Proc. Natl. Acad. Sci. USAVol. 91, pp. 12178-12182, December 1994Biochemistry

Three-dimensional structure of the platelet integrin recognitionsegment of the fibrinogen y chain obtained by carrierprotein-driven crystallization

(lysozyme/chimeric proteins/crystallography/celB adhesion/thrombosis)

JOHN P. DONAHUE*, HARESHKUMAR PATELtt, WAYNE F. ANDERSONt, AND JACEK HAWIGER*§Departments of *Microbiology and Immunology and of tBiochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232

Communicated by Russell F. Doolittle, August 1, 1994 (received for review May 4, 1994)

ABSTRACT We have developed a method for crystallizingsmall functional protein segments so that their three-dimensional structure can be determined by x-ray diffractionanalysis. This method consists of linking a small proteinsegment of unknown tertiary structure to either the amino orcarboxyl terminus of a larger carrier protein of known tertiarystructure. Crystallization of the small segment is then driven bycrystallization of the carrier protein. Using this approach, wehave obtained crystals of the human fibrinogen -chain car-boxyl-terminal segment linked to the carboxyl terminus ofchicken egg white lysozyme. The three-dimensional structureof the carboxyl-terminal segment of the fibrinogen y chain wasdetermined by x-ray diffraction analysis at a resolution of 2.4A. This segment encompasses the recognition site for theintegrin ftIbP3 receptor on activated platelets and for theclumping receptor on pathogenic staphylococci and also bearsdonor and acceptor sites for factor XIla-catalyzed crosslinkingof fibrin. Therefore, the structural information derived fromour analysis will provide a rational basis for the design ofinhibitors of these important functions of fibrinogen. More-over, carrier protein-driven crystallization will facilitate thedetermination of the three-dimensional structure of functionalsegments of other proteins that are, like fibrinogen, difficult tocrystallize in toto.

Human fibrinogen (Mr 340,000), a clottable protein in plasmaand the most abundant ligand for the integrin am,433 (glyco-proteins gpIIb/IIIa) receptor on platelets, is composed ofpairs of three nonidentical polypeptide chains (a, f3, and y)that are extensively linked by disulfide bonds to form anelongated dimeric structure (for review, see ref. 1). Thebinding of fibrinogen to the ab,833 integrin receptor on acti-vated platelets results in platelet aggregation in vitro and theformation of platelet-fibrin thrombi in vivo (for review, seeref. 2). The segment of fibrinogen responsible for binding tothe platelet aIqb,33 integrin and aggregation of activated plate-lets has been mapped to the carboxyl terminus of the y chainand pinpointed to the continuous 12-amino acid sequenceencompassing residues 400-411. This segment is both nec-essary and sufficient for optimal reactivity with platelet auqbj3(3-6). With regard to this, it is noteworthy that mutation ofboth Arg-Gly-Asp cell adhesion motifs in the a chain ofrecombinant fibrinogen does not affect the ability of thismolecule to mediate the aggregation of activated platelets (6).The fibrinogen y-chain segment y-(397-411) also serves asthe ligand for the clumping receptor on pathogenic staphy-lococci (7) and bears donor and acceptor sites for factorXIIIa-catalyzed crosslinking of fibrin (8).Two-dimensional NMR analysis of free fibrinogen -y-(400-

411) peptide in solution indicates the presence of a type II

P-turn spanning residues Gln-407 to Asp-410 (9). However,x-ray diffraction analysis of crystals derived from proteolyt-ically cleaved fibrinogen did not provide information aboutthe three-dimensional structure of this biologically importantsegment (10). We have developed a method for crystallizingsmall functional protein segments so that their three-dimensional structure can be solved by x-ray diffractionanalysis. Using this approach, we have obtained crystals ofthe fibrinogen -y-chain carboxyl-terminal segment that waslinked to the carboxyl terminus of chicken egg white (CEW)lysozyme.

MATERIALS AND METHODSConstruction of the Met-Lysozyme-Fibrinogen y(398-411)

Expression Plasmid. We constructed an expression plasmid inwhich a DNA sequence encoding the 14 carboxyl-terminalresidues of the fibrinogen y chain was inserted downstreamof the cDNA for CEW lysozyme extended at the aminoterminus by a methionine residue. Lysozyme cDNA, derivedfrom plasmid pls-1 (11) (provided by Gunther Schutz, Max-Planck-Institut fur Molekulare Genetik), was cloned into theprokaryotic expression vector pKP1500 (12) (provided byTakeyoshi Miki, Kyushu University) essentially as described(12, 13). To facilitate the insertion of a DNA fragmentencoding the carboxyl-terminal amino acid residues of thefibrinogen y chain, a Pst I site was inserted at the 3' end ofthe lysozyme coding sequence by oligonucleotide-directedmutagenesis with M13 vectors (14). This insertion resulted inthe addition of a glutamine to the carboxyl terminus oflysozyme. This residue was equivalent to Gln-398 of thefibrinogen y chain (15) in the final construct. The 575-bpEcoRI/HindIII fragment from M13mpll that contained themodified lysozyme cDNA was ligated into pKP1500. Theresulting plasmid was named pNED6.Based on the fibrinogen y-chain cDNA sequence (15), the

following complementary oligonucleotides were synthesized.G CAA CAC CAC CTA GGG GGA GCC AAA CAG GCT GGA GAC GTT TA

AC GTC GTT GTG OTG GAT CCC CCT CGG TTT GTC CGA CCT CTG CAA ATT CGA

GIn Gln His His Leu Gly Gly Ala Lys Gln Ala Gly Asp Val ***

After hybridization, the resulting double-stranded oligo-nucleotide contained a translation-termination codon (aster-isks) immediately following Val-411 and Pst I and HindIIIsticky ends at 5' and 3' termini. This DNA fragment wasligated into Pst I/HindIII-digested pNED6 to form plasmid

Abbreviation: CEW, chicken egg white.tPresent address: Department of Biological Sciences, Purdue Uni-versity, West Lafayette, IN 47907.§To whom reprint requests should be addressed at: Department ofMicrobiology and Immunology, Vanderbilt University School ofMedicine, A5321, Medical Center North, Nashville, TN 37232.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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pNED7. The DNA sequence of the lysozyme cDNA with theinserted t-chain oligonucleotide was confirmed by the dide-oxy chain-termination method (16).Met-Lysozyme-Fibrinogen y(398-411) Expression and Pu-

rification. Escherichia coli strain KP3998 (12) containingplasmid pNED7 was grown at 400C in TYG broth (1%tryptone/0.5% yeast extract/0.25% glycerol/1 mM MgSO4/0.1 M potassium phosphate, pH 7) containing 100 /lg ofampicillin per ml. Chimeric protein synthesis was induced byaddition of isopropyl (-D-thiogalactoside (0.5 mM final con-centration). Met-lysozyme-fibrinogen v-(398-411) was puri-fied from cell pellets as described (13) except that an ultra-filtration step was introduced after the third acetic acidextraction of the reduced protein. The usual yield of Met-lysozyme-fibrinogen v-(398-411) was 1.5 mg/g wet weight ofcells, with an estimated purity of at least 98% as determinedby Coomassie blue staining of these protein preparations inSDS/polyacrylamide gels.Met-Lysozyme-Fibrinogen v(398-411) Renaturation. Puri-

fied, reduced protein was renatured as described (13). Theprogress of the renaturation reaction was monitored bymeasuring the reconstitution of lysozyme enzymatic activityby using a Micrococcus lysodeikticus lysis assay performedas described by Sigma for CEW lysozyme. Maximal enzy-matic activity was recovered after 1 hr of incubation. Therenaturation reaction mixture was dialyzed against 0.1 Macetic acid and lyophilized. The residue was dissolved in 1 mlof 0.1 M acetic acid and chromatographed on a 1.5 x 47 cmcolumn of Sephadex G-25 equilibrated and run with 0.1 Macetic acid. The Met-lysozyme-fibrinogen -(398-411) thatwas contained in the excluded volume was lyophilized andthen dissolved in 50 mM NaCl. The protein concentrationwas determined by using an A280 for a 1% solution of nativeCEW lysozyme of 26.3 (17).Measmrement of Bing of Met-Lysozyme-Fibrinogen-(398-411) to Platelet Integrin amb43. Purified platelet fi-

brinogen receptor (integrin aub3, 10 Ag/ml) (provided byDavid Phillips ofCOR Therapeutics) in coating buffer (20mMTris-HCl, pH 7.4/150 mM NaCl/1 mM CaCl2/0.05% NaN3)was applied to the wells of Immulon 2 (Dynatech) microtiterplates. The plates were incubated at room temperature for 18hr and then blocked with gelatin (20 pg/ml in coating buffer).Protein-coated wells were washed with TBSCT (20 mMTris-HCl, pH 7.6/137 mM NaCl/0.5 mM CaCl2/0.05%Tween 20), and Met-lysozyme-fibrinogen v-(398-411) orCEW lysozyme were added. Plates were incubated for 2 hrat room temperature and washed with TBSCT. Chimericprotein or CEW lysozyme binding was detected with mono-clonal anti-CEW lysozyme HyHel-5 (=1.5 pg/ml; providedby Sandra Smith-Gill, National Cancer Institute) and goatanti-mouse IgG conjugated to alkaline phosphatase (A-3688,Sigma). Secondary antibody binding was detected by usingp-nitrophenyl phosphate (1 mg/ml) dissolved in 1 M Tris HCl(pH 9.5).Cryllition, Data Coilection, and Structure Determina-

tion. The purified, renatured Met-lysozyme-fibrinogenv-(398-411) (5-10 mg/ml, pH 2.5) was crystallized at 22°C inhanging drops with 1.4 M (NH4)2SO4 as precipitant bufferedwith 0.1 M Tris HCl (pH 8). Any increase in the ionic strengthor pH of the chimeric protein preparation present in hangingdrops resulted in rapid protein precipitation and the forma-tion of extremely thin needle crystals. Although the hangingdrop was equilibrated against ammonium sulfate buffered by0.1 M Tris-HCl (pH 8), the exact pH of the drop when the onecrystal large enough for data collection initially grew was notdetermined.Data were collected on a singie crystal (1.5 x 0.05 x 0.05

mm) at room temperature by using a pair of San DiegoMultiwire Systems area detectors. The crystal space groupwas P212121, and the unit cell dimensions were a = 55.9 A,

b = 74.0 A, and c = 30.8 A. The 2.4-A diffraction data werecollected with an Rmer.e of 0.140 for 15,024 observations of4625 unique reflections.The structure was determined by using the molecular

replacement method (18) with CEW lysozyme coordinates(19) and molecular replacement routines in the X-PLOR (20)program system. An initial rotation search gave a peak of 5.4a that was used for Patterson correlation refinement, whichbrought the correlation coefficient to 0.204. The refinedrotation parameters were used in a translation search thatyielded a peak of 10 a with an R value of 0.378.

Coordinates for the lysozyme portion of the structure,determined by using molecular replacement, were subjectedto an initial rigid body refinement. After rigid body refine-ment, Powell energy minim zation converged at anR value of0.242. Further refinement was carried out with a slow-cooling-simulated annealing procedure (3000K to 300 K) (21)and B factor refinement, resulting in a final R value of 0.202.

RESULTSC a rz of Met-Lysozyme-Flbrlnogn y(39-411)

Expresed in E. coli. In an effort to determine the three-dimensional structure of the fibrinogen vchain receptorrecognition segment, we constructed a plasmid that directsthe synthesis in E. coli of a chimeric protein composed ofCEW lysozyme with a methionine residue added to the aminoterminus and fibrinogen v-chain residues 398-411 added tothe carboxyl terminus. Met-lysozyme-fibrinogen y-(398-411)was purified from cytoplasmic precipitates (12) in a fullyreduced, denatured form and subsequently was renatured bysulfhydryl-disulfide exchange in vitro. The specific activityof the renatured Met-lysozyme-fibrinogen y-(398-411) was30%o of renatured native CEW lysozyme. This result wasconsistent with a previous report of Met-lysozyme renatur-ation in vitro (13).

It was important to establish that the t-chain sequencepresent as a carboxyl-terminal extension ofCEW lysozymecould adopt a biologically active conformation as measuredby auP3 receptor binding. The low solubility ofthe chimericprotein at neutral pH prevented us from using an assay inwhich inhibition of mlI-labeled fibrinogen binding toaMA onactivated platelets was measured. However, using an ELISAsystem, we demonstrated that binding of Met-lysozyme-fibrinogen v-(398-411) to immobilized Aab was wl0-foldgreater than the binding of native CEW lysozyme (Fig. 1).This binding was dependent on the presence of the a£tbP3

0.600 -

0.500 -

0.400 -

q 0.300 -

0.200 -

0.100 -o.10n -

0 10 20 30 40 50 60 70 80Protein, Ag/ml

90

FIG. 1. Binding of the Met-lysozyme-fibrinogen y-(398-411) chi-meric protein to platelet integrin rlnbP3 determined by an ELISAsystem. The assay was done as described in text, and the average oftriplicate determinations is shown. *, Met-lysozyme-fibrinogen-(398-411) with aubp3; n, Met-lysozyme-fibrinogen -t(398-411)without acvbm3; O, CEW lysozyme with atnbP3.

Biochemistry: Donahue et al.

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receptor and on the concentration of Met-lysozyme-fibrinogen y-(398-411), reaching saturation between 40 and80 pug/ml of protein.

Determination ofthe Structure ofMet-Lysozyme-Fibrinogen-(398-411). The structure of the chimeric protein was de-

termined by molecular replacement methods (18) using thecoordinates from the tetragonal CEW lysozyme structure(19). Comparison of the packing of the chimeric protein withthat ofCEW lysozyme in the orthorhombic crystal form (22)revealed that the chimeric molecules had rotated so that thecarboxyl terminus entered a space between lysozyme mole-cules rather than abutting another molecule. The addition ofthe 14 carboxyl-terminal residues of the fibrinogen y chain tothe carboxyl terminus of the CEW lysozyme did not causeany significant changes in the structure of the lysozymeportion of the chimeric protein. The rms difference in thepolypeptide backbone coordinates between the tetragonallysozyme structure and the chimeric protein was 0.63 A. Theaverage B values for the lysozyme main-chain and side-chainatoms were 14.0 and 14.4, respectively; the average B valuesfor the fibrinogen y(398-411) segment (residues 131-144 ofthe chimeric protein) main-chain and side-chain atoms were34.7 and 32.1, respectively. The higher temperature factorsfor the fibrinogen y-chain segment of the chimeric moleculesuggest that it exhibits greater mobility than the lysozyme towhich it is attached. This is not surprising given the carboxyl-terminal location of this segment and its relatively highglycine content.

Several approaches were taken to verify the validity of therefined model of the fibrinogen -(398-411) segment. Thereal-space fit correlation coefficient (23) for the y(398-411)segment to the 21F0 - FCI electron-density map ranged from65% to 79%. The same calculation for the lysozyme portionof the structure gave values from 61% to 88%. This indicatesthat the fibrinogen y(398-411) segment does not have adramatically worse fit to the electron density than the lyso-zyme portion, which was used as the model to determine thecrystal structure.Cross validation ofthe structure using the reciprocal-space

"Free R" value (20, 24) was also used to determine that theaddition of the fibrinogen y(398-411) segment to the modeltruly improved the agreement with the observed data ratherthan just adding more parameters. A randomly chosen 10%1of the data were used for the calculation of the Free R value.For the lysozyme model itself, the Free R for residues 2-130was 0.370 and for the final structure, residues 1-144, it was0.328. To determine if most of the structure could be cor-rectly placed and residues 136-144 [ry(403-411)] incorrectlyplaced, a calculation of the Free R was done for a model witha correct placement of residues 2-135 and an incorrectplacement of residues 136-144. In this case the Free R was0.392, while the Free R for residues 1-135 by themselves was

0.359. These results indicate that addition of the fibrinogen'-t(398-411) segment (residues 131-144 of the chimeric pro-tein) to the model increased the agreement with the observeddiffraction data.A slow-cooling-simulated annealing omit map (F0 - FJ)

(25) was calculated by using a model with residues 134-144[-y(401-411) segment] omitted. This map and the final modelof the fibrinogen y-(398-411) segment are shown in Fig. 2.This again demonstrated that the model agrees with experi-mental data. One final check was that the stereochemistry ofthe model was acceptable. None of the fibrinogen y-chainresidues (131-144) fall within disallowed regions of a Rama-chandran plot (26, 27).

Structure of the Fibrinogen y(398-411) Receptor Recogni-tion Segment. The model of the carboxyl-terminal extensionof the chimeric protein was built into 2FO - Fc electron-density maps in two stages. The first map was calculated byusing phases derived from the positioned CEW lysozymemodel and allowed the addition of residues 131-134 [y(398-401)]. This extended model was used to calculate phases andan electron-density map like that shown in Fig. 2. This secondmap was good enough to place the rest of the carboxyl-terminal residues. The general conformation ofthe fibrinogeny(398-411) segment is a wide turn followed by an extendedregion and ending with a wide turn (Figs. 2 and 3). In each ofthese turns, the first two residues are analogous to the firsttwo residues of a 3-turn and the last two residues areanalogous to the last two residues of a (3-turn. However, inthese turns an additional residue separates the two halves,leaving them too far apart for hydrogen-bond interactions tooccur. For residues 131-135 [y(398-402)], which composethe first turn, the side chain of Gln-132 (ychain position 399)projects into the space between the two halves and makesfour hydrogen-bond contacts with the y(398-411) segmentmain chain (Fig. 4). Residues 139-143 [y(406-410)] make upthe second wide turn, with Ala-141 (-chain position 408)separating the two halves.Because the fibrinogen -(398-411) segment does not have

a hydrophobic core that an independently folding polypeptidesegment would have, it is reasonable to ask whether theconformation that is observed is determined solely by inter-actions with the lysozyme to which it is linked or, possibly,by crystal-packing interactions. The simplified representa-tion of the structure of the fibrinogen y-,chain segmentpresented in Fig. 4 shows that there are five main-chainhydrogen-bond interactions that occur within the -(398-41l1)segment and only two main-chain hydrogen-bond interac-tions that occur between the y(398-411) segment and thelysozyme molecule to which it is attached (labeled A in Fig.4). Also, there is a close interaction between the side chainof Asp-19 of lysozyme and the carbonyl group of Gln-140(ychain position 407) (also labeled A in Fig. 4). This would

..- -'I;'i-..V..W.-

I',<

i144

FIG. 2. Stereoview of the FO - F; electron density for residues 134-144 (y-chain positions 401-411) of the Met-lysozyme-fibrinogen->'(398-411) chimeric protein. Before phases were calculated, residues 134-144 were removed from the model, and a slow-cooling-simulatedannealing (3000 K to 300 K) refinement was performed using X-PLOR (20). The map is contoured at 1.6 o, and a final model is superimposed.

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FIG. 3. Stereoview ofa ribbon diagram ofthe Met-lysozyme-fibrinogen 'y-(398-411) chimeric protein. TheCEW lysozyme molecule is shownin green, and the carboxyl-terminal fibrinogen y-t(398-411) segment (residues 131-144 of the chimeric protein) is shown in magenta.

be an unfavorable interaction unless the carboxyl group ofAsp-19 were protonated. There are six hydrogen bond inter-actions between the y(398-411) segment and symmetry-related chimeric molecules in the crystal lattice. Four hydro-gen-bond interactions occur between symmetry-related'y.(398-411) segments (labeled B in Fig. 4), and two hydro-gen-bond interactions occur between the y-(398-411) seg-ment and symmetry-related lysozyme molecules (labeled Cand D in Fig. 4). However, the observed hydrogen bondinteractions that occur between the side chain of Gln-132(y

12182 Biochemistry: Donahue et al.

protein-driven crystllization, crystal-packing interactions aremuch weaker and do not entail such extensive interactionsurfaces. Consequently, there is less danger that the observedstructure is strongly affected by the process used.

Structure ofthe Fibrinogen Carboxyl-Terminal Segment. Asexpected, the lysozyme structure is relatively unaffected bythe addition ofthe fibrinogen y-chain segment to its carboxylterminus. However, the conformation of the fibrinogen-(398-411) segment could potentially be affected by inter-actions with the lysozyme to which it is covalently attached.There are five intramolecular hydrogen-bond interactionsthat occur within the (398-411) segment and only threehydrogen-bond interactions between the v(398-411) seg-ment and the lysozyme to which it is attached. Since there aremore hydrogen bonds between atoms within the fibrinogeny-(398-411) segment than there are with the carrier lysozymemolecule, it seems unlikely that the conformation of they(398-411) segment was strongly affected by the presence ofthe lysozyme.

Crystal-packing interactions could also potentially affect theconformation of the fibrinogen ychain segment. The -t-(398-411) segment does make six crystal-packing contacts. How-ever, three of the six crystal-packing contacts are made by theside chain of Gln-140 (-chain position 407). Because this is arelatively long and flexible side chain, it is unlikely that theseinteractions strongly influence the conformation ofthe ychainsegment itself. Nevertheless, the potential influence ofcrystal-packing interactions on the structure of the y-(398-411) seg-ment can only be assessed by comparison to the structure ofthis segment in the context of different crystal packing.The three-dimensional structure of the carboxyl-terminal

segment of the human fibrinogen y chain presented in thisstudy has a number of hitherto unrecognized features. First,it is organized into a turn that is distinct from the typical/3turn suggested by NMR studies of the y(400-411) peptidein solution (9) or postulated to encompass the Arg-Gly-Aspcell attachment site of fibronectin (30). Second, this structuredoes not seem to be stabilized by a salt bridge formedbetween the E-amino group of Lys-139 (y-chain position 406)and the carboxyl group of Asp-143 or Val-144 (y-chainpositions 410 and 411) as postulated previously (3). Theuniqueness of this structure is exemplified by its selectiveinteraction with platelet integrin aIjb33, whereas a multitudeof other integrin receptors remain unengaged by this ligand(2). The only other receptor that interacts with this segmentof the human fibrinogen y chain is the staphylococcal clump-ing factor (7, 31). The apposition of acceptor and donor sitesfor enzymatic crosslinking by factor XIIIa provides anotherstructural feature for biologic function of the carboxyl-terminal segment of the y chain characterized in this study.Clearly, the three-dimensional structure presented hereinoffers powerful information for the development of models offibrinogen-aubP3 and fibrinogen-factor XIIIa interactionsand for the design of new inhibitors of these importantfunctions of fibrinogen. In addition, our success in usingCEW lysozyme as a carrier protein to drive the crystalliza-tion of the carboxyl-terminal fibrinogen -chain segmentdemonstrated the utility of this approach for determining thethree-dimensional structure of functional segments of otherproteins that are, like fibrinogen, difficult to crystallize.

J.P.D. and H.P. contributed equally to the completion of thiswork. We thank Takeyoshi Miki for plasmid pKP1500 and E. coli

strain KP3998, Gunther Schuitz for the lysozyme cDNA clone,Sandra Smith-Gill for the HyHel-5 lysozyme monoclonal antibody,and David Phillips for the platelet integrin anbP33. This research wassupported by National Institutes of Health Grants ES05355,HL49554, and HL30647.

1. Doolittle, R. F. (1984) Annu. Rev. Biochem. 53, 195-229.2. Hawiger, J. (1994) in Hemostasis and Thrombosis: Basic Prin-

ciples and Clinical Practice, eds. Colman, R. W., Hirsh, J.,Marder, V. J. & Salzman, E. W. (Lippincott, Philadelphia), 3rdEd., pp. 762-7%.

3. Kloczewiak, M., Timmons, S., Lukas, T. J. & Hawiger, J.(1984) Biochemistry 23, 1767-1774.

4. Kloczewiak, M., Timmons, S., Bednarek, M. A., Sakon, M. &Hawiger, J. (1989) Biochemistry 28, 2915-2919.

5. Peerschke, E. I. B., Francis, C. W. & Marder, V. J. (1986)Blood 67, 385-390.

6. Farrell, D. H., Thiagarajan, P., Chung, D. W. & Davie, E. W.(1992) Proc. Natl. Acad. Sci. USA 89, 10729-10732.

7. Strong, D. D., Laudano, A. P., Hawiger, J. & Doolittle, R. F.(1982) Biochemistry 21, 1414-1420.

8. Chen, R. & Doolittle, R. F. (1971) Biochemistry 10, 4486-4491.9. Blumenstein, M., Matsueda, G. R., Timmons, S. & Hawiger,

J. (1992) Biochemistry 31, 10692-10698.10. Rao, S. P. S., Poojary, M. D., Elliott, B. W., Melanson,

L. A., Oriel, B. & Cohen, C. (1991) J. Mol. Biol. 222, 89-98.11. Jung, A., Sippel, A. E., Grez, M. & Schutz, G. (1980) Proc.

Natl. Acad. Sci. USA 77, 5759-5763.12. Miki, T., Yasukochi, T., Nagatani, H., Furuno, M., Orita, T.,

Yamada, H., Imoto, T. & Horiuchi, T. (1987) Protein Eng. 1,327-332.

13. Imoto, T., Yamada, H., Yasukochi, T., Yamada, E., Ito, Y.,Ueda, T., Nagatani, H., Miki, T. & Horiuchi, T. (1987) ProteinEng. 1, 333-338.

14. Zoller, M. & Smith, M. (1983) Methods Enzymol. 100, 468-500.15. Chung, D. W., Chan, W.-Y. & Davie, E. W. (1983) Biochem-

istry 22, 3250-3256.16. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natd.

Acad. Sci. USA 74, 5463-5467.17. Saxena, V. P. & Wetlaufer, D. B. (1970) Biochemistry 9, 5015-

5023.18. Rossmann, M. G. & Blow, D. M. (1962) Acta Crystallogr. 15,

24-31.19. Kundrot, C. E. & Richards, F. M. (1988) J. Mol. Biol. 200,

401-410.20. Brunger, A. T. (1993) X-PLOR (Yale Univ., New Haven, CT),

Version 3.1.21. Brunger, A. T., Krukowski, A. & Erickson, J. (1990) Acta

Crystallogr. A 46, 585-593.22. Artymiuk, P. J., Blake, C. C. F., Rice, D. W. & Wilson, K. S.

(1982) Acta Crystallogr. 38, 778-783.23. Jones, T. A. & Kjeldgaard, M. (1993) 0 (Biomedical Center,

Uppsala, Sweden), Version 5.9.24. Brunger, A. T. (1992) Nature (London) 355, 472-474.25. Hodel, A., Kim, S. H. & Brunger, A. T. (1992) Acta Crystal-

logr. A 48, 851-858.26. Ramachandran, G. N. & Sasisekharan, V. (1968) Adv. Protein

Chem. 28, 283-437.27. Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thorn-

ton, J. M. (1993) J. Appl. Crystallogr. 26, 283-291.28. Yamada, T., Matsushima, M., Inaka, K., Ohkubo, T., Uyeda,

A., Maeda, T., Titani, K., Sekiguchi, K. & Kikuchi, M. (1993)J. Biol. Chem. 268, 10588-10592.

29. Davies, D. R., Padlan, E. A. & Sheriff, S. (1990) Annu. Rev.Biochem. 59, 439-473.

30. Ruoslahti, E. & Pierschbacher, M. D. (1987) Science 238,491-497.

31. McDevitt, D., Francois, P., Vaudraux, P. & Foster, T. J. (1994)Mol. Microbiol. 11, 237-248.

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