CA2454246A1 - Cysteine mutants and methods for detecting ligand binding to biological molecules - Google Patents

Abstract

The present invention relates generally to variants of target biological molecules ("TBMs") and to methods of making and using the same to identify ligands of TBMs. More specifically, the invention relates to individual variant TBMs and sets of variant TBMs, each of which represents a modified version of a protein of interest where a thiol has been introduced at or near a site of interest. Ligands of TBMs are identified in part through the formation of a covalent bond between a potential ligand and a reactive thiol on the TBM.

Description

CYSTEINE MUTANTS AND METHODS FOR DETECTING LIGAND BINDING TO
BIOLOGICAL MOLECULES
BACKGROUND
The drug discovery process usually beings with massive functional screening of compound libraries to identify modest affinity leads (Kd ~ 1 to 10 pM) for subsequent medicinal chemistry optimization. However, not all targets of interest are amenable to such screening. In some cases, an assay that is amenable to high throughput screening is not available. In other cases, the target can have multiple binding modes such that the results of such screens are ambiguous and difficult to interpret. Still in other cases, the assay conditions for high throughput screening are such that they are prone to artifacts. As a result, alternative methods for ligand discovery are needed that to not necessarily rely on functional assays. The present invention provides such methods.
SUMMARY
The present invention relates generally to variants of target biological molecules ("TBMs") and to methods of making and using the same to identify ligands of TBMs. More specifically, the invention relates to individual variant TBMs and sets of variant TBMs, each of which represents a modified version of a protein of interest where a thiol has been introduced at or near a site of interest. Ligands of TBMs are identified in part through the formation of a covalent bond between a potential ligand and a reactive thiol on the TBM.
DESCRIPTION OF THE FIGURES
Figure 1 schematically illustrates one embodiment of the tethering method wherein the target is a protein and the covalent bond is a disulfide. A thiol-containing protein is reacted with a plurality of ligand candidates. A ligand candidate that possesses an inherent binding affinity for the target is identified and a ligand is made comprising the identified binding determinant (represented by the circle).
Figure 2 is a representative example of a tethering experiment. Figure ZA is the deconvoluted mass spectrum of the reaction of thymidylate synthase ("TS") with a pool of 10 different ligand candidates with little or no binding affinity for TS. Figure 2B is the deconvoluted mass spectrum of the reaction of TS with a pool of 10 different ligand candidates where one of the ligand candidates possesses an inherent binding affinity to the enzyme.
Figure 3 shows three illustrative examples of the distribution pattern of the residues that are each mutated to a cysteine. Figure 3A is an example where the residues are distributed about a single site of interest. The structure is of the core domain of HIV integrase with the portion comprising the site of interest shaded in dark gray. Figure 3B is an example where the residues are distributed about two sites of interest. The structure is of the human interleukin-1 receptor with the portions comprising the two sites of interested shaded in dark gray. Figure 3C is an example where the residues are distributed throughout the surface of a protein. The structure is the trimeric structure of human TNF-a.
Figure 4 shows the side chain rotamers of cysteines in A) (3-sheets and B) a-helices.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention relates generally to variants of target biological molecules ("TBMs") and to methods of making and using the same to identify ligands of TBMs.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
References, such as Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J.
Wiley & Sons (New York, NY 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, NY 1992), provide one skilled in the art with a general guide to many of the terms used in the present application.
Definitions The definition of terms used herein include:
The term "aliphatic" or "urisubstituted aliphatic" refers to a straight, branched, cyclic, or polyeyclic hydrocarbon and includes alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties.
The term "alkyl" or "unsubstituted alkyl" refers to a saturated hydrocarbon.
The term "alkenyl" or "unsubstituted alkenyl" refers to a hydrocarbon with at least one carbon-carbon double bond.
The term "alkynyl" or "unsubstituted alkynyl" refers to a hydrocarbon with at least one carbon-carbon triple bond.
The term "aryl" or "unsubstituted aryl" refers to mono or polycyclic unsaturated moieties having at least one aromatic ring. The term includes heteroaryls that include one or more heteroatoms within the at least one aromatic ring. Illustrative examples of aryl include: phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazoly, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like.

2 The term "substituted" when used to modify a moiety refers to a substituted version of the moiety where at least one hydrogen atom is substituted with another group including but not limited to:
aliphatic; aryl, alkylaryl, F, Cl, I, Br, -OH; -NO2; -CN; -CF3; -CHZCF3; -CHZCI; -CHZOH;
-CHZCHZOH; -CHZNH2; -CHZSOZCH3; -ORX; -C(O)R'; -COOR'; -C(O)N(R")2; -OC(O)R";
-OCOOR'; -OC(O)N(R")z; -N(R")i; -S(O)ZR'; and -NR"C(O)Rx where each occurrence of R" is independently hydrogen, substituted aliphatic, unsubstituted aliphatic, substituted aryl, or unsubstituted aryl. Additionally, substitutions at adjacent groups on a moiety can together form a cyclic group.
I0 The term "antagonist" is used in the broadest sense and includes any ligand that partially or fully blocks, inhibits or neutralizes a biological activity exhibited by a target, such as a TBM. In a similar manner, the term "agonist" is used in the broadest sense and includes any ligand that mimics a biological activity exhibited by a target, such as a TBM, for example, by specifically changing the function or expression of such TBM, or the efficiency of signaling through such TBM, thereby altering (increasing or inhibiting) an already existing biological activity or triggering a new biological activity.
The term "ligand" refers to an entity that possesses a measurable binding affinity fox the target. In general, a ligand is said to have a measurable affinity if it binds to the target with a Ka or a K; of less than about 100 mM, preferably less than about 10 mM, and more preferably less than about 1 mM. In preferred embodiments, the Iigand is not a peptide and is a small molecule. A Iigand is a small molecule if it is less than about 2000 daltons in size, usually less than about 1500 daltons in size. In more preferred embodiments, the small molecule ligand is less than about 1000 daltons in size, usually less than about 750 daltons in size, and more usually less than about 500 daltons in 2.5 size.
The term "ligand candidate" refers to a compound that possesses or has been modified to possess a reactive group that is capable of forming a covalent bond with a complimentary or compatible reactive group on a target. The reactive group on either the ligand candidate or the target can be masked with, for example, a protecting group.
The term "polynucleotide", when used in singular or plural, generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotides as defined herein include, without limitation, single- and double-stranded DNA, DNA including single- and double-stranded regions, single- and double-stranded RNA, and RNA including single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-

3 stranded or include single- and double-stranded regions. In addition, the term "polynucleotide" as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. The term "polynucleotide" specifically includes DNAs and RNAs that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, are included within the term "polynucleotides" as defined herein. In general, the term "polynucleotide"
embraces all chemically, enzymatically and/or metabolically modified forms of unmodified polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells.
The phrase "protected thiol" as used herein refers to a thiol that has been reacted with a group or molecule to form a covalent bond that renders it less reactive and which may be deprotected to regenerate a free thiol.
The phrase "reversible covalent bond" as used herein refers to a covalent bond that can be broken, preferably under conditions that do not denature the target. Examples include, without limitation, disulfides, Schiff bases, thioesters, coordination complexes, boronate esters, and the like.
The phrase "reactive group" is a chemical group or moiety providing a site at which a covalent bond can be made when presented with a compatible or complementary reactive group. Illustrative examples are -SH that can react with another -SH or -SS- to form a disulfide;
an -NHZ that can react with an activated -COOH to form an amide; an -NHZ that can react with an aldehyde or lcetone to form a Schiff base and the like.
The phrase "reactive nucleophile" as used herein refers to a nucleophile that is capable of forming a covalent. bond with a compatible functional group on another molecule under conditions that do not denature or damage the target. The most relevant nucleophiles are thiols, alcohols, and amines.
Similarly, the phrase "reactive electrophile" as used herein refers to an electrophile that is capable of forming a covalent bond with a compatible functional group on another molecule, preferably under conditions that do not denature or otherwise damage the target. The most relevant electrophiles are imines, carbonyls, epoxides, aziridies, sulfonates, disulfides, activated esters;
activated carbonyls, and hemiacetals.

4 The phrase "site of interest" refers to any site on a target on which a ligand can bind. For example, when the target is an enzyme, the site of interest can include amino acids that make contact with, or lie within about 10 Angstroms (more preferably within about 5 Angstroms) of a bound substrate, inhibitor, activator, cofactor, or allosteric modulator of the enzyme. When the enzyme is a protease, the site of interest includes the substrate binding channel from S6 to S6', residues involved in catalytic function (e.g. the catalytic triad and oxy anion hole), and any cofactor (e.g. metal such as Zn) binding site. When the enzyme is a protein kinase, the site of interest includes the substrate-binding channel in addition to the ATP binding site. When the enzyme is a dehydrogenease, the site of interest includes the substrate binding region as well as the site occupied by NAD/NADH.
When the enzyme is a hydralase such as PDE4, the site of interest includes the residues in contact with CAMP as well as the residues involved in the binding of the catalytic divalent cations.
The terms "target," "Target Molecule," and "TM" are used interchangeably and in the broadest sense, and refer to a chemical or biological entity for which the binding of a ligand has an effect on the function of the target. The target can be a molecule, a portion of a molecule, or an aggregate of molecules. The binding of a ligand may be reversible or irreversible. Specific examples of target molecules include polypeptides or proteins such as enzymes and receptors, transcription factors, ligands for receptors such growth factors and cytokines, immunoglobulins, nuclear proteins, signal transduction components (e.g., kinases, phosphatases), polynucleotides, carbohydrates, glycoproteins, glycolipids, and other macromolecules, such as nucleic acid-protein complexes, chromatin or ribosomes, lipid bilayer-containing structures, such as membranes, or structures derived from membranes, such as vesicles. The definition specifically includes Target Biological Molecules ("TBMs") as defined below.
A "Target Biological Molecule" or "TBM" as used herein refers to a single biological molecule or a plurality of biological molecules capable of forming a biologically relevant complex with one another for which a small molecule agonist or antagonist has an effect on the function of the TBM.
In a preferred embodiment, the TBM is a protein or a portion thereof or that comprises two or more amino acids, and which possesses or is capable of being modified to possess a reactive group that is capable of forming a covalent bond with a compound having a complementary reactive group.
Preferred TBMs include: cell surface and soluble receptors and their ligands;
steroid receptors;
hormones; immunoglobulins; clotting factors; nuclear proteins; transcription factors; signal transduction molecules; cellular adhesion molecules, co-stimulatory molecules, chemokines, molecules involved in mediating apoptosis, enzymes, and proteins associated with DNA and/or RNA synthesis or degradation.

5 Many TBMs are those participate in a receptor.-ligand binding interaction and can be either member of a receptor-ligand pair. Illustrative examples of growth factors and their respective receptors include those for: erythropoietin (EPO), thrombopoietin (TPO), angiopoietin (ANG), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), epidermal growth factor (EGF), heregulin-a and heregulin-j3, vascular endothelial growth factor (VEGF), placental growth factor (PLGF), transforming growth factors (TGF-c~
and TGF-~), nerve growth factor (NGF), neurotrophins, fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), bone morphogenetic protein (BMP), connective tissue growth factor (CTGF), hepatocyte growth factor (HGF), and insulin-like growth factor 1 (IGF-1). Illustrative examples of hormones and their respective receptors include those for: growth hormone, prolactin, placental lactogen (LPL), insulin, follicle stimulating hormone (FSH), luteinizing hormone (LH), and neurokinin-1.
Illustrative examples of cytokines and their respective receptors include those for: ciliary neurotrophic factor (CNTF), oncostatin M (OSM), TNF-a; CD40L, stem cell factor (SCF);
interleukin-1, interleukin-2, interleukin-4, interleulein-5, interleulcin-6, interleukin-8, interleulcin-9, interleukin-13, and interleukin-18.
Other TBMs include: cellular adhesion molecules such as CD2, CDlla, LFA-l, LFA-3, ICAM-5, VCAM-1, VCAM-5, and VLA-4; costimulatory molecules such as CD28, CTLA-4, B7-l;
B7-2, ICOS, and B7RP-1; chemokines such as RANTES and MIPlb; apoptosis factors such as APAF-1, p53, bax, bak, bad, bid, and c-abl; anti-apoptosis factors such as bcl2, bcl-x(L), and mdm2;
transcription modulators such as AP-1 and AP-2; signaling proteins such as TRAF-1, TRAF-2, TRAF-3, TRAF-4, TRAF-5, and TRAF-6; and adaptor proteins such as grb2, cbl, shc, nek, and crk Enzymes are another class of preferred TBMs and can be categorized in numerous ways including as: allosteric enzymes; bacterial enzymes (isoleucyl tRNA synthase, peptide deformylase, DNA
gyrase, and the like); fungal enzymes (thymidylate synthase and the like);
viral enzymes (HIV
integrase, HSV protease, Hepatitis C helicase, Hepatitis C protease, rhinovirus protease and the like); lcinases (serinelthreonine, tyrosine, and dual specificity);
phosphatases (serine/threonine, tyrosine, and dual specificity); and proteases (aspartyl, cysteine, metallo, and serine proteases).
Notable subclasses of enzymes include: kinases such as Lck, Syk, Zap-70, JAK, FAK, ITK, BTK, MEK, MEKK, GSK-3, Raf, tgf (3-activated kinase-1 (TAK-1), PAK-l, cdlc4, Akt, PKC A, IKK j3, IKK-2, PDK, ask, nik, MAPKAPK, p90rsk, p70s6k, and PI3-K (p85 and p110 subunits);
phosphatases such as CD45, LAR, RPTP-a, RPTP-~, Cdc25A, kinase-associated phosphatase, map kinase phosphatase-1, PTP-1B, TC-PTP, PTP-PEST, SHP-1 and SHP-2; caspases such as caspases-3S 1, -3, -7, -8, -9, and -11; and cathespins such as cathepsins B, F, K, L, S, and V. Other enzymatic targets include: BACE, TALE, cytosolic phospholipase A2 (cPLA2), PARP, PDE I-VII, Rac-2, CD26, inosine monophosphate dehydrogenase, 15-lipoxygenase, acetyl CoA
carboxylase,

6 adenosylmethionine decarboxylase, dihydroorotate dehydrogenase, leukotriene A4 hydrolase, and nitric oxide synthase.
Variants of TBMs The present invention relates generally to variants of target biological molecules ("TBMs") and to methods of making and using the same to identify ligands of the TBMs. In preferred embodiments, the TBMs are proteins and the variants are cysteine mutants thereof wherein a naturally occurring non-cysteine residue of a TBM is mutated into a cysteine residue. The non-native cysteine provides a reactive group on the TBM for use in tethering.
Tethering is a method of ligand identification that relies upon the formation of a covalent bond between a reactive group on a target and a complimentary reactive group on a potential ligand, and is described in U.S. Patent No. 6,335, 155, PCT Publication Nos. WO 00!00823 and WO 02/42773, Erlanson et al., Proc. Nat. Acad. Sci. LISA 97: 9367-9372 (2000), and U.S.
Serial No. 10/121,216 entitled METHODS FOR LIGAND DISCOVERY by inventors Daniel Erlanson, Andrew Braisted, and James Wells (corresponding PCT Application No. US02/13061). The resulting covalent complex is termed a target-ligand conjugate. Because the covalent bond is formed at a pre-determined site on the target (e.g., a native or non-native cysteine), the stoichiometry and binding location are known fox ligands that are identified by this method.
Once formed, the ligand portion of the target-ligand conjugate can be identified using a number of methods. In preferred embodiments, mass spectroscopy is used. The target-ligand can be detected directly in the mass spectrometer or fragmented prior to detection.
Alternatively, the ligand can be liberated from the target-ligand conjugate within the mass spectrophotometer and subsequently identified. In other embodiments, alternate detection methods are used including to but not limited to: chromatography, labeled probes (fluorescent, radioactive, etc.), nuclear magnetic resonance ("NMR"), .surface plasmon resonance (e.g., BIACORE), capillary electrophoresis, X-ray crystallography and the like. In still other embodiments, functional assays can also be used when the binding occurs in an area essential for what the assay measures.
A schematic representation of one embodiment of the tethering method where the target is a protein and the covalent bond is a disulfide is shown in Figure 1. A thiol containing protein is reacted with a plurality of ligand candidates. In this embodiment, the ligand candidates possess a masked thiol in the fornl of a disulfide of the formula -SSR' where R' is unsubstituted C,-C,o alkyl, substituted C~-Clo alkyl, unsubstituted aryl or substituted aryl. In certain embodiments, R' is selected to enhance the solubility of the potential ligand candidates. As shown, a ligand candidate that possesses an inherent binding affinity for the target is identified and a corresponding ligand that

7

8 PCT/US02/24921 does not include the disulfide moiety is made comprising the identified binding determinant (represented by the circle).
Figure 2 illustrates two representative tethering experiments where a target enzyme, E. coli thymidylate synthase, is contacted with ligand candidates of the formula O
Rc~~'yH2 H
wherein R° is the variable moiety among this pool of library members and is unsubstituted aliphatic, substituted aliphatic, unsubstituted aryl, or substituted aryl. Like all TS
enzymes, E, coli TS has an active site cysteine (Cys146) that can be used for tethering. Although the E.
coli TS also includes four other cysteines, these cysteines are buried and were found not to be reactive in tethering experiments. For example, in an initial experiment, wild type E. coli TS and the C146S mutant (wherein the cysteine at position 146 has been mutated to serine) were contacted with cystamine, HZNCH2CHZSSCHZCHZNH2. The wild type TS enzyme reacted cleanly with one equivalent of cystamine while the mutant TS did not react indicating that the cystamine was reacting with and was selective for Cys 146.
Figure 2A is the deconvoluted mass spectrum of the reaction of TS with a pool of 10 different ligand candidates with little or no binding affinity for TS. In the absence of any binding interactions, the equilibrium in the disulfide exchange reaction between TS
and an individual ligand candidate is to the unmodified enzyme. This is schematically illustrated by the following equation.
TS-Cysl.~s-SH + R~~N~~~H2 ~ TS-Cys~ns-SS~/N~Rc -~- TS-Cyslas-SS~H2 H ~ ~O
As expected, the peak that corresponds to the unmodified enzyme is one of two most prominent peaks in the spectrum. The other prominent peak is TS where the thiol of Cys 146 has been modified with cysteamine. Although this species is not formed to a significant extent for any individual library member, the peak is due to the cumulative effect of the equilibrium reactions for each member of the library pool. When the reaction is run in the presence of a thiol-containing reducing agent such as 2-mercaptoethanol, the active site cysteine can also be modified with the reducing agent. Because cysteamine and 2-mercaptoethanol have similar molecular weights, their respective disulfide bonded TS enzymes are not distinguishable under the conditions used in this experiment. The small peaks on the right correspond to discreet library members. Notably, none of these peaks are very prominent. Figure 2A is characteristic of a spectrum where none of the ligand candidates possesses an inherent binding affinity for the target.

Figure 2B is the deconvoluted mass spectrum of the reaction of TS with a pool of 10 different ligand candidates where one of the ligand candidates possesses an inherent binding affinity to the enzyme. As can be seen, the most prominent peak is the one that corresponds to TS where the thiol of Cys146 has been modified with the N tosyl-D-proline compound. This peak dwarfs all others including those corresponding to the unmodified enzyme and TS where the thiol of Cys146 has been modified with cysteamine. Figure 2B is an example of a mass spectrum where tethering has captured a moiety that possesses a strong inherent binding affinity for the desired site.
The representative tethering experiments of Figure 2 were performed on a TBM
that already possessed a naturally occurring cysteine at a site of interest (Cys146 located in the active site of the E. coli TS enzyme). However, because TBMs do not always possess a naturally occurring cysteine at or near a site of interest, the present invention provides cysteine mutant variants of TBMs as well as methods for making the same.
Thus, in one aspect of the present invention, a set comprising at least one cysteine mutant of a protein TBM is provided wherein a naturally occurring non-cysteine residue at or near a site of interest is mutated to a cysteine residue. In one embodiment, the set comprises a plurality of cysteine mutants of a protein TBM wherein each mutant has a different naturally occurring non-cysteine residue that is mutated to a cysteine residue. In another embodiment, the set comprises at least three cysteine mutants of a protein TBM wherein each mutant has a different naturally occurring non-cysteine residue that is mutated to a cysteine residue. In yet another embodiment, the set comprises at least five cysteine mutants of a protein TBM wherein each mutant has a different naturally occurring non-cysteine residue that is mutated to a cysteine residue. In still yet another embodiment, the set comprises at least ten cysteine mutants of a protein TBM wherein each mutant has a different naturally occurnng non-cysteine residue that is mutated to a cysteine residue.
In another aspect of the present invention, methods are provided for identifying residues that are suitable for mutating into cysteines. In preferred embodiments, a model or an experimentally derived three-dimensional structure (e.g., X-ray or 3D NMR) of a TBM is used to help identify residues that are suitable for mutating into cysteines. If a structure of the TBM of interest in unavailable, then a three-dimensional structure of a related or homologous TBM
can be used as a stand-in. Once suitable residues are identified using the stand-in structure, then methods lrnown in the art, such as sequence alignment, are used to identify the corresponding residues in the TBM of interest. In general, the methods described below for identifying suitable residues for mutating into cysteines can be used alone or in any combination with each other.

9 In one method, the local backbone conformation of a candidate residue is determined and a database of experimentally solved structures is searched for examples of a disulfide-bonded cysteine having the same or similar local backbone conformation as the candidate residue. Any combination of a residue's backbone atoms (N, Ca, C and O) can be used to determine the local conformation. The likelihood that the TBM accepts the cysteine mutation improves as more examples are found in a database of known disulfide-bonded cysteines in the same or similar local backbone conformation. Experimentally solved structures are available from many sources including the Protein Databank ("PDB") which can be found on the Internet at http://www.rcsb.or and the Protein Structure Database which can be found on the Internet at http://www.pcs.com. Lists of unique, high-resolution protein chains (grouped by structures having a certain resolution and R-factor) that can be used to compile a database of experimentally solved structures are found on the Internet at htt~//www fccc edu/research/labs/dunbrack/culledpdb.htinl. In general, the local environment of a candidate residue includes the candidate residue itself and at least one residue preceding or following the candidate residue in sequence. A conformation is considered the same or similar if the root mean square deviation ("RMSD") of the atoms being compared is less than or equal to aboutl.0 Angstrom2, more preferably, less than or equal to about 0.75 Angstromz, and even more preferably, less than or equal to about 0.5 Angstrom2.
In one embodiment, the method comprises:
a) obtaining a set of coordinates of a three dimensional structure of a protein TBM
having n number of residues;
b) selecting a candidate residue i on the three dimensional structure of the TBM
wherein the candidate residue i is the ith residue where i is a number between 1 and h and residue i is not a cysteine;
c) selecting a residue j where residue j is adjacent to residue i in sequence;
d) determining a candidate reference value wherein the candidate reference value is a spatial relationship between residue i and residue j;
e) obtaining a database comprising sets of coordinates of disulfide-containing protein fragments wherein each fragment comprises at least a disulfide-bonded cysteine and a first adjacent residue where the disulfide-bonded cysteine and the first adjacent residue share the same sequential relationship as residue i and residue j;
f) determining a comparative reference value for each fragment wherein the comparative reference value is the corresponding spatial relationship between the disulfide-bonded cysteine and the first adjacent residue as the candidate reference value is between residue i and j;
and, g) determining a score wherein the score is a measure of the number of fragments in the database that possess a comparative reference value that is the same or similar to the candidate reference value.
In another embodiment, the method further comprises selecting a residue k where residue Ic is adjacent to residue i in sequence and 7t is not j; and wherein the candidate reference value is a spatial relationship between residue i, residue j, and xesidue k;
each fragment comprises at least a disulfide-bonded cysteine, a first adjacent residue, and a second adjacent residue where the disulfide-bonded cysteine and the first and second adjacent residues share the same sequential relationship as residue i, residue j, and residue lc; and the comparative reference value is the corresponding spatial relationship between the disulfide bonded cysteine, the first adjacent residue, and the second adjacent residue as the candidate reference value is between residue i, residue j, and residue k.
In another embodiment, the method comprises:
a) obtaining a set of coordinates of a three dimensional structure of a protein TBM
having ra number of residues;
b) selecting a candidate residue i on the three dimensional structure of the TBM
wherein the candidate residue i is the ith residue where i is a number between 1 and iz and residue i is not a cysteine;
c) selecting residue j and residue Ic wherein residue j and residue Jz are both adjacent in sequence to residue i;
2,5 d) determining a candidate reference value wherein the candidate reference value is a spatial xelationship of at least one backbone atom from each of residue i, residue j, and residue k;
e) obtaining a database comprising sets of coordinates of disulfide-containing protein fragments wherein each fragment comprises at least a disulfide-bonded cysteine, a first adjacent xesidue, and a second adjacent residue where the disulfide-bonded cysteine, the first adjacent residue, and the second adjacent residue share the same sequential relationship as residue i, residue j, and residue k;
f) determining a comparative reference value for each fragment wherein the comparative reference value is the corresponding spatial relationship between the disulfide-bonded cysteine, the first adjacent residue, and the second adjacent residue as the candidate reference value is between residue i, residue j, and residue k; and, g) determining a score wherein the score is a measure of the number of fragments in the database that possess a comparative reference value that is the same or similar to the candidate reference value.
In another embodiment the spatial relationship comprises a dihedral angle. In yet another embodiment, the spatial relationship comprises a pair of phi psi angles. In another embodiment, the spatial relationship comprises a distance between atoms of two residues. An illustrative example of a computer algorithm for identifying disulfide bonded pairs in a database such as the PDB and matching them with a residue that is a candidate far cysteine mutation is described in Example 1.
In another method, a site of interest is defined on a TBM and suitable residues for cysteine mutation are identified based on the location of the residue from the site of interest.
In one embodiment, a suitable residue is a non-cysteine residue that is located within the site of interest. In another embodiment, a suitable residue is a non-cysteine residue that is located within about 5 t~ from the site of interest. In yet another embodiment, a suitable residue is a non-cysteine residue that is located within about 10 ~ from the site of interest. For the purposes of these measurements, any non-cysteine residue having at least one atom falling within about 5 ~ or about 10 1~ respectively from any atom of an amino acid within the site of interest is a suitable residue for mutating into a cysteine. A TBM can have one or multiple sites of interests. In some cases, a TBM has one site of interest and the set of residues that are each being mutated to a cysteine is clustered around this site of interest. In other cases, a TBM has at least two different sites of interest and the set of residues that are each being mutated to a cysteine is clustered around the at least two different sites of interest. Still in other cases, a TBM either does not possess a distinct site of interest or possesses multiple sites of interests such that the set of residues that are being mutated to a cysteine is dispersed throughout the protein surface. Figure 3 shows three illustrative examples of the distribution pattern of the residues that are each mutated to a cysteine Tn another method, solvent accessibility is calculated for each non-cysteine residue of a TBM and used to identify suitable residues for cysteine mutation. Solvent accessibility can be calculated using any number of known methods including using standard numeric methods (Lee, B. &
Richards, F. M. J. Mol. Biol 55:379-400 (1971); Shrake, A. & Rupley, J. A. J.
Mol. Biol. 79:351-371 (1973)) and analytical methods (Connolly, M. L. Science 221:709-713 (1983); Richmond, T. J.
J. Mol. Biol. 178:63-89 (1984)). In one embodiment, suitable residues for mutation include residues where the combined surface area of the residue's atoms is equaled to or greater than about 3 S 20 h2. In another embodiment, suitable residues for mutation include residues where the combined surface area of the residue's atoms is equaled to or greater than about 30 t~z. In yet another embodiment, suitable residues for mutation include residues where the combined surface area of the residue's atoms is equaled to or greater than about 40 A2.
In another method, suitable residues for cysteine mutation are identified by hydrogen bond analysis.
Tn one embodiment, a suitable residue is a non-cysteine residue that does not participate in any hydrogen bond interaction. In another embodiment, a suitable residue is a non-cysteine residue whose side chain does not participate in any hydrogen bond interaction. Tn yet another embodiment, a suitable residue is a non-cysteine residue whose side chain does not participate in a hydrogen bond interaction with a backbone atom.
In another method, suitable residues for cysteine mutation are identified by rotamer analysis. In one embodiment, the method comprises:
a) obtaining a three dimensional structure of a TBM having n number of residues and a site of interest;
b) selecting a candidate residue i that is at or near the site of interest wherein the candidate residue i is the ith residue where i is a number between 1 and n and residue i is not a cysteine;
c) generating a set of mutated TBM structures wherein each mutated TBM
structure possesses a cysteine residue instead of residue i and wherein the cysteine residue is placed in a standard rotamer conformation; and, d) evaluating the set of mutated TBM structures.
In another embodiment, a standard rotamer conformation for cysteine comprises the set of cysteine rotamers enumerated by Ponders and Richards as described by Ponder, J. W. and Richards, F. M. J.
Mol. Biol. 193: 775-791 (1987).
In another embodiment, a standard rotamer conformation for cysteine comprises a chil angle selected from the group consisting of about 60°, about 180°, and about 300° and a chit angle selected from the group consisting of about 60°, about 120°, about 180°, about 270°, and about 300°.
In another embodiment, the method further comprises determining whether residue i is part of an a-helix or a (3-sheet and then selecting a standard rotamer conformation based on the assigned secondary structure. As shown in Figure 4, a different set of rotamers is preferred depending on the secondary structure that is assigned to the cysteine. Residue i is considered to be part of an a-helix if the phi psi angles of residues i-1, i, and i-~-1 are about 30030°
and 31530° respectively, and is considered to be part of a (3-sheet if the phi psi angles of residues i-1, i, and i+1 are about 24030°

and 12030°. If residue i is part of an a-helix, then a standard rotamer conformation for cysteine comprises a chil chit pair selected from the group consisting of about 180° and about 60°; about 180° and about 270°; and about 300° and about 300°. If residue i is part of an (3-helix, there a standard rotamer conformation for cysteine comprises a chil chit pair selected from the group consisting of about 180° and about 60°; about 180° and about 180°; about 180° and about 270°; and about 300° and about 300°.
In another embodiment, the set of mutated TBM structures are evaluated based upon whether an unfavorable steric contact is made. A residue is considered to be a suitable candidate for cysteine mutation if it can be substituted with at least one cysteine rotamer for which no unfavorable steric contact is made. An unfavorable steric contact is defined as interatomic distances that are less than about 80% of the sum of the van der Waals radii of the participating atoms. In one variation, only the backbone atoms of the TBM are considered for the purposes of determining whether the rotamers malce an unfavorable contact with the TBM. In another variation, the backbone atoms and C~ of the TBM are considered for the purposes of determining whether the rotamers make an unfavorable contact with the TBM.
In another embodiment, the set of mutated TBM structures are evaluated based on a force field calculation. Illustrative force field methods are described by, for example, Weiner, S. J. et al. .J.
Comput. Chem. 7: 230-252 (1986); Nemethy, G. et al. J. Phys. Chem. 96: 6472-6484 (1992); and Brooks, B.R, et al. J. Conaput. Cherra. 4: 187-217 (1983). All minimized conformations within about 10 kcal/mol or more preferably within about 5 kcal/mol, of the lowest-energy conformation are considered accessible.
In another embodiment, each mutated TBM structure possesses a cysteine that is capped with a S-methyl group (side chain is -CHzSSCH3) instead of residue i and wherein the capped cysteine residue is placed in a standard rotamer conformation for cysteine. A residue is considered to be a suitable candidate for cysteine mutation if it can be substituted with at least one rotamer that places the methyl carbon of the S-methyl group closer to the site of interest than the C~, In addition to adding one or more cysteines to a site of interest, it may be desirable to delete one or more naturally occurring cysteines (and replacing them with alanines for example) that are located outside of the site of interest. These mutants wherein one or more naturally occurring cysteines are deleted or "scrubbed" comprise another aspect of the present invention.
Various recombinant, chemical, synthesis and/or other techniques can be employed to modify a target such that it possesses a desired number of free thiol groups that are available for tethering. Such techniques include, for example, site-directed mutagenesis of the nucleic acid sequence encoding the target polypeptide such that it encodes a polypeptide with a different number of cysteine residues.
Particularly preferred is site-directed mutagenesis using polymerase chain reaction (PCR) amplification (see, for example, U.S. Pat. No. 4,683,195 issued 28 July 1987;
and Current Protocols In Molecular Biology, Chapter 15 (Ausubel et al., ed., 1991). Other site-directed mutagenesis techniques are also well known in the art and are described, for example, in the following publications: Ausubel et al., supra, Chapter 8; Molecular Cloning: A
Laboratory Manual., 2nd edition (Sambrook et al., 1989); Zoller et al., Methods Enzymol. 100:468-500 (1983); Zoller &
Smith, DNA 3:479-488 (1984); Zoller et al., Nucl. Acids Res., 10:6487 (1987);
Brake et al., Proc.
Natl. Acad. Sci. USA 81:4642-4646 (1984); Botstein et al., Science 229:1193 (1985); Kunkel et al., Methods Enzymol. 154:367-82 (1987), Adelman et al., DNA 2:183 (1983); and Carter et al., Nucl.
Acids Res., 13:4331 (1986). Cassette mutagenesis (Wells et al., Gene, 34:315 [1985]), and restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:41S [1986]) may also be used.
Amino acid sequence variants with more than one amino acid substitution may be generated in one of several ways. If the amino acids are located close together in the polypeptide chain, they may be mutated simultaneously, using one oligonucleotide that codes for all of the desired amino acid substitutions. If, however, the amino acids are located some distance from one another (e.g.
separated by more than ten amino acids), it is more difficult to generate a single oligonucleotide that encodes all of the desired changes. Instead, one of two alternative methods may be employed.
In the first method, a separate oligonucleotide is generated for each amino acid to be substituted.
The oligonucleotides are than annealed to the single-stranded template DNA
simultaneously, and the second strand of DNA that is synthesized from the template will encode all of the desired amino acid substitutions. The alternative method involves two or more rounds of mutagenesis to produce the desired mutant.
The invention is further illustrated by the following, non-limiting examples.
Unless otherwise noted, all the standard molecular biology procedures are performed according to protocols described in (Molecular Cloning: A Laboratory Manual, vols. 1-3, edited by Sambrook, J., Fritsch, E.F., and Maniatis, T., Cold Spring Harbor Laboratory Press, 1989; Current Protocols in Molecular Biology, vols. 1-2, edited by Ausbubel, F., Brent, R., Kingston, R., Moore, D., Seidman, J.G., Smith, J., and Struhl, K., Wiley Interscience, 1987).

This example provides an illustrative computer algorithm written in FORTRAN
for identifying disulfide pairs from the PDB that align with potential tethering mutants. A
stepwise description of the program and the source code are described below.
First, a user supplies the name of the PDB file for the template protein, the residues of the fragment to match, and the relative position of the cysteine within that fragment.
Preferred values are 1-2 residues N- and C-terminal to a potential mutant site. For example, if residue Glu 200 of PTP1B is a candidate residue, then the user would specify the fragment from residues 198 to 202 with the cysteine at relative position 3.
Second, the program reads the template file, extracts the coordinates of the N,Ca,C,O atoms for the template residues, and determines the values of ~ (C'-N-Ca C torsion) and ~ (N-Ca C-N') for each of the template residues Third, the program generates a "residue filter" based on the template ~/~
values. This filter is used to identify contiguous segments of a test protein that have ~/~r values matching those of the template residues to within a coarse (~60°) tolerance. The filter also requires that the fragment must contain a cysteine at the appropriate position. In the PTP1B example above, the filter would identify 5-residue fragments of a test protein that roughly matched the backbone conformations of residues 198-202 of PTP1B and contained a cysteine in position 3.
Fourth, the rest of the program operates iteratively on a user-supplied list of test proteins provided in a simple text file. In one embodiment, this file contains approx. 2500 culled PDB chains. For each test structure:
a) The program reads the coordinates, determines the sequence and ~/1~ values for each residue, and identifies any contiguous chains that match the residue filter specified in step (3).
b) The program checks to see that the cysteine residue in this fragment is participating in a disulfide bond. This is done by simple distance-and angle-based searching from the Sr atom.
Fragments containing unpaired cysteines are rejected.
c) For each fragment, the N,Ca,C,O atoms of the backbone are overlaid onto the corresponding atoms from the template molecule (e.g. 198-202 of PTP1B). If the backbone fits with an RMSD within a user-specified tolerance (typically 0.5-0.75 A), the overlaid coordinates of this fragment along with its disulfide-bound partner are written to a file in PDB format. A log file is maintained of each "hit", along with its RMSD value. The hits are viewed with a graphic program like Insight II or PyMOL.

Source Code P
c parameter(MAX HITS = 10000) S C
$INCLUDE tk.inc $INCLUDE tk functions.inc $INCLUDE rsm.inc $INCLUDE rsm functions.inc c Record /hndlrec/ data handle, fragment handle, template handle Record /atom rec/ AtomRec Record /res rec/ ResRec Record /res filter/ FragmentFilter(MAX_RMS ATOMS), 1S TemplateFilter(MAX_RMS_ATOMS) Record /vec/ TemplateVecArray, FragmentVecArray, T1, T2 Dimension TemplateVecArray(MAX_RMS ATOMS), FragmentVeCArray(MAX_RMS ATOMS) c -Integer*4 numTemplateRes, TemplateResList(MAX HITS), numHitRes, HitResList(MAX_HITS), numTemplateVec, CysIndex, FrameIndex, numSS, SS-1(MAX_RES), SS-2(MAX-RES), min element, max_element, num_res, icnt, jcnt, numFragAtom, FragAtomList(MAX RES), 2.S FragAtomIndex(MAX_RES),ires, fires, icys, cys_idx, jcys, iatom, jatom, LISTin, PDBout, LOGout, len_name, len_root Real*8 temp min, temp max, R2(3, 3), RMS cutoff, RMSwalue, RMS_WT(MAX_RES), angle_tol Character listfile*80, full name*80, file-path*80, file name*80, file_root*80, file_ext*80, structure_name*15, full_structure name*23, first resnumber*7, charl*1, char3*1, dine*80, token*80 C
3S LISTin = 9 PDBout = 10 LOGout = 11 FrameIndex = 1 RMS_cutoff = 0.5 angle tol = 60.
do fires = 1, MAX_RES
RMS_WT(ires) - 1.0 end do c 4S c...Get template information.
c write (6,'(/, " Enter template PDB filename : " ,$)') read (5,'(a)') tline if (.not.readPDBFile(tline, template_handle)) then S0 write (6,'(" ERROR: Unable to read template PDB file ***** " )') return end if if (get num total residues(template handle, num_res)) continue c...get template residue numbers and convert to residue indeces S5 10 write (6,'(5x, " Enter beginning, ending template residues . " ,$)') read (5,'(a)') tline if (.not.get token(tline, token)) goto 10 do icnt = 1, num_res if (getResData(template_handle, FrameIndex, icnt, ResRec)) 60 continue 1'7 if (ljust(ResRec.residue number)) continue if (compstr(ResRec.residue-number, token)) then fires = icnt goto 20 $ end if end do write (6, ' ( "ERROR: Unable to find residue ", a50) ' ) token goto 10 20 if (.not.get token(tline, token)) goto 10 do icnt = l,~num_res if (getResData(template handle, FrameIndex, icnt, ResRec)) continue if (ljust(ResRec.residue number)) continue if (compstr(ResRec.residue number, token)) then 1$ fires = icnt goto 30 end if end do write (6,'(" ERROR: Unable to find residue " ,a50)') token goto to continue c numTemplateRes = fires - fires + l do icnt = Z, numTemplateRes 2$ TemplateResList(icnt) = fires + icnt-1 end do if (numTemplateRes .eq. 1) then cys-idx = 1 else 30 write (6,'(5x, " Enter relative position of cysteine : " $)') read(5,*) cys idx end if write (6, ' (5x, ' ' Enter the RMS cutoff : ' ' , $) ' ) read (5,*) RMS cutoff 3$ c -c...Collect template residue atoms for fitting (N/CA/C/O).
c numTemplateVec = 0 do icnt = 1, numTemplateRes fires = TemplateResList(icnt) if (.not.getAtomOfRes(template handle, FrameIndex, fires, 'N', AtomRec)) then write (6,'(" ERROR: Unable to get N of template residue ",i4)') fires 4$ call exit else numTemplateVec = numTemplateVec + 1 TemplateVecArray(numTemplateVec) = AtomRec.vector end if if (. not.getAtomOfRes(template handle, Framelndex, fires, 'CA', AtomRec)) then write (6,'(" ERROR: Unable to get CA of template residue " ,i4)') fires call exit $$ else numTemplateVec = numTemplateVec + 1 TemplateVecArray(numTemplateVec) = AtomRec.vector end i f if (.not.getAtomOfRes(template handle, FrameIndex, fires, 'C', AtomRec)) then write (6,'(" ERROR: Unable to get C of template residue " , i4 ) ' ) fires call exit else $ numTemplateVec = numTemplateVec + 1 TemplateVecArray(numTemplateVec) = AtomRec.vector end if if (.not.getAtomOfRes(template_handle, FrameIndex, fires, 'O', AtomRec)) then write (6,'(" ERROR: Unable to get 0 of template residue " , i4 ) ' ) fires call exit else numTemplateVec = numTemplateVec + 1 1$ TemplateVecArray(numTemplateVec) = AtomRec.vector end if end do C
c...Construct residue filter based on internal angles from the template.
c if (.not. initializeResFilter(FragmentFilter, MAX_RMS_ATOMS)) then write(6, '(2X, " ERROR: Unable to make residue-filter record" ) r ) call exit end if FragmentFilter(1).seq-len = numTemplateRes FragmentFilter(1).start_residue = 2 do icnt = l, numTemplateRes fires = TemplateResList(icnt) if (.not.GetR.esData(template'handle, FrameTndex, fires, ResRec)) then fires write (6,'(" ERROR: Unable to get record for residue " ,i4)') call exit 3$ end if FragmentFilter(icnt).phi_val = ResRec.phi_val FragmentFilter(icnt).phi_tol = angle tol FragmentFilter(icnt).psi_val = ResRec.psi_val FragmentFilter(icnt).psi_tol = angle_tol end do FragmentFilter(cys_idx).residue name = 'CYS' if (returnTrajectory(template_handle)) continue C
call getenv ('RSM PDB_LISTFILE', listfile) 4$ if (listfile.eq.'~') then write (6,'(/, " Enter structure listfile : " ,$)') read (5,'(a)') listfile end if open (file=listfile, unit=LISTin, status="old") SO C
write (6,'(/, " Enter output logfile : " ,$)~) read (5,'(a)') tline open (file=tline, unit=LOGout, status="unknown") write (6,'(" Enter output PDBfile : ",$)') $$ read (5,'(a)') tline open (file=tline, unit=PDBout, status="unknown') C
c...Main loop c 60 50 read (LISTin,'(a)',end=999) full_name if (full name(1:l).eq.'#') goto 50 if (parse filename(full name, file_path, file name, file root, file_ext)) continue len name = index(file root,' ') - 1 c if (.not. readPDBFile(full_name, data handle)) then write(6, '(2X, " **Unable to read -PDB file ")') go to 100 end if C
c...Select only fragments containing cysteines.
c if (selectResByFilter(data handle, FrameIndex, FragmentFilter, numHitRes, HitResList)) continue if (numHitRes .eq. 0) goto 100 c c...Get list of cysteines participating in disulfide bonds.
C
call find_disulfide_pairs(data handle, FrameIndex, MAX_RES, numSS, SS_1, SS_2) if (numSS .eq. 0) goto l00 c c...Loop through fragments. Test whether: (a) cys idx'th residue is participating in a disulfide and c (b) whether the fragment has an acceptable RMS overlap with the template coordinates.
C
do 90, icnt = 1, numHitRes icys = HitResList(icnt) + cys_idx - 1 jcys = 0 do jcnt = 1, numSS
if (SS_l(jcnt).eq.icys) then jcys = SS_2 (jcnt) else if (SS 2(jcnt) .eq.icys) then joys = SS-1(jcnt) end if end do if (joys .eq. 0) goto 90 c c...Extract coordinates for RMS test c numFragAtom = 0 do jcnt = 1, numTemplateRes fires = HitResList(icnt) + jcnt - 1 if (.not.getAtomOfRes(data handle, FrameIndex, fires, 'N', 4$ AtomRec)) then write (6,'(" ERROR: Unable to get N of fragment residue ",i4)') fires goto 90 else numFragAtom = numFragAtom + 1 FragAtomList(numFragAtom) = AtomRec.index end if if (.not.getAtomOfRes(data handle, FrameIndex, fires, 'CA', AtomRec)) then write (6,'(" ERROR: Unable to get CA of fragment residue ",i4)') fires goto 90 else numFragAtom = numFragAtom + 1 FragAtomList(numFragAtom) = AtomRec.index end if if (. not.getAtomOfRes(data handle, FrameIndex, fires, 'C', AtomRec)) then write (6,'(" ERROR: Unable to get C of fragment residue ",i4)') fires goto 90 else numFragAtom = numFragAtom + 1 FragAtomList(numFragAtom) = AtomRec.index end if if (. not.getAtomOfRes(data handle, FrameIndex, fires, '0', AtomRec)) then ",i4)') fires write (6,'(" ERROR: Unable to get O of fragment residue goto 90 else numFragAtom = numFragAtom + 1 FragAtomList(numFragAtom) = AtomRec.index end if do iatom = Z, numFragAtom jatom = FragAtomList(iatom) if (. not.getAtomData(data-handle, FrameIndex, jatom, AtomRec)) then write (6,'(" ERROR: Unable to get record for fragment atom ",i6)') jatom goto 90 else FragmentVecArray(iatom) = AtomRec.vector end if end do end do C
c...RMS Fit to template.
C
call RMS_FIT(numTemplateVec, TemplateVecArray, FragmentVecArray, RMS WT, RMS_VALUE, t1, t2, r2) t2.x = -1.0 * t2.x t2.y = -1.0 * t2.y t2.z = -1.0 * t2.z if (RMS VALUE ,gt. RMS cutoff) goto 90 c c...Extract remaining atoms for fragment.

C

if (. not.getAtomOfRes(data'handle, FrameIndex, icys,'CB', AtomRec)) then write (6,'(" ERROR: Unable to get CB of fragment residue " ,i4)') icys goto 90 else numFragAtom = numFragAtom + 1 $0 FragAtomList(numFragAtom) = AtomRec.index end if if (. not.getAtomOfRes(data'handle, FrameIndex, icys,'SG', AtomRec)) then write (6,'(" ERROR: Unable to get CB of fragment residue ",i4)') icys goto 90 else numFragAtom = numFragAtom + 2 FragAtomList(numFragAtom) = AtomRec.index end if if (. not.getAtomOfRes(data_handle, FrameIndex, jcys, 'CA', AtomRec)) then write (6,'(" ERROR: Unable to get CA of fragment residue ",i4)') joys S goto 90 else numFragAtom = numFragAtom + 1 FragAtomList(numFragAtom) = AtomRec.index end if if (. not.getAtomOfRes(data_handle, FrameIndex, jcys, 'CB', AtomRec)) then write (6,'(" ERROR: Unable to get CB of fragment residue ",i4)') jcys goto 90 1S else numFragAtom = numFragAtom + 1 FragAtomList(numFragAtom) = AtomRec.index end if if (. not.getAtomOfRes(data_handle, FrameIndex, jcys, 'SG', 2,0 AtomRec) ) then write (6,'(" ERROR: Unable to get CB of fragment residue ",i4)') joys goto 90 else ZS numFragAtom = numFragAtom + 1 FragAtomList(numFragAtom) = AtomRec.index end if call index_int_array(numFragAtom, FragAtomList, FragAtomIndex) call reorder int array(numFragAtom, FragAtomList, FragAtomIndex) 30 c c...Construct fragment object and apply transformations.
C
if (getResData(data handle, 1, icys, ResRec)) continue if (ResRec.ChainID.ne.' ') then 35 first_resnumber = ResRec.ChainID //
ResRec.residue_number(1:6) else first_resnumber = ResRec.residue_number(1:6) end if 40 full_structure_name =
file root(l:len name)//'-'//first resnumber c if (make trj_from-atom_list(data handle, INT ONE, INT ONE, numFragAtom, FragAtomList, 45 . fragment handle)) continue call rsm_translate_frame(fragment_handle, INTONE, t2) call rsm_rotate_frame(fragment handle, INT ONE, r2) call rsm_translate_frame(fragment_handle, INTONE, t1) call append_fragment(fragment handle, full_structure name, SO PDBout, .FALSE.) write (LOGout,'(a22,1x,f5.2)') full_structure name, RMS_value if (returnTrajectory(fragment handle)) continue c 90 end do SS 100 if (returnTrajectory(data~handle)) continue goto 50 999 close(LISTin) close(PDBout) close(LOGout) 60 call exit end Interleukin-2 (IL-2) (accession number SWS P01585) is a cytolcine with a predominant role in the proliferation of activated T helper lymphocytes. Mitogenic stimuli or interaction of the T cell receptor complex with antigen/MHC complexes on antigen presenting cells causes synthesis and secretion of IL-2 by the activated T cell, followed by clonal expansion of the antigen-specific cells.
These effects are known as autocrine effects. In addition, IL-2 can have paracrine effects on the growth and activity of B cells and natural killer (NK) cells. These outcomes are initiated by interaction of IL-2 with its receptor on the T cell surface. Disruption of the IL-2/IL-2R interaction can suppress immune function, which has a number of clinical indications, including graft vs. host disease (GVHD), transplant rejection, and autoimmune disorders such as psoriasis, uveitis, rheumatoid arthritis, and multiple sclerosis. There is structural information available of the C12SA
mutant [3INK, Mc Kay, D. B. & Brandhuber, B. J., Science 257: 412 (1992)].
Cloning of Human IL-2 Numbering of the wild type and mutant IL-2 residues follows the convention of the first amino acid residue (A) of the mature protein being residue number 1 independent of any presequence e.g. met for the E. coli produced protein [see Taniguchi, T., et al., Nature 302: 305-310 (1983) and Devos, R., et al., Nucleic Acids Res. 11: 4307-4323 (1983)].
The DNA sequence encoding human Interleukin-2 (IL-2) was isolated from plasmid pTCGF-11 (ATCC). PCR primers were designed to contain resfiriction endonuclease sites NdeI and XhoI for subcloning into a pRSET expression vector (Invitrogen).
IL2 GGAATTCCATATGGCACCTACTTCAAGTTCTACAAAGAAAACA SEQID NO:1 Forward IL2 CCGCTCGAGTCAAGTTAGTGTTGAGATGATGCTTTGACA SEQ ID NO:2 Reverse Double-stranded IL-2/pRSET was prepared by the following procedure. The PCR
product containing the IL-2 sequence and pRSET were both cut with restriction endonucleases (1 ~l PCR
product, 1 p1 each endonuclease, 2 pM appropriate lOx buffer, 15 ~,l water;
incubated at 37 C for 2 hours). The products of nuclease cleavage were isolated from an agarose gel (1% agarose, TAE
buffer) and ligated together using T4 DNA Iigase (80 ng IL-2 sequence, 160 ng pRSET vector, 4 ItI
SX ligase buffer [300 mM Tris pH 7.5, 50 mM MgCl2, 20% PEG 8000, 5 mM ATP, S
mM DTT], 1 p,1 ligase; incubated at 15 C for 1 hour). 10 ~,l of the ligase reaction mixture was transformed into XL1 blue cells (Stratagene) (10 ~,l reaction mixture, 10 ~l SX KCM [0.S M KCI, 0.15 M CaCl2, 0.25 M MgCl2], 30 ~l water, SO p1 PEG-DMSO competent cells; incubated at 4 C
for 20 minutes, 25 C for 10 minutes), and plated onto LB/agar plates containing 100 p,g/ml ampicillin. After incubation at 37 C overnight, single colonies were grown in 5 ml 2YT media for 18 hours. Cells were then isolated and double-stranded DNA extracted from the cells using a Qiagen DNA
miniprep kit.
Generation of IL-2 Cys Mutations Site-directed mutants of IL-2 were prepared by the single-stranded DNA method (modification of Kunkel, T. A., Proc. Natl. Acad. Sci. U. S. A. 83: 488-492 (1985).
Oligonucleotides were designed to contain the desired mutations and 1 S-20 bases of flanking sequence.
The single-stranded form of the IL-2/pRSET plasmid was prepared by transformation of double-stranded plasmid into the CJ236 cell line (1 p1 IL-2/pRSET double-stranded DNA, 2 ~,1 2x KCM
salts, 7 ~1 water, 10 p1 PEG-DMSO competent CJ236 cells; incubated at 4 C for 20 minutes and 2S
IS C for 10 minutes; plated on LB/agar with 100 pg/ml ampicillin and incubated at 37 C overnight).
Single colonies of CJ236 ceps were then grown in SO ml 2YT media to midlog phase; S p,1 VCS
helper phage (Stratagene) were then added and the mixture incubated at 37 C
overnight. Single-stranded DNA was isolated from the supernatant by precipitation of phage (1/5 volume 20% PEG
8000/2.5 M NaCI; centrifuge at 12K for 1 S minutes.). Single-stranded DNA was then isolated from phage using Qiagen single-stranded DNA kit. Sequencing identified a leucine-2S
to serine mutation, which was corrected by mutagenesis using the "SZSL" oligonucleotide.
525L TAATTCCATTCAAAATCATCTGTA SEQ ID NO:3 Mutagenic Oligonucleotides N30C GGTGAGTTTGGGATTCTTGTAACAATTAATTCCATTCAAAATCATCTG SEQID NO:4 Y31C GGTGAGTTTGGGATTCTTACAATTATTAATTCCATTC SEQID NO:S
K32C GGTGAGTTTGGGATTACAGTAATTATTAATTCC SEQ ID NO:6 N33C CCTGGTGAGTTTGGGACACTTGTAATTATTAATTCC SEQID NO:7 K3SC GCATCCTGGTGAGACAGGGATTCTTGTAATTATTAATTCC SEQID NO:8 R38C CTTAAATGTGAGCATACAGGTGAGTTTGGGATTC SEQ ID NO:9 F42C GGGCATGTAAAACTTACATGTGAGCATCCTGG SEQ ID NO:10 K43C CTTGGGCATGTAAAAACAAAATGTGAGCATCC SEQ ID NO:11 Y4SC GGCCTTCTTGGGCATACAAAACTTAAATGTGAGC SEQ ID NO:12 E68C CTCAAACCTCTGGAGTGTGTGCTAAATTTAGC SEQ ID NO:13 L72C GTTTTTGCTTTGAGCACAA.TTTAGCACTTCCTCC SEQ ID NO:14 N77C CCTGGGTCTTAAGTGAAAACATTTGCTTTGAGCTAAATTTAGC SEQ ID NO:1S

GGGCATGTAAAAACAAAATGTGAGCATCCTGGTGAGTTTGGGATTCTTACAATTATTAATTCC
SEQ ID N0:16 S There was an additional double mutant made, L72C K43C, using the oligonucleotides corresponding to K43C and L72C single mutants (SEQ ID NO:11 and SEQ ID N0:14 respectively).
Site-directed mutagenesis was accomplished as follows: Mutagenesis oligonucleotides were dissolved to a concentration of 10 OD and phosphorylated on the S' end (2 ~1 oligonucleotide, 2 ~l

10 mM ATP, 2 p.1 lOX Tris-magnesium chloride buffer, 1 ~l 100 mM DTT, 10 p.1 water, 1 ~1 T4 PNK; incubate at 37 C for 4S minutes.). Phosphorylated oligonucleotides were then annealed to single-stranded DNA template (2 ~l single-stranded plasmid, 1 ~1 oligonucleotide, 1 ~.1 lOx TM
buffer, 6 ~.1 water; heat at 94 C for 2 minutes, SO C for S minutes, cool to room temperature).
1 S Double-stranded DNA was then prepared from the annealed oligonucleotide/template (add 2 ~1 10X
TM buffer, 2 ~12.S mM dNTPs, 1 ~l 100 rnM DTT, 1.S ~1 10 mM ATP, 4 ~l water, 0.4 ~l T7 DNA
polymerase, 0.6 ~.1 T4 DNA ligase; incubate at room temperature for 2 hours).
E. eoli (XLl blue, Stratagene) was then transformed with the double-stranded DNA (1 ~,1 double-stranded DNA, 10 ~1 Sx KCM, 40 ~tl water, SO ~l DMSO competent cells; incubate 20 minutes at 4 C, 10 minutes at room temperature), plated onto LB/agar containing 100 ~,g/ml ampicillin, and incubated at 37 C
overnight. Approximately four colonies from each plate were used to inoculate S ml 2YT
containing 100 pg/ml ampicillin; these cultures were grown at 37 C for 18-24 hours. Plasmids were then isolated from the cultures using Qiagen miniprep kit. These plasmids were sequenced to determine which IL-2/pRSET clones contained the desired mutation.

Sequencing primers Forward primer, "T7" AATACGACTCACTATAG SEQ ID N0:17 Reverse primer, "RSET TAGTTATTGCTCAGCGGTGG SEQ ID N0:18 REV"

Expression of IL-2 Mutants Mutant proteins were expressed as follows: IL-2/pRSET clones containing the mutation were transformed into BL21 DE3 pLysS cells (Invitrogen) (1 p1 double-stranded DNA, 2 ~,l Sx KCM, 7 Etl water, 10 p.1 DMSO competent cells; incubate 20 minutes at 4 C, 10 minutes at room temperature), plated onto LB/agar containing 100 ~g/ml ampicillin, and incubated at 37 C
overnight. 10 ml cultures in 10 ml 2YT with 100 ~g/ml ampicillin were grown overnight from single colonies. 100 ml 2YT/ampicillin (100 ~g/ml) was inoculated with these overnight cultures and incubated at 37 C for 3 hours. This culture was then added to 1.5 L
2YT/ampicillin (100 pg/ml) and incubated until late-log phase (absorbance at 600 nm ~0.8), at which time IPTG was added to a anal concentration of 1 mM. Cultures were incubated at 37 G for another 3 hours and then cells were pelleted (10 Krpm, 10 minutes) and frozen at -20 C overnight.
IL-2 mutants were then purified from the frozen cell pellets. First, cells were lysed in a microfluidizer (100 ml Tris EDTA buffer, 3 passes through a Microfluidizer [Microfluidics 110S]) and inclusion bodies were isolated by precipitation (10 Krpm, 10 minutes).
Following cell lysis, 50 E~1 of cell material was saved for analysis by SDS-PAGE. All mutants expressed as determined by gel but several (e.g. E68C) precipitated on refolding. Inclusion bodies were then resuspended in 45 ml guanidine HCl and spun at 10 Krpm for 10 minutes. The supernatant was added to refolding buffer (45 ml guanidine HCI, 36 ml Tris pH 8, 231 mg cysteamine, 46 mg cystamine, 234 ml water) and incubated at room temperature for 3-5 hours. The mixture was then spun at 10 Krpm for 20 minutes. and the supernatant dialyzed 4-5 times in 5 volumes of buffer (10 mM
ammonium acetate pH 6, 25 mM NaCI). The protein solution was then filtered through cellulose and injected onto an S Sepharose fast flow column (2.5 cm diameter x 14 cm long) at 5 ml/min. The protein was then eluted using a gradient of 0 - 75% Buffer B over 60 minutes (Buffer A: 25 mM
NH40Ac, pH 6, 25 mM NaCI; Buffer B: 25 mM NH4OAc, pH 6, 1 M NaCI). Purified protein was then exchanged into the appropriate buffer for the TETHER assay (typically 100 mM Hepes, pH
7.4). Average yields were 0.5 to 4 mg/L culture.

IL-4 (accession number SWS P05112) is a cytokine that is critical for early immune response and allergic response; its interaction with the IL-4R is involved in the generation of Th2 cells. IL-4 recruits and activates B-cells that produce IgE (immunoglobulin E), eosinophils, and mast cells.
These cells in turn tag and attack parasites in skin and in mucosal tissues and eject them from these tissues. The role of the IL-4/IL4R interaction in immune and allergic responses suggests that disruption of this interaction may alleviate such conditions as asthma, dermatitis, conjunctivitis, and rhinitis. 'There are crystal structures of IL-4 in isolation and in co-complex with a receptor molecule [1HIK, Muller, T. & Buehner, M., JMoI Biol 247: 360-372 (1995); with receptor alpha, IIAR, Hage, T., et aL, Cell 97: 271-281 (1999)].
Cloning of human IL-4 Numbering of the wild type and mutant IL-4 residues follows the convention of the first amino acid residue (H) of the mature protein being residue number 1 independent of any presequence e.g. met for the E. coli produced protein [Yokota, T., et al., Proc. Natl. Acad. Sci.
U. S. A. 83: 5894-5898 (1986)]. IL-4 lacking the secretion signal and containing an additional N-terminal methionine was expressed intracellularly in E. coli from the Sunesis RSET.IL4 plasmid.
The DNA sequence encoding human interleukin-4 (IL4) was isolated by PCR from the plasmid pcD-hIL-4 (ATCC Accession No. 57592) using PCR primers:
IL4 ForRse 5' GGGTTTCATATGCACAAGTGCGATATCACCTT SEQ ID N0:19 IL4 RevRse 5' CCGCTCGAGTCAGCTCGAACACTTTGAATA SEQ ID N0:20 These primers correspond to extracellular domain of the protein and which were designed to contain restriction endonuclease sites Nde I and XhoI for subcloning into a pRSET vector (Invitrogen). The PCR reaction was purified on a Qiaquick PCR purification column (Qiagen).
The PCR product containing the IL4 sequence was out with restriction endonucleases (41 p1 PCR
product, 2 p.1 each endonuclease, 5 p,1 appropriate lOx buffer; incubated at 37 C for 90 minutes).
The pRSET vector was cut with restriction endonucleases (6 ~g DNA, 4 p1 each endonuclease, 10 p1 appropriate lOx buffer, water to 100 p1; incubated at 37 C for 2 hours; add 2 p,1 CIP and incubated at 37 C for 45 minutes). The products of nuclease cleavage were isolated from an agarose gel (1% agarose, TBE buffer) and ligated together using T4 DNA ligase (200 ng pRSET
vector, 150 ng IL4 PCR product, 4 p.l Sx ligase buffer [300 mM Tris pH 7.5, 50 mM MgCl2, 20%
PEG 8000, 5 mM ATP, 5 mM DTT], 1 ~1 Iigase; incubated at IS C for 1 hour). 101 of the ligation reaction was transformed into XL1 blue cells (Stratagene) (10 p1 reaction mixture, 10 p,1 Sx KCM
[0.5 M KCI, 0.15 M CaCh, 0.25 M MgCl2], 30 p1 water, 50 p1 PEG-DMSO competent cells;
incubated at 4 C for 20 minutes, 25 C for IO minutes), and plated onto LB/agar plates containing 100 pg/ml ampicillin. After incubation at 37 C overnight, single colonies were grown in 3 ml 2YT
media for 18 hours. Cells were then isolated and double-stranded DNA extracted from the cells using a Qiagen DNA miniprep kit.

Generation of IL-4 Cysteine Mutations Mutations were generated using as previously described [Kunlcel, T. A., et al., Metlzods Ehzymol.
154:367-82 (1987)]. DNA oligonucleotides used are shown below and were designed to hybridize with sense strand DNA from plasmid. Sequences were verified using primers with SEQ ID N0:17 and SEQ ID N0:18.
Mutagenic Oligonucleotides QgC TTGATGATCTCACATAAGGTGA SEQ ID NO:21 E9C AGTTTTGATGATACACTGTAAGGTGAT SEQID NO:22 K12C GCTGTTCAAAGTGCAGATGATCTCCTG SEQID NO:23 S16C CTGCTCTGTGAGGCAGTTCAAAGT SEQID NO:24 K37C CAGTTGTGTTACAGGAGGCAGCAAAG SEQID NO:2S

N38C CCTTCTCAGTTGTGCACTTGGAGGC SEQID NO:26 K42C GCAGAAGGTTTCACACTCAGTTGTG SEQID NO:27 QS4C GGCTGTAGAAACACCGGAGCACAGTCG SEQID NO:28 Q78C GAATCGGATCAGACACTTGTGCCTGTG SEQID NO:29 R81C GCCGTTTCAGGAAGCAGATCAGCTGC SEQID NO:3O

R8SC CCTGTCGAGACATTTCAGGAATCG SEQID NO:31 R88C CCCAGAGGTTGCAGTCGAGCCG SEQID NO:32 N89C CCCAGAGGCACCTGTCGAGCCG SEQ ID NO:33 N97C CACAGGACAGGAACACAAGCCCGCC SEQID NO:34 K1O2C CTGGTTGGCTTCACACACAGGACAGG SEQID NO:3S

K117C CTCTCATGATCGTGCATAGCCTTTCC SEQID NO:36 R121C GAATATTTCTCACACATGATCGTC SEQID NO:37 Expression of IL-4 Mutants BL21 DE3 cells (Stratagene) were transformed with RSET.IL4 plasmids containing the described cysteine mutations and plated onto LB agar containing 100 ~.g/ml ampicillin.
After overnight growth fresh individual colonies were used to inoculate a 37 C overnight shake flask culture with 30 ml 2YT (with 50 p,g/ml ampicillin) media. In the morning this overnight culture was used to inoculate 1.5 L of 2YT/ampicillin (50 ~.g/ml), which was further cultured at 37 C and 200 rpm in a 4.0 L dented bottom shake flask. When the optical density of the culture at ~, = 600 reached 0.8 it was induced to produce IL-4 protein by the addition of 1 mM IPTG. After 4 more hours of incubation the cultures were harvested, the cells pelleted by centrifugation at 7K rpm for 10 minutes (K-9 Komposite Rotor), and frozen at-20 C.
The cell pellet was then thawed and resuspended in I00 ml of 10 mM Tris pH 8, 50 mM NaCI and 1 mM EDTA. This solution was kept chilled and run through a microfluidizer twice (model 1105 Microfluidics Corp, Newton Massachusetts), and centrifuged at 7K rpm for 15 minutes). The pellet containing the IL-4 inclusion bodies was then resuspended in a 50 ml solution of 5 M guanidine HCI, 50 mM Tris pH 8, 50 mM NaCI, 2.5 mM reduced glutathione, and 0.25 mM
oxidized glutathione, and incubated for one hour at room temperature with gentle mixing. The solubilized protein solution was then centrifuged at 7.5K rpm for 15 minutes and the supernatant 0.45 ~m filtered to remove insoluble debris.
The IL-4 was refolded by slowly adding the filtered solution to 9 volumes (450 ml) of 50 mM Tris pH 8, 50 mM NaCI, 2.5 mM reduced glutathione and 0.25 mM oxidized glutathione over a 30 minute period. The resulting solution was further incubated with slow stirring for 3 hours at room temperature, then placed in a 3000 mwco dialysis bag and exchanged 3 times against 20 L of 0.5x PBS (phosphate-buffered saline).
The refolded mutant proteins were then purified using a Hi-S Column Cartridge (Bio-Rad). After clarifying the protein solution by centrifugation and filtration it was loaded onto the column at a 5 ml/min flow rate. The column was next washed with buffer A (0.5x PBS) for 15-20 minutes, and 1.5 minute 7.5 ml fractions were collected over a 0-100% gradient between Buffer A and Buffer B
(PBS, 1M NaCI). The fractions that contained the IL-4 protein as determined by SDS-PAGE and optical density as 280 nm were pooled, concentrated with a 5K mwco filter, and their buffer exchanged to PBS. This solution was then 0.2 pm filtered, frozen in ethanol dry ice bath, and stored at -80 C.

CLONING AND MUTAGENESIS OF HUMAN TUMOR NECRO IS FACTOR-ALPHA fTNF
..

Tumor necrosis factor-a (TNF-a) (accession number SWS P01375) is a cytokine produced mainly by activated macrophages, and it plays a critical role in immune responses including septic shock, inflammation, and cachexia. This protein can interact with two receptors, TNF
Rl and TNF R2.
These two receptors share no similarity in their intracellular domains, which suggests that they are involved in different signal transduction pathways. A structure of TNF-a is available [1TNF, Eck, M. J., et al., JBiol ClZem 264: 17595-17605(1989)]; TNF-a is an elongated beta sheet, and it forms a trimer. Mutation of some of the intersubunit residues of the trimer indicates that they form part of the binding site to the receptor. However, there is no structure of TNF bound to a receptor to date.
Cloning of human TNF-a The DNA sequence encoding human Tumor Necrosis Factor (TNF) was isolated by PCR from the plasmid pUC-RI-4large (ATCC #65947) using PCR primers listed below corresponding to extracellular domain of the protein and which were designed to contain restriction endonuclease sites Nde I and XhoI for subcloning into a pRSET vector (Invitrogen).
TNF RSET For GGGTTTCATATGGTCCGTTCATCTTCTCGAAC SEQ ID N0:38 5' TNF RSET Rev CCGCTCGAGTCACAGGGCAATGATCCCAA SEQ ID N0:39 5' The PCR reaction was purified on a Qiaquick PCR purification column (Qiagen).
The PCR product containing the TNF sequence was cut with restriction endonucleases (41 p1 PCR
product, 2 ~l each endonuclease, 5 ~1 appropriate lOx buffer; incubated at 37 C for 90 minutes).
The pRSET vector was cut with restriction endonucleases (6 p.g DNA, 4 p.1 each endonuclease, 10 p.1 appropriate lOx buffer, water to 100 p.1; incubated at 37 C for 2 hours; added 2 p1 CIP and incubated at 37 C for 45 minutes). The products of nuclease cleavage were isolated from an agarose gel (1% agarose, TBE
buffer) and ligated together using T4 DNA ligase (200 ng pRSET vector, 150 ng TNF PCR product, 4 p1 5x ligase buffer [300 mM Tris pH 7.5, 50 mM MgCl2, 20% PEG 8000, 5 mM
ATP, 5 mM
DTT], 1 ~1 ligase; incubated at 15 C for 1 hour). 10 p1 of the ligation reaction was transformed into XL1 blue cells (Stratagene) (10 ~,1 reaction mixture, 10 p1 5x KCM [0.5 M
KCI, 0.15 M CaClz, 0.25 M MgCla], 30 ~1 water, 50 ~tl PEG-DMSO competent cells; incubated at 4 C
for 20 minutes, 25 C for 10 minutes), and plated onto LB/agar plates containing 100 p.g/ml ampicillin. After incubation at 37 C overnight, single colonies were grown in 3 ml 2YT media for 18 hours. Cells were then isolated and double-stranded DNA extracted from the cells using a Qiagen DNA
miniprep kit. Sequencing of TNF genes was accomplished using primers having SEQ ID N0:17 and SEQ ID NO:18.

Generation of TNF-a Cysteine Mutations Mutations were generated using as previously described [Kunlcel, T, A., et al., Methods Entzymol.
154: 367-82 (1987)]. DNA oligonucleotides used are shown below and were designed to hybridize with sense strand DNA from plasmid. Sequences of the mutants were verified using primers with S SEQ ID NO:17 and SEQ ID N0:18.
Mutagenic Oligonucleotides NO:40 A33C CAGGAGGGCATTGCACCGGCGGTTCAG SEQID NO:41 N34C GGCCAGGAGGGCACAGGCCCGGCGGTTC SEQID NO:42 R44C CAGCTGGTTATCACACAGCTCCACGCC SEQID NO:43 Q47C TGGCACCACCAGGCAGTTATCTCTCAG SEQID NO:44 T72C GAGGAGCACATGGCAGGAGGGGCAGCC SEQID NO:4S

H73C GGTGAGGAGCACACAGGTGGAGGGGCAG SEQID NO:46 L7SC GGTGTGGGTGAGGCACACATGGGTGGAG SEQID NO:47 T77C GCTGATGGTGTGGCAGAGGAGCACATG SEQID NO:48 V91C CAGAGAGGAGGTTGCACTTGGTCTGGTAG SEQID NO:49 N92C GGCAGAGAGGAGGCAGACCTTGGTCTG SEQID NO:SO

S95C GCTCTTGATGGCACAGAGGAGGTTGAC SEQID NO:Sl E104C CCTCAGCCCCCTCTGGGGTGCACCTCTGGCAGGGG SEQID NO:S2 T1OSC CCTCAGCCCCCTCTGGGCACTCCCTCTGGCAGGGG SEQID NO:S3 ElO7C GGCCTCAGCCCCGCATGGGGTCTCCCTCTGGC SEQID NO:S4 EIIOC CCAGGGCTTGGCGCAAGCCCCCTCTGGGG SEQID NO:SS

AlllC ATACCAGGGCTTGCACTCAGCCCCCTC SEQID NO:S6 K112C GGGTAGTTTCTGGCAAAATATGGCTTG SEQ ID NO:S7 QI25C CACCCTTCTCCAGGCAGAAGACCCCTCC SEQ ID NO:S$

R138C GCTGAGATCAATTGTCCCGACTATCTC SEQ ID NO:59 E146C GACCTGCCCAGAGCAGGCAAAGTCGAG SEQ ID N0:60 S147C GTAGACCTGCCCACACTCGGCAAAGTC SEQ ID NO:61 Expression of TNF-a Mutant Proteins BL21 DE3 cells (Stratagene) were transformed with RSET TNF-a plasmids containing the described cysteine mutations and plated onto LB agar containing 100 ~g/ml ampicillin. After overnight growth fresh individual colonies were used to inoculate a 37 C
overnight shalce flask culture with 30 ml 2YT (with 50 ~g/ml ampicillin) media. In the morning this overnight culture was used to inoculate 1.5 L of 2YT/ampicillin (50 ~,g/ml), which was further cultured at 37 C and 200 rpm in a 4.0 L dented bottom shake flask. When the optical density of the culture at ~. = 550 reached 0.8 it was induced to produce TNF-a protein by the addition of 1 mM
IPTG. After 4 more hours of incubation the cultures were harvested, the cells pelleted by centrifugation at 7K rpm for 10 minutes (K-9 Komposite Rotor), and frozen at -20 C.
The cell pellet was then thawed and resuspended in 100 ml of 25 mM ammonium acetate pH 6, 1 mM DTT and 1 mM EDTA. This solution was kept chilled and run through a microfluidizer twice (model 1105 Microfluidics Corp, Newton Massachusetts), centrifuged at 9K rpm for 15 minutes to remove insoluble material and further clarified by 0.45 ~m filtration. This solution was then loaded onto an S-Sepharose ff Column (Bio-Rad) column at a 5 ml/min flow rate. The flow rate was then increased to 7.5 mL/min for the following steps. The column was next washed with Buffer A (0.2 M ammonium acetate pH 6, 1 mM DTT) until the ODZSO approached zero (15-20 minutes), and fractions were collected over a 0-100% gradient in 60 minutes between Buffer A
and Buffer B (1 M
ammonium acetate pH 6, 1 mM DTT). The fractions that contained the TNF-a protein as determined by SDS-PAGE and optical density at 280 nm were pooled and placed in a 3000 mwco dialysis bag and dialyzed overnight at 4 C against 4 L of 10 mM Tris pH 7.5, 10 mM NaCI, and 1 mM DTT. The dialyzed protein solution was then clarified by centrifuging at 13.5K rpm for 10 minutes filtering through a 0.2 ~m filter.
The mutant proteins were then loaded onto a Q-Sepharose Column (Bio-Rad) at a 5 ml/min flow rate. The flow rate was increased to 7.5 mLlmin for the following steps. The column was next washed with Buffer A (10 mM Tris pH 7.5, 10 mM NaCI, 1 mM DTT) until the ODZBO
approached zero (15-20 minutes), and fractions were collected over a 0-100% gradient in 40 minutes between Buffer A and Buffer B (10 mM Tris pH 7.5, 0.5 M NaCI, 1 mM DTT). The fractions that contained the TNF-a protein as determined by SDS-PAGE and optical density at 280 nm were pooled and concentrated with a SK mwco filter, and their buffer exchanged to PBS. This solution was then 0.2 pm filtered, frozen in ethanol dry ice bath, and stored at -80 C.

CLONING AND MUTAGENESIS OF HUMAN INTERLEUKIN-1 RECEPTOR TYPE I fIL 1RI) Binding of the IL-1 receptor (accession number SWS P14778) to IL-lalpha or IL-lbeta is another important mediator of immune and inflammatory responses. This interaction is controlled by at least three mechanisms. Firstly, the protein IL-R2 binds to IL-lalpha and IL-lbeta but does not signal. Secondly, proteolytically processed IL-1R1 and IL-1R2 are soluble and bind to IL-1 in circulation. Finally there exists a natural IL-1R antagonist called IL-lra, that functions by binding IL-1R1 and thereby blocking IL-1Rl binding of IL-lalpha and IL-lbeta.
Inhibition of these interactions with an orally available small molecule would be desirable in treatment of diseases such as rheumatoid arthritis, autoimmune disorders, and ischemia. Two structures of IL-1R have been solved [with a antagonist peptide, 1GOY, Vigers, G. P. A., et aL, J.
Biol. Cl2em. 275:36927-36933 (2000); with receptor antagonist, LIRA, Schreuder, H., et al., Nature 386: 194-200 (1997)].
Cloning of human TL-1 receptor type I
The IL-1 receptor has three regions: an N-terminal extracellular region, a transmembrane region, and a C-terminal cytoplasmic region. The extracellular region itself contains three irnmunoglobin Like C2-type domains. The constructs used here contain the two N-terminal domains of the extracellular region. Numbering of the wild type and mutant IL1R residues follows the convention of the first amino acid residue (L) of the mature protein being residue number 1 after processing of the signal sequence [Sims, J. E., et al., Pi~oc. Natl. Acad. Sci. U. S. A. 86:
8946-8950 (1989)]. The sequence of the 2 domain protein is shown below as SEQ ID N0:62.

In brief, cysteine mutants were made in the context of a 2 domain receptor and a 2 domain receptor with a his tag. In addition, the constructs possessed a mutation at a glycosylation site, and one construct possessed a mutation at a glycosylation site in addition to a deletion at the C-terminal residue of the 2 domain region. The assembly of these constructs is described below.
The DNA sequence encoding human Interleukin-1 receptor (IL1R) was isolated by PCR from a HepG2 cDNA library (ATCC) using PCR primers (ILlRsigintFor 5'; ILlRintRev 5') corresponding to the signal sequence and the end of the extracellular domain of the protein.

ILlRsigintFor TTACTCAGACTTATTTGTTTCATAGCTCTA SEQ ID N0:63 ILlRiritReV GAAATTAGTGACTGGATATATTAACTGGAT SEQ ID NO:C)4 The appropriate sized band was isolated from an agarose gel and used as the template for a second round of PCR using oligos (ILlRsigFor; IL1R319Rev), which were designed to contain restriction endonuclease sites EcoRI and XhoI for subcloning into a pFBHT vector.
ILlRsig For CCGGAATTCATGAAAGTGTTACTCAGACTTATTTGTTTC SEQ ID
NO:65 IL1R319 Rev CCGCTCGAGTCACTTCTGGAAATTAGTGACTGGATATATTAA SEQ ID
NO:G6 The pFBHT vector is modified from the original pFastBacl(Gibco/BRL) by cloning the sequence for TEV protease followed by (His) tag and a stop signal into the XhoI and HinDIII sites. The PCR product containing the IL1R sequence was cut with restriction endonucleases (41 ~tl PCR
product, 2 ~1 each endonuclease, 5 ~tl appropriate lOx buffer; incubated at 37 C for 90 minutes).
The pFBHT vector was cut with restriction endonucleases (6 ~,g DNA, 4 ~,1 each endonuclease, 10 ~l appropriate lOx buffer, water to 100 q1; incubated at 37 C for 2 hours; add 2 ~.l CIP and incubated at 37 C for 45 minutes). The products of nuclease cleavage were isolated from an a.garose gel (1% agarose, TBE buffer) and ligated together using T4 DNA ligase (200 ng pFBHT
vector, 150 ng IL1R PCR product, 4 ~1 5x ligase buffer [300 mM Tris pH 7.5, 50 mM MgCl2, 20%
PEG 8000, 5 mM ATP, 5 mM DTT], 1 ~1 ligase; incubated at 15 C for 1 hour). 10 ~.1 of the ligation reaction was transformed into XL1 blue cells (Stratagene) (10 ~l reaction mixture, 10 ~1 Sx I~CM [0.5 M ICI, 0.15 M CaClz, 0.25 M MgCl2], 30 ~.1 water, 50 ~l PEG-DMSO
competent cells;
incubated at 4 C for 20 minutes, 25 C for 10 minutes), and plated onto LB/agar plates containing 100 ~g/ml ampicillin. After incubation at 37 C overnight, single colonies were grown in 3 ml 2YT
media for 18 hours. Cells were then isolated and double-stranded DNA extracted from the cells using a Qiagen DNA miniprep kit.
A 2-domain version of IL1R was created by PCR using the 3-domain IL1R-FBHT
clone as a template. PCR was performed using the primers ILlRsigFor (SEQ ID N0:65) corresponding to the signal sequence, in addition to one of the following two reverse primers. The reverse primers are ILIR2Drevstop-Xho, which corresponds to the end of the second extracellular domain of the protein with a stop signal, and ILIR2Drev-Xho, which corresponds to the end of the second extracellular domain of the protein without a stop signal to create a fusion with the TEV protease site and the His tag.
ILIR2Drevstop-Xho CCGCTCGAGTCATCATTTGTTTTCCTCTAGAGTAATAAA SEQID
N0:67 ILIR2Drev-Xho CCGCTCGAGTCATTTGTTTTCCTCTAGAGTAATAAA SEQ ID
N0:68 The PCR primers contain restrictions sites (EcoRI at the 5'end and XhoI at the 3' end), which were used to ligate the 2-domain version into the pFBHT vector. The PCR product containing the IL1R2D sequence was cut with restriction endonucleases (41 p.1 PCR product, 2 ~l each endonuclease, 5 p.1 appropriate lOx buffer; incubated at 37 C for 90 minutes).
The products of nuclease cleavage were isolated from an agarose gel (1% agarose, TBE buffer) and ligated together using T4 DNA ligase (200 ng pFBHT vector, 150 ng IL1R2D PCR product, 4 ~.I 5x ligase buffer [300 mM Tris pH 7.5, 50 mM MgCl2, 20% PEG 8000, 5 mM ATP, 5 mM DTT], 1 ~tl ligase;
incubated at 15 C for 1 hour). 10 ~cl of the ligation reaction was transformed into XLl blue cells (Stratagene) (10 ~1 reaction mixture, 10 ~1 Sx KCM [0.5 M KCI, 0.15 M CaCIz, 0.25 M MgCIz], 30 p1 water, 50 p.1 PEG-DMSO competent cells; incubated at 4 C for 20 minutes, 25 C for 10 minutes), and plated onto LB/agar plates containing 100 ~cg/ml ampicillin.
After incubation at 37 C overnight, single colonies were grown in 3 ml 2YT media for 18 hours. Cells were then isolated and double-stranded DNA extracted from the cells using a Qiagen DNA miniprep leit.
Additionally, the two glycosylation sites within IL1R2D, N83 and N176, were each individually mutated to a histidine, in order to make a more homogeneous protein. Each of these single mutants were made in the context of the 2-domain protein without a his tag (sILlRd2-FB) and the 2-domain protein with a his tag (sILlRd2-FBHT). Mutation was accomplished by PCR using two sets of primers to make two fragments, followed by stitching together of the fragments using the outside primers ILlRsigFor (SEQ ID N0:65) and either ILIR2Drevstop-Xho (SEQ ID N0:67) or ILlR2Drev-Xho (SEQ ID N0:68) as described below. Brief descriptions of the 2-domain glycosylation mutants and their construction follow.
The construct fox the N83H mutant without a his tag is referred to as sILIR2D-N83H-FB, and it was created using ILlRsigFor (SEQ ID N0:65) and N83HR (SEQ ID N0:69) along with N83HF
(SEQ ID N0:70), and ILIR2Drevstop-Xho (SEQ ID N0:67) N83HR GAGGCAGTAAGATGAATGTCTTACC SEQ ID N0:69 N83HF CTATTGCGTGGTAAGACATTCATCTT SEQ ID NO:70 The construct for the N83H mutant with a his tag is referred to as sILIR2D-N83H-FBHT and was created using ILlRsigFor (SEQ ID N0:65), and N83HR (SEQ ID N0:69) along with N83HF (SEQ
ID N0:70) and ILIR2Drev-Xho (SEQ ID N0:68).
The construct for the N176H mutant without a his tag is referred to as sILIR2D-N176H-FB and it was created using ILlRsigFor (SEQ ID N0:65), N176HR (SEQ ID N0:71), N176HF
(SEQ ID
N0:72), and ILIR2Drevstop-Xho (SEQ ID N0:67).
N176HR ATGACAAGTATAGTGCCCTCTATGCTTTTCACG SEQID NO:71 N176HF GCTGAAAAGCATAGAGGGCACTATACTTGTCAT SEQID N0:72 The construct for the N176H mutant with a his tag is referred to as sILIR2D-N176H-FBHT.and it was created using ILlRsigFor (SEQ ID N0:65), and N176HR (SEQ ID N0:71), along with N176HF (SEQ ID N0:72), and ILIR2Drev-Xho (SEQ ID N0:68).
The PCR products were isolated from and agarose gel and PCR was used to sew the two fragments together using the ILlRsigFor (SEQ ID N0:65) and ILIR2Drevstop-Xho (SEQ ID
N0:67) or ILIR2Drev-Xho primers (SEQ ID N0:68). The PCR products containing the IL1R2D
sequences mutated at the glycosylation site were cut with restriction endonucleases (41 p1 PCR product, 2 ~1 each endonuclease, 5 p.1 appropriate lOx buffer; incubated at 37 C for 90 minutes). The products of nuclease cleavage were isolated from an agarose gel (1% agarose, TBE
buffer) and ligated together using T4 DNA ligase (200 ng pFBHT vector, 150 ng IL1RZD PCR product, 4 p1 Sx ligase buffer [300 mM Tris pH 7.5, 50 mM MgCl2, 20% PEG 8000, 5 mM ATP, 5 mM DTT], 1 ~l ligase;
incubated at 15 C for 1 hour). 10 p1 of the ligation reaction was transformed into XLl blue cells (Stratagene) (10 p.1 reaction mixture, 10 ~1 Sx KCM [0.5 M KCI, 0.15 M CaCl2, 0.25 M MgCl2], 30 2.5 p,1 water, 50 ~1 PEG-DMSO competent cells; incubated at 4 C for 20 minutes, 25 C for 10 minutes), and plated onto LB/agar plates containing 100 pg/ml ampicillin.
After incubation at 37 C overnight, single colonies were grown in 3 ml 2YT media for 18 hours. Cells were then isolated and double-stranded DNA extracted from the cells using a Qiagen DNA miniprep kit. The subsequent plasmids are referred to as sILIR2D-N83H-FB or sILIR2D-N83H-FBHT
and as sILIR2D-N176H-FB or as sILIR2D-N176H-FBHT.

Finally, an additional construct was made using the sILIR2D-N83H-FB construct.
The additional construct contains the 2-domain IL1R receptor without a his tag and with two mutations: a N83H
glycosylation mutation and a deletion of the C-terminal residue (K205). This construct is named sIL1R2D2M-FB, and was made using the K205de1 oligonucleotide.
K205de1 CTCGAGTCATCAGTTTTCCTCTAG SEQ ID NO:73 Generation of IL-1RI Cysteine Mutations Site-directed mutants of IL1R2D were prepared by the single-stranded DNA
method [modification of Kunkel, T. A., Proc. Natl. Acid. Sci. U. S. A. 82: 488-492 (1985)].
Oligonucleotides were designed to contain the desired mutations and 15-20 bases of flanking sequence.
The single-stranded form of the IL1R2D (sILIR2D-FBHT, sILIR2D-N176H-FB/FBHT, sILIR2D-N83H-FB/FBHT, sIL1R2D2M-FB) plasmid was prepared by transformation of double-stranded plasmid into the CJ236 cell line (1 ~l IL1R-FB double-stranded DNA, 2 p.1 2x KCM salts, 7 ~tI
water, 10 p1 PEG-DMSO competent CJ236 cells; incubated at 4 C for 20 minutes and 25 C for 10 minutes; plated on LB/agar with 100 pg/m1 arnpicillin and incubated at 37 C
overnight). Single colonies of CJ236 cells were then grown in 50 rnl 2YT media to midlog phase;
10 p1 VCS helper phage (Stratagene) were then added and the mixture incubated at 37 C
overnight. Single-stranded DNA was isolated from the supernatant by precipitation of phage (1/5 volume 20% PEG 8000/2.5 M NaCI; centrifuge at 12K for 15 minutes.). Single-stranded DNA was then isolated from phage using Qiagen single-stranded DNA kit.
Site-directed mutagenesis was accomplished as follows. Oligonucleotides were dissolved to a concentration of 10 OD and phosphorylated on the 5' end (2 p.1 oligonucleotide, 2 ~1 10 mM ATP, 2 p.1 lOx Tris-magnesium chloride buffer, 1 p.1 100 mM DTT, IO ~l water, I p1 T4 PNK; incubate at 37 C fox 45 minutes). Phosphorylated oligonucleotides were then annealed to single-stranded DNA template (2 p.1 single-stranded plasmid, I p.1 oligonucleotide, 1 ~.I lOx TM buffer, 6 p.1 water;
heat at 94 C for 2 minutes, 50 C for 5 minutes, cool to room temperature).
Double-stranded DNA
was then prepared from the annealed oligonucleotide/template (add 2 ~.1 lOx TM
buffer, 2 ~,1 2.5 mM dNTPs, 1 ~cl 100 mM DTT, 1.5 w1 10 mM ATP, 4 p.1 water, 0.4 p.1 T7 DNA
polymerise, 0.6 q1 T4 DNA ligase; incubate at room temperature for two hours). E. coli (XL1 blue, Stratagene) was then transformed with the double-stranded DNA (1 p.1 double-stranded DNA, 10 p.1 Sx KCM, 40 p,1 water, 50 u1 DMSO competent cells; incubate 20 minutes at 4 C, 10 minutes at room temperature), plated onto LBlagar containing 100 p,g/ml ampicillin, and incubated at 37 C
overnight.
Approximately four colonies from each plate were used to inoculate 5 ml 2YT
containing 100 ~,glml ampicillin; these cultures were grown at 37 C for 18-24 hours. Plasmids were then isolated from the cultures using Qiagen miniprep kit. These plasmids were sequenced to determine which ILIR2D-FB clones contained the desired mutation.
Sequencing of IL1R2D genes was accomplished as follows. The concentration of plasmid DNA
was quantitated by absorbance at 280 nm. 800 ng of plasmid was mixed with sequencing reagents (8 p,1 DNA, 3 p.1 water, 1 ~.1 sequencing primer, 8 ~.1 sequencing mixture with Big Dye [Applied Biosystems]). The sequencing primers used were FB Forward and FB Reverse, shown below.
FB Forward TATTCCGGATTATTCATACC SEQ ID N0:74 FB Reverse CCTCTACAAATGTGGTATGGC SEQ ID N0:75 The mixture was then run through a PCR cycle (96 C, 10 s; 50 C, S s; 60 C 4 minutes; 25 cycles) and the DNA reaction products were precipitated (20 ~,I mixture, 80 p,1 75%
isopropanol; incubated minutes at room temperature, pelleted at 14 K rpm for 20 minutes; wash with 250 ~.l 70%
ethanol; heat 1 minute at 94 C). The precipitated products were then suspended in Template 15 Suppression Buffer (TSB, Applied Biosystems) and the sequence read and analyzed by an Applied Biosystems 310 capillary gel sequencer. In general, 3 out of 4 of the plasmids contained the desired mutation. A listing of the constructs and their mutants) is given below, although any cysteine mutants can be made in any of the given contexts.
Construct Mutant s sILIR2D-N83H-FB El IC, II3C, VI6C, Q108C, I110C, K112C, K114C, VI17C, VI24C, YI27C, E129C
sILIR2D-N83H-FBHT ElIC, I13C, V16C, Q108C, I110C, K112C, Q1I3C, K114C, VI17C, V124C, Y127C, E129C
sIL 1 R2D-N 176H-FB E 11 C
sILIR2D-N176H-FBHT E11C, VI6C, V124C, E129C
sIL1R2D2M-FB E11C, KI2C, I13C, A107C, K112C, V124C, Y127.
Mutagenic Oligonucleotides EIIC T~TTATTTTACATTCACGTTCC SEQID NO:76 K12C CACTAAAATTATACATTCTTCACGTTC SEQID NO:77 I13C TGACACTAAAATACATTTTTCTTCACG SEQID NO:78 VICC ATTTGCAGATGAACATAAAATTATTT SEQID NO:79 A107C ~TATGGCTTGGCAATTATAACATAAG SEQID NO:80 Q108C CTTAAATATGGCGCATGCATTATAACA SEQID NO:81 I110C GTTTCTGCTTAAAGCAGGCTTGTGCATT SEQ ID NO:82 K112C GGGTAGTTTCTGACAAAATATGGC SEQID NO:83 Q113C AACGGGTAGTTTACACTTAAATATGGC SEQID NO:84 K114C CTGCAACGGGTAGGCACTGCTTAAATATG SEQID NO:85 V117C CTCCGTCTCCTGCACAGGGTAGTTTCTG SEQ ID NO:BC) V124C CATATAAGGGCAACAAAGTCCTCC SEQ ID NO:87 Y127C CTCCATACAAGGGCACACAAG SEQ ID NO:$8 E129C TTTAAAAAAACACATATAAGGGCA SEQ ID NO:89 Expression of IL-1 R mutant proteins All IL1R-FB/FBHT plasmids were site-specifically transposed into the baculovirus shuttle vector (bacmid) by transforming the plasmids into DHlObac (GibcoBRL) competent cells as follows: 1 ~l DNA at 5 ng/~1, 10 ~.l 5x KCM [0.5 M KCl, 0.15 M CaClz, 0.25 M MgCl2], 30 ~,1 water was mixed with 50 p1 PEG-DMSO competent cells, incubated at 4 C for 20 minutes, 25 C for 10 minutes, add 900 p,1 SOC and incubate at 37 C with shaking for 4 hours, then plated onto LB/agar plates containing 50 pg/ml kanamycin, 7 ~g/ml gentamycin, 10 pg/ml tetracycline, 100 ~g/ml Bluo-gal, 10 pg/ml IPTG. After incubation at 37 C for 24 hours, large white colonies were picked and grown in 3 ml 2YT media overnight. Cells were then isolated and double-stranded DNA was extracted from the cells as follows: pellet was resuspended in 250 ~.l of Solution 1 [15 mM Tris-HCl (pH 8.0), 10 mM EDTA, 100 ~g/ml RNase A]. 250 ~l of Solution 2 [0.2 N
NaOH, 1% SDS]
was added, mixed gently and incubated at room temperature for 5 minutes. 250 ~.1 3 M potassium acetate was added and mixed, and the tube placed on ice for 10 minutes. The mixture was centrifuged 10 minutes at 14,OOOx g and the supernatant transferred to a tube containing 0.8 ml isopropanol. The contents of the tube were mixed and placed on ice for 10 minutes; centrifuged 15 minutes at 14,000x g. The pellet was washed with 70% ethanol and air-dried and the DNA
resuspended in 40 p1 TE.
The bacmid DNA was used to transfect Sf9 cells. Sf7 cells were seeded at 9 x 105 cells per 35 mm well in 2 ml of Sf 900 II SFM medium containing O.Sx concentration of antibiotic-antimycotic and allowed to attach at 27 C for 1 hour. During this time, 5 ~1 of bacmid DNA was diluted into 100 ~1 of medium without antibiotics, 6 ~1 of CeIIFECTIN reagent was diluted into 100 ~1 of medium without antibiotics and then the 2 solutions were mixed gently and allowed to incubate for 30 minutes at xoorn temperature. The cells were washed once with medium without antibiotics, the medium was aspirated and then 0.8 ml of medium was added to the lipid-DNA
complex and overlaid onto the cells. The cells were incubated for 5 hours at 27 C, the transfection medium was removed and 2 ml of medium with antibiotics was added. The cells were incubated for 72 hours at 27 C and the virus was harvested from the cell culture medium.
The virus was amplified by adding 0.5 ml of virus to a 50 ml culture of Sf~
cells at 2 x lOG cells/ml and incubating at 27 C for 72 hours. The virus was harvested from the cell culture medium and this stock was used to express the various IL1R constructs in High-Five cells.
A 1 L culture of High-Five cells at 1 x 10~ cells/ml was infected with virus at an approximate MOI of 2 and incubated for 72 hours. Cells were pelleted by centrifugation and the supernatant was loaded onto an IL1R antagonist column at 1 ml/min, washed with PBS followed by a wash with Buffer A (0.2 M NaOAc pH 5.0, 0.2 M NaCI). The protein was eluted from the column by running a gradient from 0-100% of Buffer B (0.2 M NaOAc pH 2.5, 0.2 M NaCI) in 10 minutes followed by 15 minutes of 100% Buffer B at 1 ml/min collecting 2 ml fractions in tubes containing 300 ~.l of unbuffered Tris. The appropriate fractions were pooled, concentrated and dialyzed against 5 L of 50 mM Tris pH 8.0, 100 mM NaCl at 4 C and filtered through a 0.2 ~m filter.

CLONING AND MUTAGENESIS OF HUMAN CASPASE-3 fCASP-3) Caspase-3 (accession number SWS P42574) is one of a series of caspases involved in the apoptosis 3U of cells. It exists as the inactive proform, and can be processed by caspases 8, 9, or 10 to form a small subunit and a large subunit, which heterodimerize to constitute the active form. Caspases that are substrates for caspase-3 in the cascade are caspase-6, caspase-7 and caspase-9. Caspase-3 has been shown to be the important for the cleavage of amyloid-beta precursor protein 4A. This cleavage has been linked to the deposition of Abeta peptide deposition and death of neurons in Alzheimers disease and hippocampal neurons following ischemic and exitoxic brain injury. There is a crystal structure available for caspase-3 [1CP3, Mittl, P. R., et al., JBiol CIZerya 272:6539-6547 (1997)].

Cloning of Human Caspase-3 The human version of caspase-3 (also known as Yama, CPP32 beta) was cloned directly from Jurkat cells (Clone E6-1; ATCC). Briefly, total RNA was purified from Jurleat cells growing at 37 C/5% COz using Tri-Reagent (Sigma). Oligonucleotide primers were designed to allow DNA
encoding the large and small subunits of Caspase-3/Yama/CPP32 to be amplified by polymerase chain reaction (PCR). Briefly, DNA encoding amino acids 28-175 (encompassing most of the large subunit) was directly amplified from 1 ~.g total RNA using Ready-To-Go-PCR
Beads (Amersham/Pharmacia) and the following oligonucleotides:
casp-3 large for TTCCATATGTCTGGAATATCCCTGGACAACAGTTA SEQ ID NO:90 carp-3 large rev AAGGAATTCTTAGTCTGTCTCAATGCCACAGTCCAG SEQ ID N0:91 DNA 'encoding amino acids 176-277 (encompassing most of the small subunit) was directly amplified from 1 p,g total RNA using Ready-To-Go-PCR Beads (Amersham/Pharmacia) and the following oligonucleotides:
carp-3 small for TTCCATATGAGTGGTGTTGATGATGACATGGCG SEQ ID N0:92 casp-3 small rev AAGGAATTCTTAGTGATAAAAATAGAGTTCTTTTGTGAG SEQ ID N0:93 Amplified DNA corresponding to either the large subunit or the small subunit of caspase-3 was then cleaved with the restriction enzymes EcoRI and NdeI and directly cloned using standard molecular biology techniques into pRSET-b (Invitrogen) digested with EcoRI and NdeI. [See e.g., Tewari. M., et al., Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhabitable protease that cleaves the death substrate poly (ADP-ribose) polymerase, Cell 81: 801-809 (1995)x.
Generation of Casp-3 Cys Mutations Plasmids containing DNA encoding either the large or small subunits of Caspase-3 were separately transformed into E. cola K12 CJ236 cells (New England BioLabs) and cells containing each 2S construct were selected by their ability to grow on ampicillin containing agar plates. Overnight cultures of the large and small subunits were individually grown in 2YT
(containing 100 p.g/mL of ampicillin) at 37 C. Each culture was diluted 1:100 and grown to A~oo = 0.3-0.6. A 1.5 mL
sample of each culture was removed and infected with 10 ~.L of phage VCS-M13 (Stratagene), shaken at 37 C for 60 minutes, and an overnight culture of each was prepared with 1 mL of the infected culture diluted 1:100 in 2YT with 100 ~g/mL of ampicillin and 20 ~g/ml of chloramphenicol and grown at 37 C. Cells were centrifuged at 3000 rcf for 10 minutes and 1/5 volume of 20%PEG/2.5 M NaCI was added to the supernatant. Samples were incubated at room temperature for 10 minutes and then centrifuged at 4000 rcf for 15 minutes.
The phage pellet was resuspended in PBS and spun at 15 I~ rpm for 10 minutes to remove remaining particulate matter.
Supernatant was retained, and single~stranded DNA was purified from the supernatant following procedures for the QIA prep spin M13 kit (Qiagen).
Mutagenic Oligonucleotides Cysteine mutations in the small subunit were made with the corresponding primers:
Y204C TCGCCAAGAACAATAACCAGG ~ SEQ ID N0:94 S2O9C GCCATCCTTACAATTTCGCCA SEQ ID NO:95 W214C CTGGATGAAACAGGAGCCATC SEQ ID NO:96 S251C AGCGTCAAAGCAAAAGGACTC SEQ ID NO:97 F256C CTTTGCATGACAAGTAGCGTC SEQ ID NO:98 Cysteine mutations in the large subunit were made with the corresponding primers:
M61C CCGAGATGTACATCCAGTGCT SEQ ID NO:99 N0:100 NO:101 N0:102 N0:103 Approximately 100 pmol of each primer was phosphorylated by incubating at 37 C
for 60 minutes in buffer containing 1X TM Buffer (0.5 M Tris pH 7.5, 0.1 M MgClz ), 1 mM ATP, 5 mM DTT, and SLT T4 I~inase (NEB). I~inased primers were annealed to the template DNA
in a 20 ~,L reaction volume (~50 ng kinased primer, 1X TM Buffer, and 10-50 ng single-stranded DNA) by incubation at 85 C for 2 minutes, 50 C for 5 minutes, and then at 4 C for 30-60 minutes.
An extension cocktail (2 mM ATP, 5 mM dNTPs, 30 mM DTT, T4 DNA ligase (NEB), and T7 polymerase (NEB)) was added to each annealing reaction and incubated at room temperature for 3 hours.

Mutagenized DNA was transformed into E. coli XL1-Blue cells, and colonies containing plasmid DNA selected were for by growth on LB agar plates containing 100 ~.g/ml ampicillin. DNA
sequencing was used to identify plasmids containing the appropriate mutation.
Expression of Casp-3 Mutant Proteins Plasmid DNA encoding cysteine mutations in the large subunit were transformed into Codon Plus BL21 Cells and plasmid DNA encoding cysteine mutations in the small subunit were transformed into BL21 (DE3) pLysS Cells. Codon Plus BL21 Cells containing plasmids encoding wild-type and cysteine mutated versions of the large subunit were grown in 2YT
containing 150 ~,g/mL of ampicillin overnight at 37 C and immediately harvested. BL21 pLysS cells containing plasmids encoding wild-type and cysteine mutated versions of the small subunit were grown in 2YT at 37 C
with 150 ~,g/mL of ampicillin until A~oo = 0.6. Cultures were subsequently induced with 1mM
IPTG and grown for an additional 3-4 hours at 37 C. After harvesting cells by centrifuging at 4K
rpm for 10 minutes, the cell pellet was resuspended in Tris-HCl (pH 8.0)/5 mM
EDTA and micro fluidized twice. Inclusion bodies were isolated by centrifugation at 9K rpm for 10 minutes and then resuspended in 6 M guanidine hydrochloride. Denatured subunits were rapidly and evenly diluted to 100 ~g/mL in renaturation buffer (100 mM Tris/KOH (pH 8.0), 10% sucrose, 0.1% CHAPS, 0.15 M NaCI, and 10 mM DTT) and allowed to renature by incubation at room temperature for 60 minutes with slow stirring.
Renatured proteins were dialyzed overnight in buffer containing 10 mM Tris (pH
8.5), 10 mM
DTT, and 0.1 mM EDTA. Precipitate was removed by centrifuging at 9K rpm for 15 minutes and filtering the supernatant through a 0.22 pm cellulose nitrate filter. The supernatant was then loaded onto an anion-exchange column (UnoS Q-Column (BioRad)), and correctly folded caspase-3 protein was eluted with a 0-0.25 M NaCI gradient at 3 mL/min. Aliquots of each fraction were electrophoresed on a denaturing polyacrylamide gel and fractions containing Caspase-3 protein were pooled.

PTP-1B (accession number SWS P18031) is a tyrosine phosphatase that has a C-terminal domain that is associated to the endoplasmic reticulum (ER) and a phosphatase domain that faces the cytoplasm. The proteins that it dephosphorylates are transported to this location by vesicles. The activity of PTP-1B is regulated by phosphorylation on serine and protein degradation. PTP-lBis a negative regulator of insulin signaling, and plays a role in the cellular response to interferon stimulation. This phosphatase may play a role in obesity by decreasing the sensitivity of organisms to leptin, thereby increasing appetite. Additionally, PTP-1B plays a role in the control of cell growth. A crystal structure has been solved for PTP-1B [1PTY, Puius, Y. A., et al., Proc Natl Acad Sci USA 94: 13420-13425 (1997)].
Cloning of human PTP-1B
Full length human PTP-1B is 435 amino acids in length; the protease domain comprises the first 288 amino acids. Because truncated portions of PTP-1B comprising the protease domain is fully active, various truncated versions of PTP-1B are often used. A cDNA encoding the first 321 amino acids of human PTP-1B was isolated from human fetal heart total RNA
(Clontech).
Oligonucleotide primers corresponding to nucleotides 91 to 114 (For) and complementary to nucleotides 1030 to 1053 (Rev) of the PTP-1B cDNA [Genbank M31724.1, Chernoff, J., et al., Proc. Natl. Acid. Sci. U. S. A. 87: 2735-2739 (1990)] were synthesized and used to generate a DNA
using the polymerise chain reaction.
Forward GCCCATATGGAGATGGAAAAGGAGTTCGAG SEQ ID
N0:104 Rev GCGACGCGAATTCTTAATTGTGTGGCTCCAGGATTCGTTT SEQ ID
NO:105 The primer Forward incorporates an NdeI restriction site at the first ATG
codon and the primer Rev inserts a UAA stop codon followed by an EcoRI restriction site after nucleotide 1053. cDNAs were digested with restriction nucleases NdeI and EcoRI and cloned into pRSETc (Invitrogen) using standard molecular biology techniques. The identity of the isolated cDNA Was verified by DNA
sequence analysis (methodology is outlined in a later paragraph).
A shorter cDNA, PTP-1B 298, encoding amino acid residues 1-298 was generated using oligonuclotide primers Forward and Rev2 and the clone described above as a template in a polymerise chain reaction.
Rev2 TGCCGGAATTCCTTAGTCCTCGTGGGAAAGCTCC SEQ ID
N0:106 Generation of PTP-1B Cysteine Mutants Site-directed mutants of PTP-1B (amino acids 1-321), PTP-1B 298 (amino acids 1-298) and PTP-1B 298-2M (with Cys32 and Cys92 changed to Ser and Val, respectively) were prepared by the single-stranded DNA method (modification of Kunkel, 1985). 298-2M was made with the following oligonucleotides.

N0:107 N0:108 Oligonucleotides were designed to contain the desired mutations and 12 bases of flanleing sequence on each side of the mutation. The single-stranded form of the PTP-1B/pRSET, PTP-1B 298/pRSET
and PTP-1B 298-2M/pRSET plasmid was prepared by transformation of double-stranded plasmid into the CJ236 cell line (1 ~,1 double-stranded plasmid DNA, 2 ~.1 Sx KCM
salts, 7 ~tl water, 10 p1 PEG-DMSO competent CJ236 cells; incubated on ice for 20 minutes followed by 25 C for 10 minutes; plated on LB/agar with 1.00 pg/ml ampicillin and incubated at 37 C
overnight). Single colonies of CJ236 cells were then grown in 100 ml 2YT media to midlog phase; 5 ~,l VCS helper phage (Stratagene) were then added and the mixture incubated at 37 C
overnight. Single-stranded DNA was isolated from the supernatant by precipitation of phage (1/5 volume 20% PEG
8000/2.5M NaCI; centrifuge at 12K for 15 minutes). Single-stranded DNA was then isolated from phage using Qiagen single-stranded DNA lcit.
Site-directed mutagenesis was accomplished as follows. Oligonucleotides were dissolved in TE (10 mM Tris pH 8.0, 1mM EDTA) to a concentration of 10 OD and phosphorylated on the 5' end (2 p1 oligonucleotide, 2 p.1 10 mM ATP, 2 ~,1 lOx Tris-magnesium chloride buffer, 1 p1 100 mM DTT, 12.5 p.1 water, 0.5 0,1 T4 PNK; incubate at 37 C for 30 minutes).
Phosphorylated oligonucleotides were then annealed to single-stranded DNA template (2 ~1 single-stranded plasmid, 0.6 ~.l oligonucleotide, 6.4 p.1 water; heat at 94 C for 2 minutes, slow cool to room temperature). Double-stranded DNA was then prepared from the annealed oligonucleotide/template (add 2 p.1 lOx TM
buffer, 2 ~.1 2.5 mM dNTPs, 1 ~,1 100 mM DTT, 0.5 ~1 10 mM ATP, 4.6 ~tl water, 0.4 p1 T7 DNA
polymerase, 0.2 ~,1 T4 DNA ligase; incubate at room temperature for two hours). E. coli (XI,1 blue, Stratagene) were then transformed with the double-stranded DNA (5 p1 double-stranded DNA, 5 p1 Sx KCM, 15 p.1 water, 25 p1 PEG-DMSO competent cells; incubate 20 minutes on ice, 10 min. at room temperature), plated onto LB/agar containing 100 pg/ml ampicillin, and incubated at 37 C
overnight. Approximately four colonies from each plate were used to inoculate 5 ml 2YT
containing 100 pg/ml ampicillin; these cultures were grown at 37 C for 18-24 hours. Plasmids were then isolated from the cultures using Qiagen miniprep lcit. These plasmids were sequenced to determine which clones contained the desired mutation.
A listing of the constructs and the single mutations to cysteine made in each context is given below.

PTP-1B 321 H25C, D29C, R47C, D48C, SSOC, K120C, M258C
PTP-1B 298 H25G, D29C, D48C, SSOC, K120C, M258C, F280C
PTP-1B 298-2M E4C, E8C, H25C, A27C, D29C, K36C, Y46C, R47C, D48C, V49C, SSOC, FSZC, K120C, S151C, Y152C, T178C, D181C, F182C, E186C, S187C, A189C, K197C, E200C, L272C, E276C, I218C, M258C, Q262C, V287C
However, it should be understood that any of the site-directed mutants may be made in any construct of PTP-1B. For example, another construct is another truncated version of PTP-1B
having residues 1-382, shown as SEQ ID N0:109 below.

l81 DFGVPESPAS FLNFLFKVRESGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKD

Mutagenic Oligonucleotides NO:110 NO:111 N0:112 N0:113 NO: l I4 NO:115 N0:116 N0:117 N0:118 N0:119 SSOC CTATGGTCAAAGGGACAGACGTCTCTGTACC SEQID

N0:120 N0:121 N0:122 N0:123 N0:124 N0:125 N0:126 N0:127 N0:128 N0:129 N0:130 N0:131 N0:132 N0:133 N0:134 N0:135 NO;136 N0:137 Besides mutations to cysteines, mutations removing naturally occurring cysteines can also be made.
For example, two different "scrubs" of Cys 215 were made in the PTP-1B 298-2M
context using the following oligonueleotides:

N0;138 N0:139 In the PTP-1B 298 context, two quadruple mutants were made using the C92A
oligonucleotide shown below. They are C32S, C92A, V287C, C215A, which used SEQ ID N0:107 SEQ
ID
N0:140 SEQ ID N0:137 and SEQ ID N0:138 and C32S, C92A, E276C, C215A, which used SEQ
ID N0:107, SEQ ID N0:140 SEQ ID N0:135 and SEQ ID N0:138.

N0:140 Sequencing of PTP-IB clones was accomplished as follows. The concentration of plasmid DNA
was quantitated by absorbance at 280 nm. 1000 ng of plasmid was mixed with sequencing reagents (1 p,g DNA, 6 p1 water, 1 p1 sequencing primer at 3.2 pm/p.l, 8 ~l sequencing mixture with Big Dye [Applied Biosystems]). The sequencing primers are SEQ ID N0:17 and SEQ ID
N0:18. The mixture was then run through a PCR cycle (96 C,10 s; 50 C, 5 s; 60 C 4 minutes; 25 cycles) and the DNA reaction products were precipitated (20 p1 mixture, 80 p1 75%
isopropanol; incubated 20 minutes at room temperature then pelleted at 14 K rpm for 20 minutes; wash with 250 p1 75%
isopropanol; heat 1 minute at 94 C). The precipitated products were then resuspended in 20 ~1 TSB (Applied Biosystems) and the sequence read and analyzed by an Applied Biosystems 310 capillary gel sequences. In general, 1/4 of the plasrnids contained the desired mutation.
Expression of Cysteine Mutants of PTP-1B
Mutant proteins were expressed as follows. PTP-1B clones were transformed into BL21 codon plus cells (Stratagene) (1 p,1 double-stranded DNA, 2 ~,l 5x KCM, 7 ~1 water, 10 ~,1 DMSO competent cells; incubate 20 minutes at 4 C, 10 minutes at room temperature), plated onto LB/agar containing 100 ~.g/ml ampicillin, and incubated at 37 C overnight. 2 single colonies were picked off the plates or from frozen glycerol stocks of these mutants and inoculated in 100 ml 2YT with 50 p,g/ml carbenicillin and grown overnight at 37 C. 50 ml from the overnight cultures were added to 1.5 L
of 2YT/carbenicillin (50 ~.g/ml) and incubated at 37 C for 3-4 hours until late-log phase (absorbance at 600 nm ~0.8-0.9). At this point, protein expression was induced with the addition of IPTG to a final concentration of 1 mM. Cultures were incubated at 37 C for another 4 hours and then cells were harvested by centrifugation (7K rpm, 7 minutes) and frozen at-20 C.
PTP-1B proteins were purified from the frozen cell pellets as described in the following. First, cells were lysed in a microfluidizer in 100 ml of buffer containing 20 mM MES pH
6.5, 1 mM EDTA, 1 mM DTT, and 10% glycerol buffer (with 3 passes through a Microfluidizer [Microfluidics 110S]) and inclusion bodies were removed by centrifugation (10K rpm, 10 minutes).
Purification of all PTP-1B mutants was performed at 4 C. The supernatants from the centrifugation were filtered through 0.45 ~.m cellulose acetate (5 ~1 of this material was analyzed by SDS-PAGE) and loaded onto an SP Sepharose fast flow column (2.5 cm diameter x 14 cm long) equilibrated in Buffer A (20 mM MES pH 6.5, 1 mM EDTA, 1 mM DTT, 1% glycerol) at 4 ml/min.
The protein was then eluted using a gradient of 0 - 50% Buffer B over 60 minutes (Buffer B: 20 mM MES pH 6.5, 1 mM EDTA, 1 mM DTT, 1 % glycerol, 1 M NaCI). Yield and purity was examined by SDS-PAGE and, if necessary, PTP-1B was further purified by hydrophobic interaction chromatography (HIC). Protein was supplemented with ammonium sulfate until a final concentration of 1.4 M was reached. The protein solution was filtered and loaded onto an HIC
column at 4 ml/min in Buffer A2: 25 mM Tris pH 7.5, 1 mM EDTA, 1.4 M
(NHø)~SO4, 1 mM
DTT. Protein was eluted with a gradient of 0 - 100% Buffer B over 30 minutes (Buffer B2: 25 mM
Tris pH 7.5, 1 mM EDTA, 1 mM DTT, 1% glycerol). Finally, the purified protein was dialyzed at 4 C into the appropriate assay buffer (25 mM Tris pH 8, 100 mM NaCI, 5 mM
EDTA, 1 mM DTT, 1% glycerol). Yields varied from mutant to mutant but typically were within the range of 3-20 mg/L culture.

HIV IN
HIV IN is one of three key enzyme targets of the human immunodeficiency virus;
it removes two nucleotides from each 3' end of the originally blunt viral DNA, and inserts the viral DNA into the host DNA by strand transfer. The integration process is completed by host DNA
repair enzymes.
HIV IN has three distinct domains: the N-terminal domain, the catalytic core domain, and the C-terminal domain. Although the X-ray crystal structures of each of these isolated domains have been solved, it is not yet clear how they interact with each other. Integration is absolutely essential for the replication of the virus and progression of disease, and thus integrase inhibitors can be used in the treatment of HIV/AIDS. Structures of core domain of integrase are available [lEXQ, Chen, J.
C. -H., et al., Proc. Natl. Acad. Sci. U. S. A. 97: 8233-8238 (2000); 1BL3, Maignan, S., et al., JMoI
Biol 282:359-368 (1998); in complex with tetraphenyl arsonium, 1HYZ and 1HYV, Molteni, V., et al., Acta Crystallogr D Bio Crystallog., 57:536-544 (2001)].
Cloning of HIV IN
Numbering of the wild type and mutant HIV-1 integrase residues follows the convention of the first amino acid residue of the mature protein being residue number 1, and the HIV-1 integrase catalytic core domain being comprised of residues S2-210 [Leavitt, A. D., et al., J Biol Che~i 268: 2113-2119 (1993)].
A plasmid construct, pT7-7 HT-INtetta, encoding the HIV integrase core domain (residues SO-212), having an N-terminal 6x histidine tag and thrombin cleavable linker, and C56S, W131D, F139D, and F18SK mutations in the pT7-7 (Novagen) vector background [Chen, J. C. -H., et al., Proc. Natl.
Acad. Sci. U. S. A. 97: 8233-8238 (2000)] was obtained from Dr. Andy Leavitt at UCSF. Upon comparison of the crystal structure of this core domain variant [Chen, J. C. -H., et al., Proc. Natl.
Acad. Sci. U. S. A. 97: 8233-8238 (2000)] to other integrase core structures, it was noted that the F139D mutation, designed to increase solubility of the protein, caused a rotation of the side chain that transmitted a distortion to the catalytically important Asp116. The mutation was therefore reverted to the wild-type phenylalanine residue by Quiclechange mutagenesis (Stratagene), following manufacturer's instructions and using SEQ ID N0:141 and SEQ ID
N0:142.
D139F1-int GTATCAAACAGGAATTCGGTATCCCGTACAAC SEQ ID N0:141 D139F2-int GTTGTACGGGATACCGAATTCCTGTTTGATACC SEQ ID N0:142 This generated pT7-7 HT-INm, encoding the triple mutant (C56S, W131D, F185K) of the integrase core, SEQ ID NO: I43.

In preparation for making cysteine mutations at tethering sites, the two wild-type cysteines, (C130 and C65) were replaced by alanine residues and the DNA encoding the His-tagged INm core domain transferred into the pRSET A vector, containing an F1 origin of replication that allows preparation of single-stranded plasmid DNA, and thus mutagenesis by the Kunlcel method [Kunkel, T. A., et al., Methods Enzymol. 204: 125-139 (1991)]. Replacement of 0130 by alanine was accomplished by cassette mutagenesis, using the double stranded cassette composed of SEQ ID
N0:144 and SEQ ID N0:145. The cassette, containing the appropriate overhangs at each end, was ligated into pT7-7 HT-INS; digested with BsiWI and EcoRI.
C130A cassette 1 GTACGTGCTGCAGCCGACTGGGCTGGTATCAAACAGG SEQ ID N0:144 C130A cassette 2 GAATTCCTGTTTGATACCAGCCCAGTCGGCTGCAGCAC SEQ ID N0:145 The C65A mutation was carried out independently by Quickchange mutagenesis on pT7-7 HT-INM
using SEQ ID N0:146 and SEQ ID N0:147.
C65A1-irit ATCTGGCAACTGGACGCGACTCACCTCGAGGGT SEQ ID NO:146 C65A2-int ACCCTCGAGGTGAGTCGCGTCCAGTTGCCAGAT SEQ ID N0:147 The DNA encoding HT-C130A integrase core domain was subcloned into the pRSET A
vector by PCR cloning. SEQ ID N0:14~ and SEQ ID N0:149 were used as PCR primers, and the resulting amplified product was digested with NdeI and Hind III, and ligated into pRSET
A that had been digested with the same enzymes, to generate pRSET-HT-C130A-INS;.
C130 rsetF GGAGATATACATATGCACCACCATCACC SEQ ID N0:148 C130 rsetR ATCATCGATGATAAGCTTCCTAGGTCTGG SEQ ID N0:149 A BamHI fragment of pT7-7 HT-C65A-INm containing the C65A mutation was ligated into pRSET-HT-C130A-INtp, to generate pRSET-HT-IN~e",p,ate. This plasmid served as a template for further Kunlcel mutagenesis to introduce cysteine substitutions at positions chosen for tethering.
SEQ ID N0:17 was used for sequencing.
Mutagenic Oligonucleotides N0:150 NO:151 N0:152 N0:153 N0:154 N0:155 N0:156 N0:157 N0:158 N0:159 N0:160 N0:161 N0:162 N0:163 N0:164 N0:165 N0:166 N0:167 N0:168 N0:169 Expression of IN Cysteine Mutants pT7-7 and pRSET integrase core domain expression plasmids were transformed into BL2lstar E.
coli (Invitrogen) by standard methods, and a single colony from the resulting plate was used to inoculate 250 mL of 2x YT broth containing 100 ~.g/mL ampicillin. Following overnight growth at 37 C, the cells were harvested by centrifugation at 4K rpm and resuspended in 100 mL 2YT/amp.
40 mL of the washed cells was used to inoculate 1.5 L of the same media, and after growth at 37 C
to an OD at 600 nm of between 0.5 and 0.8, the culture was moved to 22 C and allowed to cool.
IPTG was added to a final concentration of 0.1 mM and expression continued 17-19 h at 22 C.
Cells were harvested by centrifugation at 4K rpm. Cell pellets were resuspended in 100 mL Wash 5 buffer (Wash 5: 20 mM Tris-HCI, 1 M MgClz, 5 mM imidazole, 5 mM (3-mercaptoethanol, pH 7.4) and lysis was accomplished by sonication for 1 minute, repeated a total of 3 times with 2 minutes rest between. Cell debris was removed by centrifugation at 14K rpm followed by filtration.
Integrase core domain was purified by affinity chromatography on Ni-NTA
superflow resin (Qiagen) at 4 C. After loading the cell lysate, the column was washed with Wash 40 buffer (Wash 40: 20 mM Tris-HCl, 0.5 M NaCI, 40 mM imidazole, 5 mM (3-mercaptoethanol, pH
7.4) and His-tagged IN core domain eluted with E400 buffer (E400: 20 mM Tris-HCI, 0.5 M
NaCI. 400 mM
imidazole, 5 mM (3-mercaptoethanol). The purified enzyme was dialyzed versus 20 mM Tris, 0.5 M NaCI, 2.5 mM CaClz, 5 mM (3-mercaptoethanol, pH 7.4 at 4 C , and aliquoted into 1,5 mL tubes.
Biotinylated thrombin (Novagen) (2U thrombin/mg of protein) was added and the tubes rotated overnight at 4 C, followed by thrombin removal using streptavidin-agarose resin (Novagen) and separation of His-tagged protein and peptides from the cleaved material by passage through a second column of Ni-NTA sepharose fast-flow. Purified, cleaved integrase core domain was dialyzed against 20 mM Tris-HCI, 0.5 M NaCI, 3 mM DTT, and 5% glycerol, pH
7.4, and stored at -20 C. Protein concentrations were determined by absorbance at 280 nm after desalting on NAP-5 columns (Pharmacia), using E28o'~° _ (1.174), and molecular weights confirmed by ESI mass spectrometry (Finnigan).

HUMAN BETA-SITE AMYLOID PRECURSOR PROTEINCLEAV1NG ENZYMEl fBACEl) BACE1 (accession number SWS 56817) is a typel integral glycoprotein that is an aspartic protease.
Found mostly in the Golgi, BACEl cleaves the amyloid precursor protein to form the Abeta peptide. A strong association has been shown between deposition of this peptide on the cerebrum and Alzheimer's disease; therefore BACEl is one of the primary targets for this disease. A crystal structure of BACE1 has been solved [1FKN, Hong, L. et al., Science 290:150-153 (2000)].
Cloning of Human BACE1 The proprotease domain gene sequence (bases 64-1362, amino acid residues 22-454) was subcloned from pFBHT into the E. coli expression vector pRSETC by PCR, to create pB22, which served as a template for mutagenesis to incorporate cysteine tethering sites. For a description of pFBHT, a modified pFastBac plasmid, see example 4 above. The subcloning was accomplished as follows.
The cDNA encoding full-length human BACE1, bases 1-1551, starting from the initiator Met codon and including an extra 48 bases of mRNA transcript following the stop codon [Vassar, R., et al., Science 286: 735-741 (1999)] was obtained by a combination of PCR cloning of the 3' 1425 bases from human cDNA libraries, and synthesis of the remaining 5' 126 bases by serial overlapping PCR. All PCR reactions were performed using Advantage2 polymerase (Clontech) according to manufacturers instructions. A fragment spanning bases 126-374 was obtained by PCR from a human cerebral cortex library and SEQ ID N0:170 and SEQ ID N0:171; a fragment spanning bases 339-770 was obtained by PCR from a Stratagene Unizap XR human brain cDNA
library, and SEQ ID N0:172 and SEQ ID N0:173; and the 3' end fragment, spanning bases 735-1551, was obtained by PCR from a human brain library, using SEQ ID NO:174 and SEQ ID
N0:175. The three fragments, having 35 by of overlap at the junctions, were gel purified and combined in one PCR reaction, using primers to the ends (SEQ ID N0:170 and SEQ ID NO:176) to amplify the 126-1551 product.
FOr2 GCTGCCCCGGGAGACCGACGAAGA SEQ ID NO:17O
midRev2 CGGAGGTCCCGGTATGTGCTGGAC SEQ ID N0:171 midFor CCAGAGGCAGCTGTCCAGCACATA SEQ ID N0:172 midRevl TCCCGCCGGATGGGTGTATACCAG SEQID N0:173 BACE14 GTACACAGGCAGTCTCTGGTATACACC SEQ ID NO:174 BACE11 GTGTGGTCCAGGGGAATCTCTATCTTCTG SEQ ID NO:17S
RACES GTCATCGTCTCGAGTCACTTCAGCAGGGAGATGTCATCAG SEQ ID NO:176 The 126-1551 piece, and the subsequent elongated products, were used as a templates for serial overlapping PCR reactions, to add the remaining 5' -126 bases using SEQ ID
N0:177, SEQ ID
NO:178 and SEQ ID N0:179 as forward primers, with SEQ ID N0:176 always at the reverse primer.
BACE fi112 CGGCTGCCCCTGCGCAGCGGCCTGGGGGGCGCCCCCCTGGGGCTGCGGCTGCCCCGGGAG
SEQ ID N0:177 BACE filll ATGGGCGCGGGAGTGCTGCCTGCCCACGGCACCCAGCACGGCATCCGGCTGCCCCTGCGC
2p SEQ ID N0:178 BACE for-EcoRI
CCGGAATTCATGGCCCAAGCCCTGCCCTGGCTCCTGCTGTGGATGGGCGCGGGAGTG
SEQ ID N0:179 SEQ ID N0:179 and SEQ ID N0:176 contained EcoRI and XhoI restriction sites, respectively, and digestion of the PCR product, along with the Baculovirus expression vector, pFBHT, with the same enzymes was followed by gel purification and ligation of the resulting DNA
fragments, yielding the construct, pFBHT-BACE. This construct was used as a template for PCR
amplification of bases 1-1362, corresponding to the preproBACE soluble protease domain, using SEQ ID
N0:180 and SEQ
ID N0:181.
S
proFor-Nde CGCCATATGGCGGGAGTGCTGCCTGCCCACGGC SEQ ID N0:180 BACErev-RI CCGGAATTCTCAGGTTGACTCATCTGTCTGTGGAAT SEQ ID N0:181 SEQ ID N0:180 and SEQ ID NO:181 contained NdeI and EcoRI restriction sites, respectively, and digestion of the PCR product, along with the E. coli expression vector, pRSETC, with the same enzymes was followed by gel purification and ligation of the resulting DNA
fragments led to the construct pB 1. Vector pB 1 was then used as a template fox Kunlcel mutagenesis (Kunkel, T. A., et al., Metlae~ds Enzymol. 154:367-382 [1987]) to delete the BACE presequence (bases 1-63), producing the construct pB22. pB22 served as a template for mutagenesis to incorporate cysteine tethering sites, using either the Kunkel method or a Quiclcchange mutagenesis kit (Stratagene).
Mutagenenic Oligonucleotides N0:182 N0:183 NO:184 NO:185 NO:186 N0:187 N0:188 N0:189 N0:190 N0:191 N0:192 Expression of Human BACEI Mutants pB22 was transformed into BL2lstar E. coli (Invitrogen) by standard methods, and a single colony from the resulting plate was used to inoculate 50 mL of 2xYT broth containing 100 ~.g/mL
ampicillin. Following overnight growth at 37 C, 40 mL of the culture was used to inoculate 1.5 L
of the same media, and after growth at 37 C to an OD at 600 nm of between 0.5 and 0.8, IPTG was added to a final concentration of 1.0 mM and expression continued 3 h at 37 C.
Cells were harvested by centrifugation at 4K rpm. Cell pellets were resuspended in 100 mL
buffer TE (10 mM
Tris-HCl, 1 mM EDTA, pH 8.0) and lysis was accomplished using a French Press microfluidizer (two passages). The crude extract, containing BACE1 as insoluble inclusion bodies, was centrifuged at 14K rpm for 15 minutes, and the resulting pellet washed by resuspension in PBS (10 mM sodium phosphate, 150 mM NaCI, pH 7.4) followed by centrifugation at 14K
rpm for 20 minutes. Washed inclusion body pellets were solubilized in 50 mM CAPS, 8 M
urea, 1 mM
EDTA, and 100 mM (3-mercaptoethanol, pH 10, and remaining insoluble debris removed by centrifugation at 20K rpm for 30 minutes. BACEl was refolded by slow injection of the urea-solubilized protein to between 50 and 100 volumes of rapidly stirred water, or 10 mM NazC03, pH
10, followed by incubation at room temperature for 3-7 days. When BACE1 enzymatic activity no longer increased over time, the pH of the refolding solution was adjusted to 8.0 by addition of 5 mM (final concentration) Tris-HCI, and loaded onto a Q-Sepharose column.
Protein was eluted using a linear gradient of 0 to 500 mM NaCI in 10 mM Tris-HCI, pH 8Ø BACEl was further purified by S-Sepharose chromatography at pH 4.5. Purified enzyme was dialyzed versus 20 mM
Tris, 0.125 M NaCI, pH 7.2 at 4 C, and stored at 4 C. Protein concentrations were determined by absorbance at 280nm, using s28o'°~° _ (0.74).

CLONING AND MUTAGENESIS OF MITO EN-ACTIVATED PROTEIN
KINASEIEXTRACELLULAR SIGNAL-REGULATED K1NASE K1NASE fMEK) Mek-1 (accession number SWS Q02750) is a dual specificity lcinase that plays a key role in cellular proliferation and survival in response to mitogenic stimuli. Melc-1 is the central component of a three-kinase cascade commonly called a MAP kinase cascade. This Raf Melc-Erk Icinase cascade transmits information from cell surface receptors (e.g. EGFR, HER2, PDGFR, FGFR, IGF, etc.) to the nucleus. This pathway is upregulated in approximately 30% of all tumor types, either through overexpression of specific cell surface receptors (e.g. HER2 in breast cancers) or through activating mutations in Ras, a key upstream component of this pathway. Disruption of Mek-1 function has dramatic anti-tumor effects, both in cell culture and in animals. Mele-2 (accession number SWS
P3G507) is a dual specificity kinase that is both highly homologous (79%
identity) to Mek-1 and coordinately expressed with Mek-1. Thus, Mek-1 and Mek-2 represent attractive targets for the development of novel anti-cancer therapeutics. There are no crystal structures to date for Melc-1 or Melc-2.
Cloning of human Melc-1 and Mek-2 Numbering of the wild type and mutant Mek-1 and Mek-2 residues begins at their respective amino termini, with residue number 1 being the initiation methionine, according to the NCBI reported sequences (NCBI accession number L05624 for Melc-1 and NCBI accession number for Mek-2). All standard cloning and mutagenesis steps were carried out according to the recommendations of the enzyme manufacturer.
The DNA encoding human Mek-1 was isolated from plasmid pUSE MEK1 (Upstate Biotechnology) and inserted into plasmid pGEX-4T-1 (Amersham) in frame with GST as follows.
First, pUSE MEKl was digested with NotI (New England Biolabs), the 3' overhang filled in with the Klenow fragment of DNA polymerase (New England Biolabs), and the 1193 by product encoding MEK1 was isolated from an agarose gel. pGEX-4T-1 was linearized by digestion with EcoRI (New England Biolabs) and the 3' overhang similarly filled in with the Klenow fragment of DNA polymerase (New England Biolabs). The MEK1 and pGEX-4T-1 DNA fragments were then ligated with T4 Iigase and amplified in E. coli strain Top 10F' (Invitrogen) to generate plasmid pGEX-MEKl .
The DNA encoding human Mek-2 was isolated from plasmid pUSE MEK2 (Upstate Biotechnology) and inserted into plasmid pGEX-4T-1 (Amersham) in frame with GST as follows.
First, pUSE MEK2 was digested with NotI (New England Biolabs), the 3' overhang filled in with the Klenow fragment of DNA polymerase (New England Biolabs), and the 1213 by product encoding MEK2 was isolated from an agarose gel. pGEX-4T-1 was linearized by digestion with EcoRI (New England Biolabs) and the 3' overhang similarly filled in with the Klenow fragment of DNA polymerase (New England Biolabs). The MEK2 and pGEX-4T-1 DNA fragments were then ligated with T4 ligase and amplified in E. coli strain ToplOF' (Invitrogen) to generate plasmid pGEX-MEK2.
Generation of Mek-1 and Mek-2 Cysteine Mutants All mutagenesis steps were performed using long range PCR. Reactions contained the parent plasmid (2 ng/~1), sense strand mutant primer (0.5 ~,M), and antisense strand mutant primer (0.5 ~.M) that are unique to each reaction. In addition, all reactions contained dNTPs (25 uM) and Pfu polymerase (0.05 Units/ql; Stratagene). Reactions were incubated for one minute at 95 C followed by 16 cycles of (0.5 minutes at 95 C, 1 minute at 55 C, and 2 minutes at 68 C
) and a final 10 minutes at 6S C. Parent plasmid DNA was then digested with DpnI (New England Biolabs) and the remaining linear PCR product was transformed into E. coli strain TopIOF' (Invitrogen).
Mutagenized plasmid DNA, the result of in vivo recombination and subsequent amplification, was purified using QIAquick (Qiagen) columns and verified by sequencing.
First, a 6xHIS epitope tag was introduced into pGEX-MEK1, at the carboxy terminus of MEKl, to generate pGEX-MEK1-HIS using the sense and antisense oligonucleotides MEK1-6HIS-s and MEK1-6HIS-as, resepectively. Similarly, a 6xHIS epitope tag was introduced into pGEX- MEK2, at the carboxy terminus of MEK2, to generate pGEX-MEK2-HIS using the sense and antisense oligonucleotides, IVIEK2-6HIS-s and MEK2-6HIS-as, resepectively.
MEK1-6HIS-s CACGCTGCCAGCATCGGCGTCGACCCAACCCTGGTT
CCGCGTGGATCCCATCACCATCACCATCACTGAGCG
GCCAATTCCCGG
SEQ ID N0:193 MEK1-6HIS-as CCGGGAATTGGCCGCTCAGTGATGGTGATGGTGATG
GGATCCACGCGGAACCAGGGTTGGGTCGACGCCGAT
GCTGGCAGCGTG
SEQ ID N0:194 MEK2-6HIS-s ACGCGTACTGCAGTGGGCGTCGACCCAACCCTGGTT
~S CCGCGTGGATCCCATCACCATCACCATCACTGAGCG
GCCAATTCCCGG
SEQ ID N0:195 MEK2-6HIS-as CCGGGAATTGGCCGCTCAGTGATGGTGATGGTGATG

TGCAGTACGCGT
SEQ ID N0:196 Subsequently, 16 individual mutations were introduced into pGEX-MEK1-HIS.
Similarly, the 35 analogous 16 individual mutations were introduced into pGEX-MEK2-HIS. Each of these mutations introduces a cysteine into the MEK1 or MEK2 protein, and each is named according to the resultant amino acid substitution. For example, primer pair MEK1-N78C-sense and MEK1-N78C-antisense were used to introduce a cysteine in place of N78 of MEK1, generating pGEX-MEK1/N78C-HIS.

Mutagenic Oligonucleotides N0:197 MEK1-N78C-as G~CACCACACCGCCGCAGCCAGCCCCCAGCTCSEQ ID .

N0:198 N0:199 MEK1-G79C-as CTTGAACACCACACCGCAATTGCCAGCCCCCAGSEQ ID

N0:200 N0:201 MEK1-I107C-asTATGATCTGGTTCCGGCATGCGGGTTTGATCTCSEQ ID

N0:202 N0:203 MEK1-R1O8C-aSCCTTATGATCTGGTTGCAGATTGCGGGTTTGATSEQ ID

N0:204 N0:205 MEK1-II l CTGCAGCTCCCTTATGCACTGGTTCCGGATTGCSEQ ID
IC-as N0:206 MEKl-EI 14C-S~CCAGATCATAAGGTGCCTGCAGGTTCTGCATSEQ ID

N0:207 MEK1-E114C-asATGCAGAACCTGCAGGCACCTTATGATCTGGTTSEQ ID

N0:208 N0:209 MEKl-LI 18C-aSAGAGTTGCACTCATGGCAAACCTGCAGCTCCCTSEQ ID

N0:2I0 MEK1-V127C-S ~CTCTCCGTACATCTGCGGCTTCTATGGTGCGSEQ ID

N0:211 MEK1-V127C-asCGCACCATAGAAGCCGCAGATGTACGGAGAGTTSEQ ID

N0:212 MEK1-M143C-s GAGATCAGTATCTGCTGCGAGCACATGGATGGASEQ ID

N0:213 as N0:214 N0:215 MEK1-S150C-asCAGGACTTGATCCAGGCAACCTCCATCCATGTGSEQ ID

N0:2I6 MEK1-L180C-s ~GGCCTGACATATTGCAGGGAGAAGCACAAG SEQ ID

N0:217 MEK1-L180C-asCTTGTGCTTCTCCCTGCAATATGTCAGGCCTTTSEQ ID

N0:218 N0:219 MEKl-I186C-asGACATCTCTGTGCATGCACTTGTGCTTCTCCCTSEQ ID

N0:220 MEKl-K192C-S ATGCACAGAGATGTCTGCCCCTCCAACATCCTASEQ ID

N0:221 MEK1-K192C-asTAGGATGTTGGAGGGGCAGACATCTCTGTGCATSEQ ID

N0:222 N0:223 MEKl-S194C-asGTTGACTAGGATGTTGCAGGGCTTGACATCTCTSEQ ID

N0:224 MEKI-LI97C-s AAGCCCTCCAACATCTGCGTCAACTCCCGTGGGSEQ ID

NO:225 MEK1-L197C-asCCCACGGGAGTTGACGCAGATGTTGGAGGGCTTSEQ ID

N0:226 N0:227 MEKl-V211C-asGATGAGCTGCCCGCTGCACCCAAAGTCACAGAGSEQ ID

N0:228 N0:229 MEK2-N82C-asGGTGACCACCCCGCCGCAGCCCGCGCCCAGCTCSEQ ID

N0:230 N0:23 I

MEK2-G83C-asTTTGGTGACCACCCCGCAGTTGCCCGCGCCCAGSEQ ID

NO:232 MEK2-I111C-sGAGATCAAGCCGGCCTGCCGGAACCAGATCATCSEQ ID

N0:233 MEK2-I111C-asGATGATCTGGTTCCGGCAGGCCGGCTTGATCTCSEQ ID

N0:234 N0:235 MEK2-R112C-asGCGGATGATCTGGTTGCAGATGGCCGGCTTGATSEQ ID

N0:236 15C-s N0:237 15C-as N0:23 N0:23 MEK2-E118C-asGTGCAGGACCTGCAGGCAGCGGATGATCTGGTTSEQ ID

N0:240 MEK2-LI22C-sCGCGAGCTGCAGGTCTGCCACGAATGCAACTCGSEQ ID

NO:241 MEK2-L122C-asCGAGTTGCATTCGTGGCAGACCTGCAGCTCGCGSEQ ID

N0:242 N0:243 MEK2-V131C-asGGCCCCGTAGAAGCCGCAGATGTACGGCGAGTTSEQ ID

N0:244 N0:245 as N0:246 N0:247 MEK2-S154C-asCAGCACCTGGTCCAGGCAGCCGCCGTCCATGTGSEQ ID

N0:248 N0:249 MEK2-L184C-aSCTGGTGCTTCTCTCGGCAGTACGCCAAGCCCCGSEQ ID

N0;250 N0:251 MEK2-I190C-asCACATCTCGGTGCATGCACTGGTGCTTCTCTCGSEQ ID

N0:252 N0:253 MEK2-K196C-asGAGGATGTTGGAGGGGCACACATCTCGGTGCATSEQ ID

N0:254 N0:255 MEK2-S198C-asGTTCACGAGGATGTTGCAGGGCTTCACATCTCGSEQ ID

N0:256 N0:257 MEK2-L201C-asCCCTCTAGAGTTCACGCAGATGTTGGAGGGCTTSEQ ID

N0:25 N0:259 MEK2-V215C-aSTATGAGCTGGCCGCTGCACCCGAAGTCACACAGSEQ ID

N0:260 Sequencing primers pGEX forward GGGCTGGCAAGCCACGTTTGGTG SEQ ID
N0:261 pGEX reverse CCGGGAGCTGCATGTGTCAGAGG SEQ ID
N0:262 Expression of Mek-1 and Mek-2 mutants Mutant alleles of Mek-1 and Mek-2 were expressed in E. coli and purified essentially as described for Mek-1 [by McDonald, O. B., et al., Analytical Biochern. 268: 3I8-329 (I999)]. Plasmids S containing the mutant Mek-1 and Mek-2 alleles were transformed into BL21 DE3 pLysS cells (Invitrogen) according to manufacturer's suggestions. Cultures were grown overnight at 37 C
from single colonies in 100 ml 2YT medium supplemented with 100 ~glml ampicillin and 100 ~,g/ml chloramphenicol. This culture was then added to 1.5 L 2YT supplemented with 100 ~,g/ml ampicillin to achieve an ODGO° of approximately 0.05 and then grown to an OD~oo of approximately 0.7 at 30 C. Expression was induced with the addition of IPTG to a final concentration of 1 mM
and the culture was incubated for four hours at 2S C. Cells were pelleted in a Sorfall GSA rotor at 6I~ rpm for 15 minutes and stored at -80 C.
Mek-1 and Mek-2 mutants were purified from cells by first resuspending cell pellets in ice cold 1S PBS containing 0.5% Triton X-100 and incubating on ice for 4S minutes, followed by extensive sonication. Lysates were clarified by centrifugation in a Sorvall GSA rotor at 12K rpm for one hour. Fusion proteins were first purified on Ni-NTA resin (Qiagen) according to manufacturer's suggestions, followed by further purification on glutathione agarose as described [by McDonald, O.
B., et al., Analytical Biocherra. 268: 318-329 (1999)]. Epitope tags were removed with thrombin cleavage and aliquots of purified protein were stored at -80 C in TBS
containing 10% glycerol.

CLONING AND MUTAGENESIS OF HUMAN CATHEPSIN S ICATS~
Cathepsin S (accession number SWS P25774) is a thiol protease located primarily in the lysosome.
2S This enzyme plays roles in antigen presentation by processing of the MHC-II
antigen receptor; thus inhibitors to the enzyme could be used for diseases such as inflammation and autoimmunity such as rheumatoid arthritis, multiple sclerosis, asthma and organ rejection. It has also been reported that cats is present in increased levels in the Alzheimer's disease and Down Syndrome brain compared with normal brain. A structural model of cathepsin S [1BXF, Fengler, A. &
Brandt W., Protein Eng 11:1007-1013(1998)] and a crystal structure of the C25S mutant [Turkenburg, J. P. et al. Acta Crystallogr D Biol Crystallog 58: 4S 1-4SS (2002)] are available.
Cloning of human cats The DNA sequence encoding human cathepsin S (cats) was isolated by PCR from the plasmid pDualGC (Stratagene #EOI089) using PCR primers listed below corresponding to the protein N-and C-termini. These primers were designed to contain restriction endonuclease sites EcoRI and XhoI, for subcloning into a modified pFastBac vector, pFBHT (c.~ example 4 above). SEQ ID

NO: 263 was used with SEQ ID NO: 264 and SEQ ID NO 265 to make cats with and without a 6xhis tag, respectively.
5' Cats EcoRI CCGGAATTCATGAAACGGCTGGTTTGTGTGCT SEQ ID N0:263 3' Cats XhoI CCCCGCTCGAGGATTTCTGGGTAAGAGGGAAAG SEQ ID N0:264 3'CatS XhoI stop CCCCGCTCGAGCTAGATTTCTGGGTAAGAGGGAAA SEQ ID N0:265 S The PCR reaction was purified on a Qiaquick PCR purification column (Qiagen). The PCR product containing the cats sequence was cut with restriction endonucleases (42 ~l PCR
product, 1 ~1 each endonuclease, 5 ~tl appropriate lOx buffer; incubated at 37 C for 3 hours).
The pFBHT vector was cut with restriction endonucleases (5 pg DNA, 1 ~1 each endonuclease, 3 ~l appropriate lOx buffer, water to 30 ~1; incubated at 37 C for 3 hours; added 1 ~1 CIP and incubated at 37 C for 6Q
minutes). The products of nuclease cleavage were isolated from an agarose gel (1% agarose, TBE
buffer) and ligated together using T4 DNA ligase (50 ng pFBHT vector and 50 ng catS PCR
product in 10 ~,1, 10 ~.l 2x ligase buffer (Roche), 1 ~.l ligase, incubated at 25 C for 15 minutes). 1 ~l of the ligation reaction was transformed into Library Efficiency Chemically Competent DHSa cells (Invitrogen) (1 ~l ligation reaction, 100 p1 competent cells; incubated at 4 C
for 30 minutes, 42 C
for 45 seconds, 4 C for 2 minutes, then 900 ~1 SOC media was added and incubated for 1 hour with shaking at 225 rpm at 37 C), and plated onto LB/agar plates containing 100 ~.g/ml ampicillin.
After incubation at 37 C overnight, single colonies were grown in 3 ml LB
media containing 100 ug/ml ampicillin for 8 hours. Cells were then isolated and double-stranded DNA
extracted from the cells using a Qiagen DNA miniprep lcit. Sequencing of cats gene was accomplished using M13/pUC Forward and Reverse Amplification Primers (Invitrogen # 18430-017).
Generation of Cats Cysteine Mutations Mutations were generated using as previously described [Kunlcel T. A., et al., Methoels_Erazynaol.
154: 367-382 (1987)]. DNA oligonucleotides used are shown below and were designed to hybridize with sense strand DNA from plasmid. Sequences were verified using primers with SEQ
ID NO:74 and SEQ ID N0:75.
Mutagenic Oligonucleotides Y1 SC CACAAGAACCTTGACATTTCACTTCAGT SEQ ID NO:266 K64C CACCATTGCAGCCACAGTTTCCATATTT SEQ ID NO:267 N67C CATGAAGCCACCACAGCAGCCTTTGTT SEQ ID NO:268 T72C CTGGAAAGCCGTGCACATGAAGCCACC SEQ ID N0:269 E11SC GCCATAAGGAAGGCAAGTGTACTTTGA SEQ ID NO:Z7O
R141C GAAAGAAGGATGACACGCATCTACACC SEQ ID NO:271 F146C ACTTCTGTAGAGGCAGAAAGAAGGATG SEQ ID NO:272 F211C TGGGTAAGAGGGACAGCTAGCAATCCC SEQID NO:273 Scrub mutations of the cysteines were also made using the following oligonucleotides.
C12A CACTTCAGTAACAGCCCCTTTCTCTCTC SEQ ID NO:274 C12Y CACTTCAGTAACATACCCTTTCTCTCTC SEQ ID NO:275 C25S CACTGAAAGCCCAGGAAGCACCACAAGA SEQ ID N0:27G
C110A CAGTGTACTTTGAAGCTGTGGCAGCACG SEQ ID NO:277 Expression of Cats Mutant Proteins All Cats-FBHT plasmids were site-specifically transposed into the baculovirus shuttle vector (bacmid) by transforming the plasmids into DHlObac (Gibco/BRL) competent cells as follows: I
~,1 DNA at 5 ng/pl, lOpl Sx KCM [0.5 M KCI, 0.15 M CaClz, 0.25 M MgCl2], 30 p1 water was mixed with 50 p,1 PEG-DMSO competent cells, incubated at 4 C for 20 minutes, 25 C for 10 minutes, added 900 p.1 SOC and incubated at 37 C with shaking for 4 hours, then plated onto LB/agar plates containing 50 ~.glml kanamycin, 7 ~.glml gentamycin, 10 pg/ml tetracycline, 100 ~,g/rnl Bluo-gal, 10 ~g/ml IPTG. After incubation at 37 C for 24 hours, large white colonies were picked and grown in 3 ml 2YT media overnight. Cells were then isolated and double-stranded DNA was extracted from the cells as follows: pellet was resuspended in 250 ~l of Solution 1 [15 mM Tris-HCl (pH 8.0), 10 mM EDTA, 100 ~,g/ml RNase A]. Added 250 ~,l of Solution 2 [0.2 N
NaOH, 1% SDS] mixed gently and incubated at room temperature for 5 minutes.
Added 250 p,1 3 M potassium acetate, mixed and placed on ice for 10 minutes. Centrifuged 10 minutes at 14,OOOx g and transferred supernatant to a tube containing 0.8 ml isopropanol. Mix and place on ice for 10 minutes. Centrifuge 15 minutes at 14,OOOx g, wash with 70% ethanol, air dry pellet and resuspended DNA in 40 ~l TE.

The bacmid DNA was used to transfect Sf9 cells. Sf9 cells were seeded at 9 x lOs cells per 3S mrn well in 2 ml of Sf 900 II SFM medium containing O.Sx concentration of antibiotic-antimycotic and allowed to attach at 27 C for 1 hour. During this time, 5 ~,l of bacmid DNA
was diluted into 100 ~1 of medium without antibiotics, 6 ~l of CeIIFECTIN reagent was diluted into 100 ~l of medium without antibiotics and then the 2 solutions were mixed gently and allowed to incubate for 30 minutes at room temperature. The cells were washed once with medium without antibiotics, the medium was aspirated and then 0.8 ml of medium was added to the lipid-DNA
complex and overlaid onto the cells. The cells were incubated for 5 hours at 27 C, the transfection medium was removed and 2 ml of medium with antibiotics was added. The cells were incubated for 72 hours at 27 C and the virus was harvested from the cell culture medium.
The virus was amplified by adding 1.0 ml of virus to a 50 ml culture of Sf~
cells at 2 x 10~ cells/ml and incubating at 27 C for 72 hours. The virus was harvested from the cell culture medium and this stock was used to express the various cats constructs in High-Five cells.
A 1 L culture of High-Five cells at 2 x 10~ cells/ml was infected with virus at an approximate MOI of 2 and incubated for 72 hours. Cells were pelleted by centrifugation and the supernatant was dialyzed against 20 L Load buffer (50 mM NaHZP04, pH 8.0, 300 mM NaCI, 10 mM
imidazole), filtered and loaded onto a Ni-NTA (Superflow Ni-NTA, Qiagen) column at 1 ml/min, washed with Load buffer at 2 ml/min and eluted with 50 mM NaH2P04, pH 8.0, 300 mM NaCI, 250 mM
imidazole.

Caspase-1 (accession number SWS P25774), like other caspases exists as an inactive proform, and is proteolytically processed into a large subunit and a small subunit, which then combine to form the active enzyme. An important substrate of caspase-1 is the proform of interleukin-1 (beta).
Caspase-1 produces the active form of this cytokine, which plays a role in processes such as inflammation, septic shock and wound healing. Additionally, active capase-1 induces apoptosis, and plays a role in the progression Huntington's disease. The structure of caspase-1 has been solved [1BMQ, Okamoto, Y., et al., Chern Phar~rn Bull (Tolcyo), 47:11-21 (1999)].

IL-13 (accession number SWS P35225), which is produced mainly by activated Th2 cells, shows structural and functional similarities to IL-4. Like IL-4, it increases the secretion of immunoglobulin E by B cells and is involved in the expulsion of parasites. In addition, IL-13 downregulates the production of cytokines including IL-lb, IL-6, TNF-alpha and IL-8 by stimulated monocytes. IL-13 also prolongs monocyte survival, increases the expression of MHC
class II and CD23 on the surface of monocytes, and increases expression of CD23 on B cells.

Furthermore, IL-2 and IL-13 synergize in the regulation interferon-gamma synthesis. Due to these effects, IL-13 plays a role in conditions such as allergy and asthma. In particular, a polymorphism at position 130 (Q) increases the risk of asthma development. The structure of IL-13 has been solved by nuclear magnetic resonance (NMR) [1GA3, Eissenmesser, E. Z. et al., J. Mol. Biol. 310:
231-241 (2001)].

CD40L (accession number SWS P29965) is a protein that is found in two forms, a transmembrane form and also an active, proteolytically processed, extracellular soluble form. The transmembrane form is expressed on the surface of CD4+ T lymphocytes. Like other members of the TNF family, it is forms a homotrimer. CD40L mediates the proliferation of B cells, epithelial cells, fibroblasts, and smooth muscle cells. Binding of CD40L to the CD40 receptor on T cells provides a critical signal for isotype class switching and production of immunoglobulin antibodies. Defects in CD40L
lead to an elevation in IgM levels, and an deficiency in all other immunoglobulin subtypes.
Inhibitors to CD40L would find use in the treatment of autoimmune disease and graft rejection. In addition, reduced interaction between CD40L and its receptor reduces the degree of tau hyperphosphorylation in a mouse model of Alzheimer's disease. The crystal structure of CD40L
has been solved [lALY, Karpusas, M., et al., Structure 3:1031-1039(1995), erratum in Str~ueture 3:1046 (1995)].
HUMAN B-CELL ACTIVATING FACTOR (BAFFI
A member of the TNF superfamily, BAFF (accession number SWS Q9Y275) is a homotrimer and found in both transmembrane and soluble forms. The transmembrane form is processed by the furin family of proprotein convertases. BAFF is upregulated by interferon-gamma and downregulated by PMA/ionomycin treatment. BAFF binds to three different receptors. When it binds to the B-cell specific receptor (BAFFR), it promotes survival of B-cells and the B-cell response. Furthermore, both BAFF and a proliferation-inducing ligand (APRIL) bind to the receptors transmembranc activator and CAML interactor (TACT) and B cell maturation antigen (BCMA), forming a 2 ligands-2 receptors pathway that is responsible for stimulation of T-cell and B-cell function and humoral immunity. Inhibitors of BAFF would serve as therapeutics for autoimmune diseases characterized by abnormal B-cell activity, such as systemic lupus erythematosis (SLE) and rheumatoid arthritis (RA). A structure of the soluble protein is available [1JH5, Liu, Y., et al., Cell, 108: 383-394 (2002)].

P53 (accession number SWS P04637), a transcription factor that can suppress tumor growth, binds DNA as a homotetramer and is activated by phosphorylation of a serine residue.
There are two mechanisms of tumor suppression, depending upon the cell type: induction of growth arrest and activation of apoptosis. PS3 controls cell growth by regulating expression of a set of genes; for example, it increases the transcription of an inhibitor of cyclin-dependent kinases. Apoptosis results from the p53-mediated stimulation of Bax or Fas expression, or the decrease in Bcl2 expression. P53 is mutated or inactivated in about 60% of known cancers, and is also often overexpressed in a variety of tumor tissues. Reversible inhibitors of pS3 could be used as an adjunct to conventional radio- and chemotherapy to prevent damage to nornlal tissues during treatment and its severe side effects. Such an inhibitor was shown to protect mice from lethal doses of radiation without the promotion of tumor formation. There is a crystal structure of human p53 bound to Xerzopus laevis mdm2 protein [lYCQ, Kussie, P. H., et al., Scie~zce 274: 948-9S3 (1996)].

In response to DNA damage, p53 increases the transcription of the protein mdm2 (accession number SWS Q00987). In a form of negative feedback, mdm2 inhibits pS3-induced cell cycle 1 S arrest and apoptosis by two means. Firstly, mdm2 binds the transcriptional activation domain of pS3, reducing its transcriptional activation activity. Secondly, in the presence of ubiquitin E1 and E2, mdm2 serves as an ubiquitin protein ligase E3 for both itself and pS3. The ubiquitination of pS3 allows its export from the nucleus to the proteasome, where it is destroyed. There are eight isoforms of mdm2 that are produced by alternative splicing. They are mdm2, mdm2-A, mdm2-Al, mdm2-B, mdm2-C, mdm2-D, mdm2-E, and mdm2-alpha. Of these, mdm2-A, mdm2-B, mdm2-C, mdm2-D, and mdm2-E are observed in human cancers but not in normal tissues.
Mdm2 amplification has also been observed in certain tumor types, including soft tissue sarcoma, osteosarcoma, and glioblastoma. These tumors often contain wild type pS3.
Small molecule inhibitors of mdm2 could promote the proapoptotic activity of the wild type pS3 and find use in cancer therapy. The structure of ~e~zopus laevis mdm2 in complex with human pS3 has been solved [IYCR, Kussie, P. H. et al., Science 274: 948-9S3 (1996)].
BB CL-XX
BcI-x (accession number SWS Q07817) is a member of the Bcl2 family of proteins and has two major isoforms produced by alternative splicing, bcl-x(L), bcl-x(S). The Iong isoform, bcl-x(L) is found in long-lived postmitotic cells and inhibits apoptosis, whereas the short isoform, bcl-x(S), is found in cells with a high turnover rate and promotes apoptosis. The long isoform inhibits apoptosis by binding to voltage-dependent anion channel (VDAC) and preventing the release of apopotosis activator cytochrome c from fihe mitochondrial membrane. This antiapoptotic activity is 3S dependent upon the BH4 (bcI-2 homology) domain of Bcl-x(L); binding of this protein to other Bcl2 family members is dependent upon the BH1 and BH2 domains. Expression of Bcl-x(L) has been observed to be expressed primarily by the neoplastic cells in a majority of lymphoma cases.

Inhibition of bcl-x(L) expression in several cell lines resulted in apoptosis.
Thus, due to its antiapoptotic effects, bcl-x(L) is a target for cancer therapeutics.
Interestingly, binding of Bcl-x(L) to another Bcl2 family member, the proapoptotic protein Bax, results in an increase in apoptosis (see below). A crystal structure of Bcl-x(L) has been solved [1MAZ, Muchmore, S. W., et al.
Nature 381: 335=341 (1996)].
BAX
Bax [accession number SWS Q07812 (BAX alpha); SWS Q07814 (BAX beta); SWS

(BAX gamma); SWS P55269 (BAX delta)] promotes apoptosis by binding to the antiapoptotic protein bcl-x(L), inducing the release of cytochrome c, and activating caspase-3. Bax has several isoforms produced by alternative splicing; some are membrane bound and others axe cytoplasmic.
The BH3 domain of Bax is necessary for its binding to members of the anti-apoptotic Bcl2 family.
Defects in Bax are observed in some cell lineages from hematopoietic cancers.
Bax agonists could be used in cancer therapies, while Bax inhibitors could be used to counteract neuronal cell death resulting from ischemia, spinal cord injury, Parkinson's disease and Alzheimer's disease. An NMR
structure of BAX has been solved (1F16, Suzuki, M., et al., Cell 103:645-654 (2000)].

CDC25A (accession number SWS P30304) is a dual-specificity phosphatase also lrnown as M-phase inducer phosphatase 1 (MPIl). Induced by cyclin B, CDC25A is required for progression of the cell cycle, and induces mitosis in a dosage-dependent manner. CDC25 directly dephosphorylates CDC2, thereby decreasing its activity. It has also been demonstrated in vitro that CDC25 dephosphorylates CDK2 in complex with cyclinE. Elevated levels of CDC25 can trigger uncontrolled cell growth and are linked with increased mortality in breast cancer patients.
Activated CDC25A is also observed in degenerating neurons of the Alzheimer's diseased brain. A
structure of the catalytic core has been solved [1C25, Fauman, F. B., et al., Cell 93: 617-625 (1998)].

CD28 (accession number SWS P10747) is a disulfide-linked homodimeric transmembrane protein expressed on activated B-cells and a subset of T-cells. This protein can bind three others: B7-1, B7-2, and CTLA-4. The interaction of CD28 with B7-1 and B7-2 present on the surface of antigen presenting cells (APCs) results in a co-stimulation of naive T-cell activation, whereas subsequent interaction of the same B7-1 and B7-2 molecules with CTLA-4 leads to an attenuation of the T-cell stimulation. CD28-associated signaling pathways are important therapeutic targets for autoimmune disease, graft vs. host disease (GVHD), graft rejection, and promotion of immunity against tumors.
The structure of CD28 has not been solved to date.

There are 2 B7 proteins: B7-1 (accession number SWS P33681), also known as CD80, and B7-2 (accession number SWS P42081), also known as CD86. Both are highly glycosylated transmembrane proteins expressed on activated B-cells. ~ Early events in immune response are controlled by the interactions of these molecules with CD28 and CTLA-4 (see above). Thus B7-1 and B7-2 make significant targets for therapeutics treating autoimmune disease. A structure of the soluble form of B7-1 has been solved [1DR9, Ikemizu, S., et al., Immunity 12:
51-60 (2000)] in addition to a structure of B7-1 in complex with CTLA-4 [1I8L, Stamper, C. C., et al., Nature 410:
608-611 (2001)]. In addition, a structure of B7-2 in complex with CTLA-4 has been solved [1I85, Schwartz, J.-C. D., et al., Nature 410: 604-608 (2001)].
The immune system comprises in part the complement cascade, which is a set of more than 20 proteins. CSa is one of these complement proteins; it is a cytolcine-like activation product of C5.
CSa effects inflammation, and specifically has a role in the recruitment of neutrophils in response to bacterial infection. In sepsis, the life threatening spread of bacterial toxins through the blood, the effects of CSa are exhausted, due to an overexposure of the neutrophils to excessive amounts of this complement protein. Furthermore, expression levels of CSa receptor (accession number SWS
P21730) are increased in certain vital organs during sepsis. Thus inhibitors of CSa or the CSa receptor could help in treating sepsis. Inhibitors of CSa could also be used in the treatment of bullous pemphigoid, the most common autoimmune blistering disease. Another effect of CSa is its synergy with the Abeta peptide to promote secretion of IL-1 and IL-6 in human macrophage-like THP-1 cells; CSa may therefore be involved in the pathogenesis of Alzheimer's disease. Although the structure of CSa has been solved by NMR [1KJS, Zhang, X, et al., Proteins 28: 261-267 (1997)], there is no structure of the CSa receptor to date.
AKT
Akt is an important component of the signaling pathway of growth factor receptors. There are three highly related Akt genes, Akt 1-3 (accession numbers SWS P31749, Aktl; SWS
P31751, Akt2;
SWS Q9Y243), which show compensatory effects for one another. However, they have different expression patterns, suggesting that each may have unique functions as well.
Each Alct is activated by phosphorylation of multiple residues and is activated by the kinase ILK.
Binding of activated Akt to PI3K (phosphatidyl inositol 3-Icinase) causes the translocation of the active Akt to the plasma membrane. Akt has pleiotropic effects leading to cell survival.
Additionally, Akt amplification and elevated levels of Akt have been found in some types of cancers. A crystal structure of the kinase domain of Akt2, also known as PKB-(3, has recently been obtained [Yang, J., et al., Molecular Fell 9: 1227-1240 (2002)]."

CD45 (accession number SWS P08575) is a receptor protein tyrosine phosphatase that is primarily Located in the plasma membrane of leukocytes; it has several isoforms differing in the extracellular domain, the significance of which is presently unknown. Substrates for CD45 include the kinases lyc, fyn, and other src kinases. Additionally, CD45 engages in noncovalent interactions with the lymphocyte phosphatase associated protein (LPAP). CD45 is critical for activation through the antigen receptor on T cells and B cells, and may also be important for the antigen-mediated activation in other leukocytes. Dimerization of CD45 disables its function.
Inhibitors of CD45 could be used to prevent allograft rejection. There is no structure of CD45 to date.

HER-2 (accession number SWS P04626), otherwise lrnown as ErbB2 is a receptor tyrosine kinase that is related to EGFR (ErbB 1). Although there are no known ligands for HER-2 in isolation, when HER-2 dimerizes with other members of the ErbB family, i.e., ErbBl, ErbB3 and ErbB4, the dimeric complex can bind to a number of ligands. These Iigands include heregulins, EGF, betacellulin, and NRG, although binding depends upon which ErbB proteins are in the heterodimer.
Ligand binding increases the phosphorylation of HER-2, and effects subsequent intracellular signaling steps. HER-2 is frequently overexpressed in breast cancer cells, and this overexpression may mediate their proliferation. Breast cancer cells overexpressing HER-2 are also more responsive to HER-2 inhibitors. HER-2 is also implicated in a number of other cancers, such as ovarian, prostate, lung, fallopian tube, osteosarcoma, and childhood medulloblastoma. The structure of this receptor has not yet been solved.
HUMAN GLYCOGEN SYNTHASE K1NASE-3 fGSK-3~
GSK-3 (accession numbers SWS P49840, GSK-3a; SWS P49841, GSK-3~3) is involved in the hormonal control of Myb, glycogen synthase, and c jun. The phosphorylation of c jun by GSK-3 decreases the affinity of c-jun for DNA. . Additionally, GSK-3 is phosphorylated by ILK-1 and Akt-1. Phosphorylation by Aktl causes the inhibition of catalytic activity of GSK-3, which normally phosphorylates cyclin D, thereby targeting cyclin D for destruction.
The net effect of this phosphorylation of GSK-3 is the promotion of cell survival. Increased GSK-3 activity has been found in tissue from diabetic patients, consistent with its role in the development of insulin resistance. Furthermore, GSK-3~ is overexpressed in the Alzheimer's disease brain, and this overexpression is associated with tau protein hyperphosphorylation, a hallmark of the disease.
Finally, the effects of some mood-stabilizing drugs such as lithium appear to be mediated by inhibition of GSK. Therefore it is possible that GSK-3 inhibitors would increase the effectiveness of some psychoactive drugs. There is a structure available for GSK-3(3 [IH$F, Dajani,. R., et al., Cell 105: 721-732 (2001)].

The protein complex alpha-E/beta-7 is a transrnembrane integrin that plays a role in lymphocyte migration and homing. Specifically, the complex serves as a receptor for E-cadherin. Alpha-E
(accession number SWS P38570) is made up of two subunits, a and a, the oe-subunit itself is composed of a light chain and a heavy chain linked by a disulfide bond.
Likewise, beta-7 (accession number SWS P26010) is also composed of a- and (I- subunits. The alpha-E/beta-7 complex normally mediates the adhesion of infra-epithelial T lymphocytes to mucosal epithelial cell layers; it also plays a role in the dissemination of non-Hodgkin's lymphoma.
Furthermore, a possible mechanism of inflammation involves migration of lymphocytes from the gut epithelium to other parts of the body. Changes in alpha-E/beta-7 levels have been observed in a variety of diseases. Elevated levels of this integrin have been observed in patients with Systemic Lupus Erythematosus (SLE), in the lung epithelium of patients with interstitial lung disease, and in the sinovial fluid of patients with rheumatoid arthritis. Altered patterns of alpha-E/beta-7 expression have been observed in patients with Crohn's disease, and antibodies to this complex were shown to prevent immunization-induced colitis in a mouse model. Hence, inhibitors to this complex would be valuable in the treatment of inflammation, especially mucosal inflammation.
'There are no structures available for alpha-E or beta-7.
TISSUE FACTOR
Human tissue factor (accession number SWS P13726), also lrnown as thromboplastin, is an integral transmembrane protein that is normally located at the extravascular cell surface. Upon injury to the skin, tissue factor is exposed to blood and complexes with the active form of coagulation enzyme Factor VII, known as Factor VIIA (see below). Tissue factor can bind both the inactive and active forms of coagulation Factor VII, and is an obligate cofactor for Factor VIIA
in triggering the coagulation cascade. Furthermore, since Tissue Factor plays a major role in thrombosis, inhibition of this factor would be expected to decrease the risk for clinical outcomes of tlu-ombosis such as atherosclerosis, arterial occlusion, stroke, and myocardial infarction. A
structure of the extracellular domain of tissue factor has been solved [2HFT, Muller, Y. A., et al., J Mol Biol 256:144-159 (1996)].
FACTOR VII
Factor VII (accession number SWS P08709) is the zymogen (inactive precursor) form of the serine protease coagulation Factor VIIa. More than 99% of this protease circulates in the inactive single-chain form; upon cleavage of an Arg-Ile peptide bond by one of several factors, the active two-chain form is produced. This two-chain form comprises a heavy chain and a light chain, linked by a disulfide bond. Enzymatic carboxylation of Glu residues in Factor VII, which is dependent upon vitamin K, allows the protein to bind calcium. In the presence of calcium and the cofactor human tissue factor (see above), Factor VIIa cleaves Factor X and Factor IX to produce their respective active forms, which propagate the coagulation cascade. Defects in Factor VII
can lead to bleeding disorders, where recombinant Factor VIIa finds use as a treatment. Conversely, some polymorphisms of the Factor VII gene have been associated with an increased risk for myocardial infarction, which is often caused by blood clots. Factor VII inhibitors are expected to find use in preventing heart disease. A structure of the zymogen form of factor VII in complex with an inhibitory peptide has been solved [1JBU, Eigenbrot, C., et al., Structure 9:627-636 (2001)].

Claims (22)

What is claimed is:

1. A method comprising:
a) obtaining a set of coordinates of a three dimensional structure of a protein TBM
having n number of residues;
b) selecting a candidate residue i on the three dimensional structure of the TBM
wherein the candidate residue i is the ith residue where i is a number between 1 and n and residue i is not a cysteine;
c) selecting a residue j where residue j is adjacent to residue i in sequence;
d) determining a candidate reference value wherein the candidate reference value is a spatial relationship between residue i and residue j;
e) obtaining a database comprising sets of coordinates of disulfide-containing protein fragments wherein each fragment comprises at least a disulfide-bonded cysteine and a first adjacent residue where the disulfide-bonded cysteine and the first adjacent residue share the same sequential relationship as residue i and residue j;
f) determining a comparative reference value for each fragment wherein the comparative reference value is the corresponding spatial relationship between the disulfide-bonded cysteine and the first adjacent residue as the candidate reference value is between residue i and j;
and, g) determining a score wherein the score is a measure of the number of fragments in the database that possess a comparative reference value that is the same or similar to the candidate reference value.

2. The method of claim 1 further comprising:
selecting a residue k where residue k is adjacent to residue i in sequence and k is not j; and wherein the candidate reference value is a spatial relationship between residue i, residue j, and residue k;
each fragment comprises at least a disulfide-bonded cysteine, a first adjacent residue, and a second adjacent residue where the disulfide-bonded cysteine and the first and second adjacent residues share the same sequential relationship as residue i, residue j, and residue k; and the comparative reference value is the corresponding spatial relationship between the disulfide bonded cysteine, the first adjacent residue, and the second adjacent residue as the candidate reference value is between residue i, residue j, and residue k.

3. A method comprising:

a) obtaining a set of coordinates of a three dimensional structure of a protein TBM
having n number of residues;
b) selecting a candidate residue i on the three dimensional structure of the TBM
wherein the candidate residue i is the ith residue where i is a number between 1 and n and residue i is not a cysteine;
c) selecting residue j and residue k wherein residue j and residue k are both adjacent in sequence to residue i;
d) determining a candidate reference value wherein the candidate reference value is a spatial relationship of at least one backbone atom from each of residue i, residue j, and residue k;
e) obtaining a database comprising sets of coordinates of disulfide-containing protein fragments wherein each fragment comprises at least a disulfide-bonded cysteine, a first adjacent residue, and a second adjacent residue where the disulfide-bonded cysteine, the first adjacent residue, and the second adjacent residue share the same sequential relationship as residue i, residue j, and residue k;
f) determining a comparative reference value for each fragment wherein the comparative reference value is the corresponding spatial relationship between the disulfide-bonded cysteine, the first adjacent residue, and the second adjacent residue as the candidate reference value is between residue i, residue j, and residue k; and, g) determining a score wherein the score is a measure of the number of fragments in the database that possess a comparative reference value that is the same or similar to the candidate reference value.

4. The method of any one of claims 1-3 wherein the spatial relationship comprises a dihedral angle.

5. The method of any one of claims 1-3 wherein the spatial relationship comprises a pair of phi psi angles.

6. The method of any one of claims 1-3 wherein the spatial relationship comprises a plurality of distances between atoms of two residues.

7. The method of any one of claims 1-3 wherein residue i is at least partially surface accessible.

8. The method of claim 7 wherein residue i has an accessible surface area of at least about 20 .ANG.2

9. The method of any one of claims 1-3 wherein residue i does not participate in a hydrogen bond interaction with a backbone atom of the TBM.

A method comprising:
a) obtaining a three dimensional stricture of a TBM having n number of residues and a site of interest;
b) selecting a candidate residue i that is at or near the site of interest wherein the candidate residue i is the ith residue where i is a number between 1 and n and residue i is not a cysteine;
c) generating a set of mutated TBM structures wherein each mutated TBM
structure possesses a cysteine residue instead of residue i and wherein the cysteine residue is placed in a standard rotamer conformation; and, d) evaluating the set of mutated TBM structures.

11. The method of claim 10 wherein the cysteine residue is capped with a S-methyl group.

12. The method of claim 10 wherein the standard rotamer conformation for cysteine comprises:
a chil angle selected from the group consisting of about 60°, about 180°, and about 300°;
and a chi2 angle selected from the group consisting of about 60°, about 120°, about 180°, about 270°, and about 300°.

13. The method of claim 10 wherein evaluation step comprises determining whether each rotamer conformation makes an unfavorable steric contact with the TBM.

14. The method of claim 10 wherein the evaluation step comprises a force field calculation.

15. The method of claim 11 wherein the evaluation step comprises determining whether each rotamer conformation places the methyl carbon of the S-methyl group closer to the site of interest than the C .beta..

16. A set of variant proteins, said proteins each being a mutated version of a TBM wherein a naturally occurring non-cysteine residue of the TBM is mutated into a cysteine.

17. The set of claim 16 comprising at least 3 cysteine mutants.

18. The set of claim 16 wherein one or more naturally occurring cysteines of the TBM is mutated to a non-cysteine residue.

19. The set of claim 16 wherein the TBM is a cell surface or soluble receptor.

20. The set of claim 16 wherein the TBM is a cytokine.

21. The set of claim 16 wherein the TBM is an enzyme.

22. The set of claim 16 wherein the TBM is selected from the group consisting of IL-2; IL-4;
TNF-.alpha.; IL-1 receptor; caspase-3; PTP-1B; HIV integrase; BACE1; MEK-1;
Cat-S; caspase-1; IL-13; CD40L; BAFF; P53; mdm2; bcl-x; bax; CDC25A; CD28; B7; C5A; AKT; CD45;
HER2; GSK-3; alpha-E/beta-7; tissue factor; and Factor VII.