus EF-P-like binding surface, as defined by the sets of structure coordinates described above.
- X-ray structure coordinates define a unique configuration of points in space.
- a set of structure coordinates for protein or a protein/ligand complex, or a portion thereof define a relative set of points that, in turn, define a configuration in three dimensions.
- a similar or identical configuration can be defined by an entirely different set of coordinates, provided the distances and angles between coordinates remain essentially the same.
- a scalable configuration of points can be defined by increasing or decreasing the distances between coordinates by a scalar factor while keeping the angles essentially the same.
- the present invention thus includes the scalable three- dimensional configuration of points derived from the structure coordinates of at least a portion of an S. aureus EF-P molecule or molecular complex, as listed in Figure 4, as well as structurally equivalent configurations, as described below.
- the scalable three-dimensional configuration includes points derived from structure coordinates representing the locations of a plurality of the amino acids defining the S. aureus EF-P binding surface.
- the scalable three-dimensional configuration includes points derived from structure coordinates representing the locations the backbone atoms of a plurality of amino acids defining the S. aureus EF-P binding surface, preferably those amino acids listed in Table 1.
- the scalable three-dimensional configuration includes points derived from structure coordinates representing the locations of the side chain and the backbone atoms (other than hydrogens) of a plurality of the amino acids defining the S. aureus EF-P binding surface, preferably those amino acids listed in Table 1.
- the scalable three-dimensional configuration of points are derived from structure coordinates representing the locations of backbone and, optionally, side chain atoms (other than hydrogens) of amino acids within 4 A, preferably within 7 A, more preferably within 10 A, and most preferably within 15 A of Lys33 as shown in Table 2.
- the invention also includes the scalable three- dimensional configuration of points derived from structure coordinates of molecules or molecular complexes that are structurally homologous to S. aureus EF-P, as well as structurally equivalent configurations.
- Structurally homologous molecules or molecular complexes are defined below.
- structurally homologous molecules can be identified using the structure coordinates of S. aureus EF-P ( Figure 4) according to a method of the invention.
- the configurations of points in space derived from structure coordinates according to the invention can be visualized as, for example, a holographic image, a stereodiagram, a model or a computer-displayed image, and the invention thus includes such images, diagrams or models.
- Various computational analyses can be used to determine whether a molecule or the binding surface portion thereof is "structurally equivalent,” defined in terms of its three-dimensional structure, to all or part of S. aureus EF-P or its binding surfaces.
- Such analyses may be carried out in current software applications, such as the Molecular Similarity application of QUANTA (Molecular Simulations Inc., San Diego, CA) version 4.1, and as described in the accompanying User's Guide.
- the Molecular Similarity application permits comparisons between different structures, different conformations of the same structure, and different parts of the same structure.
- the procedure used in Molecular Similarity to compare structures is divided into four steps: (1) load the structures to be compared; (2) define the atom equivalences in these structures; (3) perform a fitting operation; and (4) analyze the results.
- Each structure is identified by a name.
- One structure is identified as the target (i.e., the fixed structure); all remaining structures are working structures (i.e., moving structures).
- equivalent atoms are defined as protein backbone atoms (N, C ⁇ , C, and O) for all conserved residues between the two structures being compared.
- a conserved residue is defined as a residue that is structurally or functionally equivalent. Only rigid fitting operations are considered.
- the working structure is translated and rotated to obtain an optimum fit with the target structure.
- the fitting operation uses an algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atom is an absolute minimum. This number, given in angstroms, is reported by QUANTA.
- Particularly preferred structurally equivalent molecules or molecular complexes are those that are defined by the entire set of structure coordinates in Figure 4, ⁇ a root mean square deviation from the conserved backbone atoms of those amino acids of not more than 1.9 A. More preferably, the root mean square deviation is less than about 1.0 A.
- root mean square deviation means the square root of the arithmetic mean of the squares of the deviations. It is a way to express the deviation or variation from a trend or object.
- the "root mean square deviation” defines the variation in the backbone of a protein from the backbone of S. aureus EF-P or a binding surface portion thereof, as defined by the structure coordinates of S. aureus EF-P described herein.
- Transformation of the structure coordinates for all or a portion of S. aureus EF-P or the S. aureus EF-P/ligand complex, for structurally homologous molecules as defined below, or for the structural equivalents of any of these molecules or molecular complexes as defined above, into three- dimensional graphical representations of the molecule or complex can be conveniently achieved through the use of commercially-available software.
- the nvention thus further provides a machine-readable storage medium comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a graphical three-dimensional representation of any of the molecule or molecular complexes of this invention that have been described above.
- the machine-readable data storage medium comprises a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a graphical three-dimensional representation of a molecule or molecular complex comprising all or any parts of an S. aureus EF-P binding surface or an S. aureus EF-P-like binding surface, as defined above.
- the machine-readable data storage medium is capable of displaying a graphical three-dimensional representation of a molecule or molecular complex defined by the structure coordinates of all of the amino acids in Figure 4, ⁇ a root mean square deviation from the backbone atoms of said amino acids of not more than 1.9 A.
- the machine-readable data storage medium comprises a data storage material encoded with a first set of machine readable data which comprises the Fourier transform of the structure coordinates set forth in Figure 4, and which, when using a machine programmed with instructions for using said data, can be combined with a second set of machine readable data comprising the x-ray diffraction pattern of a molecule or molecular complex to determine at least a portion of the structure coordinates corresponding to the second set of machine readable data.
- a system for reading a data storage medium may include a computer comprising a central processing unit (“CPU"), a working memory which may be, e.g., RAM (random access memory) or “core” memory, mass storage memory (such as one or more disk drives or CD-ROM drives), one or more display devices (e.g., cathode-ray tube (“CRT”) displays, light emitting diode (“LED”) displays, liquid crystal displays (“LCDs”), electroluminescent displays, vacuum fluorescent displays, field emission displays (“FEDs”), plasma displays, projection panels, etc.), one or more user input devices (e.g., keyboards, microphones, mice, touch screens, etc.), one or more input lines, and one or more output lines, all of which are interconnected by a conventional bidirectional system bus.
- CPU central processing unit
- working memory which may be, e.g., RAM (random access memory) or “core” memory, mass storage memory (such as one or more disk drives or CD-ROM drives), one or more display devices (e
- the system may be a stand-alone computer, or may be networked (e.g., through local area networks, wide area networks, intranets, extranets, or the internet) to other systems (e.g., computers, hosts, servers, etc.).
- the system may also include additional computer controlled devices such as consumer electronics and appliances.
- Input hardware may be coupled to the computer by input lines and may be implemented in a variety of ways. Machine-readable data of this invention may be inputted via the use of a modem or modems connected by a telephone line or dedicated data line. Alternatively or additionally, the input hardware may comprise CD-ROM drives or disk drives. In conjunction with a display terminal, a keyboard may also be used as an input device.
- Output hardware may be coupled to the computer by output lines and may similarly be implemented by conventional devices.
- the output hardware may include a display device for displaying a graphical representation of a binding surface of this invention using a program such as QUANTA as described herein.
- Output hardware might also include a printer, so that hard copy output may be produced, or a disk drive, to store system output for later use.
- a CPU coordinates the use of the various input and output devices, coordinates data accesses from mass storage devices, accesses to and from working memory, and determines the sequence of data processing steps.
- a number of programs may be used to process the machine-readable data of this invention. Such programs are discussed in reference to the computational methods of drug discovery as described herein. References to components of the hardware system are included as appropriate throughout the following description of the data storage medium.
- Machine-readable storage devices useful in the present invention include, but are not limited to, magnetic devices, electrical devices, optical devices, and combinations thereof.
- Examples of such data storage devices include, but are not limited to, hard disk devices, CD devices, digital video disk devices, floppy disk devices, removable hard disk devices, magneto-optic disk devices, magnetic tape devices, flash memory devices, bubble memory devices, holographic storage devices, and any other mass storage peripheral device.
- these storage devices include necessary hardware (e.g., drives, controllers, power supplies, etc.) as well as any necessary media (e.g., disks, flash cards, etc.) to enable the storage of data.
- the structure coordinates set forth in Figure 4 can be used to aid in obtaining structural information about another crystallized molecule or molecular complex.
- the method of the invention allows determination of at least a portion of the three-dimensional structure of molecules or molecular complexes which contain one or more structural features that are similar to structural features of S. aureus EF-P. These molecules are referred to herein as "structurally homologous" to S. aureus EF-P.
- Similar structural features can include, for example, regions of amino acid identity, conserved active site or binding site motifs, and similarly arranged secondary structural elements (e.g., ⁇ helices and ⁇ sheets).
- structural homology is determined by aligning the residues of the two amino acid sequences to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order.
- two amino acid sequences are compared using the Blastp program, version 2.0.9, of the BLAST 2 search algorithm, as described by Tatiana et al., FEMS Microbiol Lett 174, 247-50 (1999), and available at http://www.ncbi.nlm.nih.gov/gorf/bl2.html.
- a structurally homologous molecule is a protein that has an amino acid sequence sharing at least 65% identity with a native or recombinant amino acid sequence of S. aureus EF-P (for example, SEQ ID NO: 1). More preferably, a protein that is structurally homologous to S.
- aureus EF-P includes at least one contiguous stretch of at least 50 amino acids that shares at least 80% amino acid sequence identity with the analogous portion of the native or recombinant S. aureus EF-P (for example, SEQ ID NO: 1).
- Methods for generating structural information about the structurally homologous molecule or molecular complex are well- known and include, for example, molecular replacement techniques.
- this invention provides a method of utilizing molecular replacement to obtain structural information about a molecule or molecular complex whose structure is unknown comprising the steps of:
- Figure 4 to the x-ray diffraction pattern to generate a three-dimensional electron density map of the molecule or molecular complex whose structure is unknown.
- all or part of the structure coordinates of S. aureus EF-P or the S. aureus EF-P/ligand complex as provided by this invention can be used to determine the structure of a crystallized molecule or molecular complex whose structure is unknown more quickly and efficiently than attempting to determine such information ah initio.
- Molecular replacement provides an accurate estimation of the phases for an unknown structure. Phases are a factor in equations used to solve crystal structures that cannot be determined directly. Obtaining accurate values for the phases, by methods other than molecular replacement, is a time- consuming process that involves iterative cycles of approximations and refinements and greatly hinders the solution of crystal structures. However, when the crystal structure of a protein containing at least a structurally homologous portion has been solved, the phases from the known structure provide a satisfactory estimate of the phases for the unknown structure.
- this method involves generating a preliminary model of a molecule or molecular complex whose structure coordinates are unknown, by orienting and positioning the relevant portion of S. aureus EF-P according to Figure 4 within the unit cell of the crystal of the unknown molecule or molecular complex so as best to account for the observed x-ray diffraction pattern of the crystal of the molecule or molecular complex whose structure is unknown. Phases can then be calculated from this model and combined with the observed x-ray diffraction pattern amplitudes to generate an electron density map of the structure whose coordinates are unknown.
- Structural information about a portion of any crystallized molecule or molecular complex that is sufficiently structurally homologous to a portion of S. aureus EF-P can be resolved by this method.
- a molecule that shares one or more structural features with S. aureus EF-P as described above a molecule that has similar bioactivity, such as the same substrate specificity or ligand binding activity as S. aureus EF-P, may also be sufficiently structurally homologous to S. aureus EF-P to permit use of the structure coordinates of S. aureus EF-P to solve its crystal structure.
- the method of molecular replacement is utilized to obtain structural information about a molecule or molecular complex, wherein the molecule or molecular complex comprises at least one S. aureus EF-P subunit or homolog.
- a "subunit" of S. aureus EF-P is an S. aureus EF-P molecule that has been truncated at the N-terminus or the C-terminus, or both.
- a "homolog" of S. aureus EF-P is a protein that contains one or more amino acid substitutions, deletions, additions, or rearrangements with respect to the amino acid sequence of S.
- structurally homologous molecules can contain deletions or additions of one or more contiguous or noncontiguous amino acids, such as a loop or a domain.
- Structurally homologous molecules also include "modified" S.
- aureus EF-P molecules that have been chemically or enzymatically derivatized at one or more constituent amino acid, including side chain modifications, backbone modifications, and N- and C- terminal modifications including acetylation, hydroxylation, methylation, amidation, and the attachment of carbohydrate or lipid moieties, cofactors, and the like.
- a heavy atom derivative of S. aureus EF-P is also included as an
- S. aureus EF-P homolog refers to derivatives of S. aureus EF-P produced by chemically modifying a crystal of S. aureus EF-P.
- a crystal is soaked in a solution containing heavy metal atom salts, or organometallic compounds, e.g., lead chloride, gold thiomalate, thiomersal or uranyl acetate, which can diffuse through the crystal and bind to the surface of the protein.
- the location(s) of the bound heavy metal atom(s) can be determined by x-ray diffraction analysis of the soaked crystal. This information, in turn, is used to generate the phase information used to construct three-dimensional structure of the protein (T.L. Blundell and N.L. Johnson, Protein Crystallography, Academic Press (1976)).
- the structure coordinates of S. aureus EF-P as provided by this invention are particularly useful in solving the structure of other crystal forms of S. aureus EF-P or S. aureus EF-P complexes.
- the structure coordinates of S. aureus EF-P as provided by this invention are particularly useful in solving the structure of S. aureus EF-P mutants. Mutants may be prepared, for example, by expression of S. aureus EF- P cDNA previously altered in its coding sequence by oligonucleotide-directed mutagenesis.
- Mutants may also be generated by site-specific inco ⁇ oration of unnatural amino acids into EF-P proteins using the general biosynthetic method of C. J. Noren et al., Science, 244: 182- 188 (1989).
- the codon encoding the amino acid of interest in wild-type S. aureus EF-P is replaced by a "blank" nonsense codon, TAG, using oligonucleotide-directed mutagenesis.
- a suppressor tRNA directed against this codon is then chemically aminoacylated in vitro with the desired unnatural amino acid.
- the aminoacylated tRNA is then added to an in vitro translation system to yield a mutant S. aureus EF-P with the site-specific inco ⁇ orated unnatural amino acid.
- Selenocysteine or selenomethionine may be inco ⁇ orated into wild-type or mutant S. aureus EF-P by expression of S. aureus EF-P-encoding cDNAs in auxotrophic E. coli strains (W.A. Hendrickson et al., EMBO J., 9(5):1665-1672 (1990)).
- the wild-type or mutagenized S. aureus EF-P cDNA may be expressed in a host organism on a growth medium depleted of either natural cysteine or methionine (or both) but enriched in selenocysteine or selenomethionine (or both).
- selenomethionine analogues may be prepared by down regulation methionine biosynthesis. (T.E. Benson et al., Nat. Struct. Biol., 2:644-53 (1995); G.D. Van Duyne et al., J. Mol. Biol. 229:105-24 (1993)).
- the structure coordinates of S. aureus EF-P in Figure 4 are also particularly useful to solve the structure of crystals of S. aureus EF-P, S. aureus EF-P mutants or S. aureus EF-P homologs co-complexed with a variety of chemical entities.
- This approach enables the determination of the optimal sites for interaction between chemical entities, including candidate S. aureus EF-P inhibitors. Potential sites for modification within the various binding site of the molecule can also be identified. This information provides an additional tool for determining the most efficient binding interactions, for example, increased hydrophobic interactions, between S. aureus EF-P and a chemical entity.
- All of the complexes referred to above may be studied using well-known x-ray diffraction techniques and may be refined versus 1.5-3 A resolution x-ray data to an R value of about 0.20 or less using computer software, such as X-PLOR (Yale University, 81992, distributed by Molecular Simulations, Inc.; see, e.g., Blundell & Johnson, supra; Meth. Enzymol., Vol. 114 & 115, H.W. Wyckoff et al., eds., Academic Press (1985)).
- This information may thus be used to optimize known S. aureus EF-P inhibitors, and more importantly, to design new S. aureus EF-P inhibitors.
- the invention also includes the unique three-dimensional configuration defined by a set of points defined by the structure coordinates for a molecule or molecular complex structurally homologous to S. aureus EF-P as determined using the method of the present invention, structurally equivalent configurations, and magnetic storage media comprising such set of structure coordinates.
- the invention includes structurally homologous molecules as identified using the method of the invention.
- EF-P homolog can be built or refined without crystallizing the homolog.
- a preliminary model of the S. aureus EF-P homolog is created by sequence alignment with S. aureus EF-P, secondary structure prediction, the screening of structural libraries, or any combination of those techniques.
- Computational software may be used to carry out the sequence alignments and the secondary structure predictions.
- Structural incoherences e.g., structural fragments around insertions and deletions, can be modeled by screening a structural library for peptides of the desired length and with a suitable conformation.
- a side chain rotamer library may be employed. If the S.
- the final homology model can be used to solve the crystal structure of the homolog by molecular replacement, as described above.
- the preliminary model is subjected to energy minimization to yield an energy minimized model.
- the energy minimized model may contain regions where stereochemistry restraints are violated, in which case such regions are remodeled to obtain a final homology model.
- the homology model is positioned according to the results of molecular replacement, and subjected to further refinement comprising molecular dynamics calculations.
- Computational techniques can be used to screen, identify, select and/or design chemical entities capable of associating with S. aureus EF-P or structurally homologous molecules. Knowledge of the structure coordinates for S. aureus EF-P permits the design and/or identification of synthetic compounds and/or other molecules which have a shape complementary to the conformation of the S. aureus EF-P binding site.
- computational techniques can be used to identify or design chemical entities, such as inhibitors, agonists and antagonists, that associate with an S. aureus EF-P binding surface or an S. aureus EF-P-like binding surface. Inhibitors may bind to or interfere with all or a portion of the binding surface of S.
- aureus EF-P can be competitive, non-competitive, or uncompetitive inhibitors; or interfere with dimerization by binding at the interface between the two monomers.
- inhibitors that are bound to a binding surface of S. aureus EF-P may interfere with binding of S. aureus EF-P to a ribosomal protein or ribosomal RNA during translation.
- these inhibitors/agonists/antagonists may be used therapeutically or prophylactically to block S. aureus EF-P activity and, thus, inhibit growth of the bacteria or cause its death.
- Structure-activity data for analogs of ligands that bind to or interfere with S. aureus EF-P or S. aureus EF-P-like binding surfaces can also be obtained computationally.
- chemical entity refers to chemical compounds, complexes of two or more chemical compounds, and fragments of such compounds or complexes.
- Chemical entities that are determined to associate with S. aureus EF-P are potential drug candidates.
- Data stored in a machine-readable storage medium that is capable of displaying a graphical three-dimensional representation of the structure of S. aureus EF-P or a structurally homologous molecule, as identified herein, or portions thereof may thus be advantageously used for drug discovery.
- the structure coordinates of the chemical entity are used to generate a three- dimensional image that can be computationally fit to the three-dimensional image of S. aureus EF-P or a structurally homologous molecule.
- the three- dimensional molecular structure encoded by the data in the data storage medium can then be computationally evaluated for its ability to associate with chemical entities.
- the molecular structures encoded by the data is displayed in a graphical three-dimensional representation on a computer screen, the protein structure can also be visually inspected for potential association with chemical entities.
- One embodiment of the method of drug design involves evaluating the potential association of a known chemical entity with S. aureus EF-P or a structurally homologous molecule, particularly with an S. aureus EF- P binding surface or S. aureus EF-P-like binding surface.
- the method of drug design thus includes computationally evaluating the potential of a selected chemical entity to associate with any of the molecules or molecular complexes set forth above. This method comprises the steps of: (a) employing computational means to perform a fitting operation between the selected chemical entity and a binding surface of the molecule or molecular complex; and (b) analyzing the results of said fitting operation to quantify the association between the chemical entity and the binding surface.
- the method of drug design involves computer-assisted design of chemical entities that associate with S. aureus EF- P, its homologs, or portions thereof.
- Chemical entities can be designed in a step-wise fashion, one fragment at a time, or may be designed as a whole or "de novo.”
- the chemical entity identified or designed according to the method must be capable of structurally associating with at least part of an S. aureus EF-P or S. aureus EF-P-like binding surfaces, and must be able, sterically and energetically, to assume a conformation that allows it to associate with the S. aureus EF-P or S. aureus EF-P-like binding surface.
- Non-covalent molecular interactions important in this association include hydrogen bonding, van der Waals interactions, hydrophobic interactions, and electrostatic interactions.
- Conformational considerations include the overall three-dimensional structure and orientation of the chemical entity in relation to the binding surface, and the spacing between various functional groups of an entity that directly interact with the S. aureus EF-P-like binding surface or homologs thereof.
- the potential binding of a chemical entity to an S. aureus EF-P or S. aureus EF-P-like binding surface is analyzed using computer modeling techniques prior to the actual synthesis and testing of the chemical entity. If these computational experiments suggest insufficient interaction and association between it and the S. aureus EF-P or S. aureus EF-P-like binding surface, testing of the entity is obviated. However, if computer modeling indicates a strong interaction, the molecule may then be synthesized and tested for its ability to bind to or interfere with an S. aureus EF-P or S. aureus EF-P- like binding surface, binding assays to determine if a compound actually binds to S.
- aureus EF-P can also be performed and are well known in the art.
- binding assays may employ kinetic or thermodynamic methodology using a wide variety of techniques including, but not limited to, microcalorimetry, circular dichroism, capillary zone electrophoresis, nuclear magnetic resonance spectroscopy, fluorescence spectroscopy, and combinations thereof.
- One skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with an S. aureus EF- P or S. aureus EF-P-like binding surface.
- This process may begin by visual inspection of, for example, an S. aureus EF-P or S. aureus EF-P-like binding surface on the computer screen based on the S. aureus EF-P structure coordinates in Figure 4 or other coordinates which define a similar shape generated from the machine-readable storage medium.
- Selected fragments or chemical entities may then be positioned in a variety of orientations, or docked, within the binding surface. Docking may be accomplished using software such as QUANTA and SYBYL, followed by energy minimization and molecular dynamics with standard molecular mechanics forcefields, such as CHARMM and AMBER.
- Specialized computer programs may also assist in the process of selecting fragments or chemical entities. Examples include GRID (P.J.
- S. aureus EF-P binding compounds may be designed "de novo" using either an empty binding site or optionally including some portion(s) of a known inhibitor(s).
- LUDI H.-J. Bohm, J. Comp. Aid. Molec. Design. 6:61-78 (1992); available from Molecular Simulations Inc., San Diego, CA); LEGEND (Y. Nishibata et al., Tetrahedron, 47:8985 (1991); available from Molecular Simulations Inc., San Diego, CA); LEGEND (Y. Nishibata et al., Tetrahedron, 47:8985 (1991); available from Molecular
- an effective S. aureus EF-P or S. aureus EF-P-like binding surface inhibitor must preferably demonstrate a relatively small difference in energy between its bound and free states (i.e., a small deformation energy of binding).
- the most efficient S. aureus EF-P or S. aureus EF-P-like binding surface inhibitors should preferably be designed with a deformation energy of binding of not greater than about 10 kcal/mole; more preferably, not greater than 7 kcal/mole.
- S. aureus EF-P or S. aureus EF-P-like binding surface inhibitors may interact with the binding surface in more than one conformation that is similar in overall binding energy. In those cases, the deformation energy of binding is taken to be the difference between the energy of the free entity and the average energy of the conformations observed when the inhibitor binds to the protein.
- aureus EF-P-like binding surface may be further computationally optimized so that in its bound state it would preferably lack repulsive electrostatic interaction with the target enzyme and with the surrounding water molecules.
- Such non-complementary electrostatic interactions include repulsive charge-charge, dipole-dipole, and charge-dipole interactions.
- Another approach encompassed by this invention is the computational screening of small molecule databases for chemical entities or compounds that can bind in whole, or in part, to a S. aureus EF-P or S. aureus EF-P-like binding surface.
- the quality of fit of such entities to the binding site may be judged either by shape complementarity or by estimated interaction energy (E.C. Meng et al., J. Comp. Chem., 13, pp. 505-524 (1992)).
- This invention also enables the development of chemical entities that can isomerize to short-lived reaction intermediates in the chemical reaction of a substrate or other compound that binds to or interferes with or with S. aureus EF-P. Time-dependent analysis of structural changes in S.
- aureus EF-P during its interaction with other molecules is carried out.
- the reaction intermediates of S. aureus EF-P can also be deduced from the reaction product in co-complex with S. aureus EF-P.
- Such information is useful to design improved analogs of known S. aureus EF-P inhibitors or to design novel classes of inhibitors based on the reaction intermediates of the S. aureus EF-P and inhibitor co-complex. This provides a novel route for designing S. aureus EF-P inhibitors with both high specificity and stability.
- Yet another approach to rational drug design involves probing the S. aureus EF-P crystal of the invention with molecules comprising a variety of different functional groups to determine optimal sites for interaction between candidate S. aureus EF-P inhibitors and the protein. For example, high resolution x-ray diffraction data collected from crystals soaked in or co- crystallized with other molecules allows the determination of where each type of solvent molecule sticks. Molecules that bind tightly to those sites can then be further modified and synthesized and tested for their EF-P inhibitor activity (J. Travis, Science, 262:1374 (1993)). In a related approach, iterative drug design is used to identify inhibitors of S. aureus EF-P.
- Iterative drug design is a method for optimizing associations between a protein and a compound by determining and evaluating the three-dimensional structures of successive sets of protein/compound complexes.
- iterative drug design crystals of a series of protein/compound complexes are obtained and then the three-dimensional structures of each complex is solved.
- Such an approach provides insight into the association between the proteins and compounds of each complex. This is accomplished by selecting compounds with inhibitory activity, obtaining crystals of this new protein/compound complex, solving the three dimensional structure of the complex, and comparing the associations between the new protein compound complex and previously solved protein/compound complexes. By observing how changes in the compound affected the protein/compound associations, these associations may be optimized.
- a compound that is identified or designed as a result of any of these methods can be obtained (or synthesized) and tested for its biological activity, e.g., inhibition of S. aureus EF-P activity.
- compositions of this invention comprise an inhibitor of S. aureus EF-P activity identified according to the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.
- pharmaceutically acceptable carrier refers to a carrier(s) that is “acceptable” in the sense of being compatible with the other ingredients of a composition and not deleterious to the recipient thereof.
- the pH of the formulation is adjusted with pharmaceutically acceptable acids, bases, or buffers to enhance the stability of the formulated compound or its delivery form.
- compositions of the invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. Oral administration or administration by injection is preferred.
- parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.
- Dosage levels of between about 0.01 and about 100 mg kg body weight per day, preferably between about 0.5 and about 75 mg/kg body weight per day of the S. aureus EF-P inhibitory compounds described herein are useful for the prevention and treatment of S. aureus EF-P mediated disease.
- the pharmaceutical compositions of this invention will be administered from about 1 to about 5 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy.
- the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
- a typical preparation will contain from about 5% to about 95% active compound (w/w).
- such preparations contain from about 20% to about 80% active compound.
- the Escherichia coli construct Ml 5 (pREP4) (pQE-60 EF-P) which expresses S. aureus EF-P was obtained from Human Genome Sciences. Various genes and polypeptides derived from S. aureus are published in WO 0012678.
- the construct was grown in a minimal salts medium, M9, which contained glucose and NH 4 C1 as the sources of carbon and nitrogen. Endogenous methionine biosynthesis was then inhibited while adding an excess of selenomethionine to the growth medium just prior to IPTG induction of EF-P synthesis (T.E. Benson et al., Nat. Struct.
- basal M9 media was Na 2 HPO 4 , 6 g; KH 2 PO 4 , 3 g; NH 4 C1, 1.0 g; and NaCl, 0.5 g per L of deionized water.
- the pH was adjusted to 7.4 with concentrated KOH and the medium was sterilized by autoclaving.
- IM MgSO 4 1.0 ml
- IM CaCl 2 0.1 ml
- trace metal salts solution 0.1 ml, 10 mM thiamin, 1.0 ml
- 20% glucose 20 ml.
- the trace metal salts solution contained per L of deionized water: MgCl 2 ' 6H 2 O, 39.44 g; MnSO 4 H 2 O, 5.58 g; FeSO 4 - 7H 2 O, 1.11 g; Na ⁇ MoO," 2H 2 O, 0.48 g; CaCl 2 , 0.33 g; NaCl, 0.12 g; and ascorbic acid, 1.0 g.
- Filter sterilized ampicillin and kanamycin were added to the medium at final concentrations of 100 ⁇ g/ml and 30 ⁇ g/ml, respectively.
- Fermentations were prepared in 100 ml volumes of M9 medium contained in 500 ml wide mouth flasks. A 0.1 ml aliquot of the stock culture was inoculated into the medium and allowed to grow at 37°C for 18 - 20 hours with a shaking rate of 200 ⁇ m. The seed culture was harvested by centrifugation and then resuspended in an equal volume of M9 medium. The resuspended seed was used to inoculate expression fermentations at a rate of 3%. For expression, the culture was grown under the same conditions to an A 600 of 0.6.
- methionine biosynthesis was down regulated by the addition of L-lysine, L-threonine, and L-phenylalanine at a final concentration for each of 100 ⁇ g/ml and L-leucine, L-iso leucine, and L-valine at 50 ⁇ g/ml each.
- D,L-selenomethionine was added simultaneously to a final concentration of 100 ⁇ g/ml.
- protein expression was induced by addition of IPTG (isopropyl thio- ⁇ -D-galactosidase, Gibco BRL) to 1 mM. Growth of the culture was continued for an additional 3 hours until an A 600 of 2.0 - 2.2. Cells were then harvested by centrifugation and frozen at -80°C. Under these conditions, the average yield of cell paste was 4.0 g/L and approximately 20 mg/L of selenomethionine EF-P was produced.
- E. coli cell paste resulting from 1 liter of induced cell culture was resuspended in 25 mL of buffer A (50 mM Tris pH 7.6 containing 300 mM NaCl and 10% glycerol). 10 mg of DNAse I and 1 CompleteTM protease inhibitor tablet was added to the suspension. The cells were then lysed by passing the suspension three times through a French Press (Spectronic
- the soluble cytosol was prepared from the lysate by ultracentrifugation at 100,000 x g for 60 min at 4°C. Benzonase (25 ⁇ L) was added to the cytosol to remove any residual polynucleotides. The clarified cytosol was injected onto a HR10/10 FPLC column (Pharmacia Biotech,
- the gradient consisted of 0-8% buffer B in 20 minutes, followed by a 10 minute segment of 8% buffer B, and then a linear gradient of 8-100% buffer B over 20 minutes.
- the column was washed with 100% buffer B for 30 minutes. 4 mL fractions were collected starting at the beginning of the gradient. Selected fractions were analyzed by SDS-PAGE for purity before making the final pool for crystallographic studies.
- S. aureus EF-P was prepared and purified.
- the final purified EF-P protein sample was exchanged into 10 mM Tris pH 7.6 (5 mM DTT was added for selenomethionine EF-P) and concentrated to 12 mg/mL for crystallization experiments.
- Initial crystals of EF-P were obtained from a standard library of crystallization screening solutions - Hampton Screen 1, condition 37, 0.1 M sodium acetate pH 4.6, 8% PEG 4000. Optimization of the crystallization condition was conducted using EF-P at a concentration of 12 mg/ml. Crystals grew best at 0.1 M sodium acetate pH 5.2-5.4 and 1-4% PEG 4000. The crystals were grown by vapor diffusion in hanging drops by the addition of a 1 :1 starting ratio of protein to well solution.
- Multiwavelength anomalous dispersion data was collection at the Advanced Photon Source located at Argonne National Labs in Argonne, IL on beamline 17-ID on the sodium acetate crystal form.
- Complete data sets were collected at three different wavelengths - one remote, low energy wavelength (1.0332 A), one wavelength at the inflection point of the selenium X-ray fluorescence spectrum where the dispersive differences would be maximal (0.979746 A) and one wavelength at the peak of the X-ray fluorescence spectrum where the anomalous differences would be maximal (0.979617 A).
- Each of these individual data sets was indexed and integrated separately (see Table 1 for integration statistics).
Abstract
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AU64012/00A AU6401200A (en) | 1999-08-06 | 2000-08-04 | Crystallization and structure determination of staphylococcus aureus elongation factor |
EP00951000A EP1200468A1 (en) | 1999-08-06 | 2000-08-04 | CRYSTALLIZATION AND STRUCTURE DETERMINATION OF i STAPHYLOCOCCUS AUREUS /i ELONGATION FACTOR P |
CA002376065A CA2376065A1 (en) | 1999-08-06 | 2000-08-04 | Crystallization and structure determination of staphylococcus aureus elongation factor p |
JP2001515713A JP2003510250A (en) | 1999-08-06 | 2000-08-04 | Crystallization and structure determination of Staphylococcus aureus elongation factor P |
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US7968523B2 (en) | 2001-07-23 | 2011-06-28 | Senesco Technologies, Inc. | Method for inducing apoptosis using apoptosis-specific EIF5-A |
US7381708B2 (en) | 2001-07-23 | 2008-06-03 | Sensco Technologies, Inc. | Suppression of eIF5A1 expression by the use of antisense oligonucleotides for the prevention of retinal cell death in the glaucomatous eye |
US7217517B2 (en) | 2001-07-23 | 2007-05-15 | Senesco Technologies, Inc. | Nucleic acids, polypeptides, and methods for modulating apoptosis |
CN100558410C (en) * | 2002-05-07 | 2009-11-11 | 森尼斯科技术公司 | Regulate the nucleic acid of apoptosis, polypeptide, and method |
KR20050098954A (en) | 2003-03-05 | 2005-10-12 | 세네스코 테크놀로지스 인코포레이티드 | Use of antisense oligonucleotides or sirna to suppress expression of eif-5a1 |
US8703929B2 (en) | 2008-03-07 | 2014-04-22 | Senesco Technologies, Inc. | Compositions comprising siRNA and plasmids |
US8445638B2 (en) | 2008-09-03 | 2013-05-21 | Senesco Technologies, Inc. | Use of a truncated eIF-5A1 polynucleotide to induce apoptosis in cancer cells |
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