the structure of a designed peptidomimetic inhibitor complex of α-thrombin

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  • Protein Engineering vol.6 no.5 pp.471-478, 1993

    The structure of a designed peptidomimetic inhibitor complex ofa-thrombin

    Tswei-Ping Wu, Vivien Yee2, A.Tulinksy4,R.Alan Chmsciel1, Hiroshi Nakanishi1, Richard Shen1,Cheryl Priebe1 and Mkhael Kahn1-3-4

    Michigan State University, Department of Chemistry, East Ijnsing, MI48824, and 'Department of Chemistry, University of Illinois, PO Box 4348,Chicago, IL 60680, USA2Present address: Department of Biochemistry, SJ-70, University ofWashington, Seattle, WA 98195, USA3Present address: Department of Pathobiology, SC38, University ofWashington, Seattle, WA 98195, USA4To whom correspondence should be addressed

    Thrombin displays remarkable specificity, effecting theremoval of flbrinopeptides A and B of fibrinogen through theselective cleavage of two Arg-Gly bonds between the 181Arg/Lys-Xaa bonds in fibrinogen. Significant advances havebeen made in recent years towards understanding the originof the specificity of cleavage of the Argl6 - Glyl7 bond of theAa-chain of human fibrinogen. We have previously proposeda model for the bound structure of fibrinopeptide A7_16(FPA), based upon NMR data, computer-assisted molecularmodeling and the synthesis and study of peptidomimeticsubstrates and inhibitors of thrombin. We now report thestructure of the ternary complex of an FPA mimetic (FPAM),hirugen and thrombin at 2.5 A resolution (/{-factor = 0.138)and specificity data for the inhibition of thrombin and relatedtrypsin-like proteinases by FPAM. The crystallographicstructures of FPA and its chloromethyl ketone derivativebound to thrombin were determined. Although there aredifferences between these structures in the above modeledFPA structure and that of the crystal structure of FPAMbound to thrombin, the 4>, \p angles in the critical region ofP 1 - P 2 - P 3 in all of the structures are similar to those ofbovine pancreatic trypsin inhibitor (BPTI) in theBPTI-trypsin complex and D-Phe-Pro-Arg (PPACK) inthe PPACK-thrombin structure. A comparison betweenthese and an NMR-derived structure is carried out anddiscussed.Key words: fibrinopeptide A/inrubitor/mimetic/teniplate/thrombin

    IntroductionTrypsin-like serine proteases form a large and highly selectivefamily of enzymes involved in hemostasis/coagulation (Davie andFujikawa, 1975) and complement activation (Muller-Eberhard,1975). Sequencing of these proteases has shown the presence ofa homologous trypsin-like core with insertions that can modifyspecificity and which are generally responsible for interactionswith other macromolecular components (Magnusson et al.,1976).

    Limited proteolysis is an important regulatory event and thepotential for controlled intervention of it to ameliorate specificdisease states has been recognized for a long time. The trypsin-like serine protease thrombin effects such cleavages and playsa critical role in thrombosis and hemostasis (Mann, 1987). Oxford University Press

    Thrombin exhibits remarkable specificity in the removal offibrinopeptides A and B of fibrinogen through the selectivecleavage of two Arg-Gly bonds of the 181 Arg/LysXaasequences in fibrinogen (Blomback, 1967). Recently, utilizingcrystallographic (Rydel et al., 1990, 1991) and NMR (Ni et al.,1989) data, computer-assisted molecular modeling andpeptidomimetic substrates and inhibitors (Kahn et al., 1988), weproposed a model for the thrombin-bound structure of FPA(Nakanishi et al., 1992).

    Subsequently, the X-ray crystal structures of human FPAbound to bovine thrombin (Martin et al., 1992) and achloromethyl ketone FPA-derived thrombin complex (Stubbset al., 1992) were determined. Notwithstanding some variancebetween these and also the predicted structure, the latter provedto be similar to the bound structure of FPA in many respects.In particular, the utilization of BPTI as a template to orient thePI, P2 and P3 residues in the active site was very effective inthe modeling. Such a conformation is likely to be a common motifamong extended peptide substrates of trypsin-like proteinases(Hubbard et al., 1991). The turn of FPA, although for the mostpart not in direct contact with the enzyme, plays a critical rolein appositely creating a hydrophobic cluster consisting of residuesPhe8, Leu9 and Vall5 (Martin et al., 1992; Stubbs et al., 1992).In an effort to understand better the interplay between the primarysequence and conformation required for thrombin substrates andinhibitors, we undertook a crystallographic investigation of our 'previously described and modeled mimetic FPAM (Nakanishiet al., 1992) (Figure 1) complexed with human a-uirombin. Wewish to report here the structure of the ternary complex of FPAM,hirugen and human a-thrombin at 2.5 A resolution formedthrough a FPAM chloromethyl ketone, a comparison of thestructure with the two FPAbovine thrombin complexes and theresults of specificity studies of FPAM with thrombin and relatedtrypsin-like serine proteinases.

    Experimental proceduresThe synthesis of chloromemyl ketone inhibitor FPAM is oudinedin Figure 1. The enantiospecific synthesis of 1 was accomplishedas described (Kahn et al., 1988), except that azetidinone (2) wasprepared enantiospecificalry by the Evans protocol (Evans et al.,1981). Coupling to the valylarginyl chloromemyl ketone (Kettnerand Shaw, 1979) and deprotection of the guanidinium group withanhydrous HF in-the presence of anisole afforded FPAM. Allnew compounds were characterized by *H NMR (400 MHz)and fast atom bombardment or plasma desorption mass spectro-metry. Enantiomeric purity was assessed by 19F NMR of theMosher esters (Dale et al., 1969), I3C NMR and HPLC whereappropriate (Priebe, 1991).

    Protease inactivation assays [human thrombin, plasmin,urokinase, porcine trypsin, kallikrein and bovine factor Xa (allpurchased from Sigma)] by the affinity labels FPAM and PPACKwere conducted in 50 mM MOPS buffer, pH 7.0, at 25C ina volume of 0.5 ml. The reactions were initiated by the additionof the enzymes. A minimum of five 40 /J aliquots were removed

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    ^

    PPr*.DEAD J-**^*

    (2)

    Ptr-AN

  • Mimetic thromMn inhibitor

    Table I. Final least-squares parameters and deviations of FPAM-thrombin

    Target a 6 Root mean square

    Table n . Root mean square deviations between the hirugen-thrombin andFPAMhirugen thrombin complexes

    Distances (A)Bond distanceAngle distancePlanar 1,4 distance

    Non-bonded distances (A)Single torsionMultiple torsionPossible H-bond

    Torsion angles ()PlanarStaggeredOrthonormal

    Plane groups (A)

    Chiral centers (A3)

    Thermal restraints (A2)Main chain bondMain chain angleSide chain bondSide chain angle

    Mean bond ansle (dea)

    0.0200.0300.050

    0.500.500.50

    31520

    0.02

    0.15

    1.52.02.52.5

    117.2 2.7

    0.0160.0440.049

    0.230.320.31

    22431

    0.01

    0.18

    1.11.91.72.6

    Number of a(\F\)reflections

    /{-valueshell

    A-valuesphere

    4.603.853.423.132.902.722.40

    1388140614231387148215211532

    39343128262321

    80645651444137

    0.1460.1110.1180.1410.1570.1650.163

    0.1460.1280.1250.1280.1320.1350.138

    o(\F\) = 26-180 (sin 0/X-1/6); < | |Fp | - |F c | |> = 53.0.40 x 0.27 x 0.10 mm employing a Siemens multiwire areadetector with graphite monochromated CuK^ radiation and aRigaku RU200 rotating anode X-ray generator operating at 50 kVand 150 mA. The crystal-detector distance was 11.65 cm, thedetector swing angle was set at 12, the scan range was 0.2per frame of measurement and each frame was measured for 90 s.The raw data were reduced and scaled with XENGEN programs(Howard et al., 1987) yielding 37 533 reflections of which11 675 were unique. After removing reflections with I/ail) < 2,a set containing 10 918 independent reflections remained (93%observed, R^^ = 0.039).

    The FPAMthrombin crystal structure was solved usingisomorphous thrombin coordinates of the hirugen-thrombincomplex (Skrzypczak-Jankun et al., 1991). Since no electrondensity was found for the autolysis insertion loop from Thrl47to Lysl49E in other isomorphous hirugen complexes, the initialmodel only included the A-chain from ThrlH to Argl5 andHel6-Glul46 and Glyl50-Glu247 of the B-chain of thrombin.The structure was refined employing the restrained least-squaresmethod implemented in the program PROLSQ (Hendrickson andKonnert, 1980) with intermittent model building performed onan Evans and Sutherland PS390 interactive stereographies usingthe program FRODO (Jones, 1982). The refinement proceededin two stages, initially including data from (7.02.8 A) and then(7.0-2.5 A) resolution difference maps. Each major round

    (A) Atomnumber

    All protein atomsMain chainCarbonyl oxygensSide chainsSulfurs (Cys, Met)Carbon alphas

    0.540.280.350.700.360.29

    197273824698814

    246

    of refinement was followed by model building using(2|F0| - |FC |) and (|FO| - |FC|) electron density maps and an ex-amination of the Ramachandran plot. The lvalue started at 0.28,with hirugen not being included in the calculations, with an overallthermal parameter of 25 A2. The first (2|FO| - |FC|) map at 2.8A resolution showed good density for most of thethrombinhirugen residues and Vall5-Argl6 of FPAM (Figure2). Hirugen and FPAM were fitted to the density and graduallyincluded throughout further calculations. The special aromaticgroups in FPAM, not being regular amino acid residues, did nothave normal peptide bonds connecting with the bicyclic ring(Figure 2). In order for the PROLSQ program to recognize andrefine the aromatic rings, modifications in the amino aciddictionary were made and some additional restraints were appliedin a control file (Figure 2). The /?-value decreased to 0.192 afterthe first refinement stage and water molecules were located at2.5 A resolution. Peaks were considered to be possible watermolecules by comparing (7.0-2.5) A and (8.0-2.5) A resolutiondifference maps. In addition, hirugen and the FPAM wereupdated according to the electron density maps. The finalFPAM-thrombin-hirugen structure has a crystallographicfl-value of 0.138 for 10 139 reflections between 7.0 and 2.5 Aresolution with 234 water molecules and a mean thermalparameter of 29 A2. The mean occupancy of the watermolecules is approximately 0.67 and their mean thermalparameter is also 29 A2. The final reflection weight and R-values in each range are given in Table I along with a summaryof refinement parameters. The distribution of main chain torsionangles (

  • T.-P.Wu et ai.

    complexes have been compared in detail and a stereoview of thesuperposition of the active site regions is shown in Figure 3, fromwhich it can be seen that the only significant change in the activesite induced by the binding of FPAM to thrombin is associatedwith Trp 60D. Another large deviation in the region occurs atthe side chain of He 174, which results in the side chain pointingtowards the face of the phenyl ring of Phal of FPAM butbasically, the residues in the catalytic site have practically thesame conformations as those in the hirugen-thrombin complex(Skrzypzcak-Jankun etal., 1991), where the active site isunoccupied. Thus, the conformation of the active site of thrombin

    is conserved and binding of FPAM or PPACK does not inducemuch change in the region (Bode et al., 1992).Structure of FPAMThe FPAM is almost completely defined by electron density inthe active site of the thrombin. Only the density of the two phenylrings lacks continuity to the two aromatic groups. The two phenylrings are ~ 7.8 A from each other in the apolar binding site regionof thrombin (Figure 4) and each of them interacts with thrombinthrough a number of hydrophobic contacts. The conformationof Argl6 to Rng3 of FPAM (Figure 2) is very similar to that

    Fig. 3. Stereoview of the comparison of the active site of thrombin in the FPAM and the hirugen complexes. Hirugen-thrombin, dashed lines; active site isunoccupied.

    Fig. 4. Stereoview of the comparison of FPAM and PPACK in their thrombin complexes. FPAM in bold.

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  • of PPACK in the PPACK-thrombin complex (Figure 4) (Bodeet al., 1992), with the former making an appproximately helicalturn between Glyl4 and Argl6, which is followed by the mimetic/3-bend of the bicyclic ring. There are some minor differencesin the main chain positions between the two structures in the helix-like turn. The main chain nitrogen atoms of Glyl4 and Argl6make a two-strand antiparallel /3-sheet with the thrombinSer214-Gly216 segment (Figure 5) so this region, like that ofPPACK, also possesses very favorable interactions.

    FPAM-thrombin interactionThe active site of thrombin, which displays a preference forarginyl and lysyl side chains, has the form of an elongated channeland is defined by peptide segments Tyr60ATrp60D,Arg97-Leu99, Thrl72-Argl75, Cysl91-Glyl96 andSer214-Glu217 (Figures 3 and 5). The density observed for thecatalytic triad indicates an intermediate hemiketal is formedbetween the carbonyl group of Argl6 and Serl95 OG (2.2 A)(Figure 5). Both FPAM and PPACK, being chloromethyl ketonederivatives, have the same contacts in this region. The SIspecificity pocket of the FPAM complex is occupied with anarginyl group with similar geometry to that of arginine in PPACK(Bode et al., 1992), hirulog 1 (Skrzypzcak-Jankun et al., 1991)and /3-homoarginine in hirulog 3 -thrombin (Qiu et al., 1992):the guanidinium group of Argl6 forms a doubly hydrogen-bondedsalt bridge with the carboxyl oxygen atoms of Asp 189 (2.5 Aand 3.1 A) (Figure 5). Moreover, the guanidinium group makes

    Mimetic thrombin inhibitor

    a close contact with the carbonyl oxygen of Gly219 (3.1 A) andthe main chain nitrogen atom of Argl6 may be involved in ahydrogen bond with the carbonyl oxygen of Ser214 (3.1 A). Theside chain of Vail5 in the S2 subsite is buried within ahydrophobic cage which is the apolar binding site of thrombin(Figure 5). The valyl group makes hydrophobic contacts withside chains of Tyr60A, Trp60D and Leu99 (3.8 A, 4.3 A and3.7 A respectively) and occupies a spatial region similar to Proof PPACK in the PPACK-thrombin complex (Bode et al.,1992). The P3 interaction observed in the present structure isdue primarily to an antiparallel /3-sheet hydrogen bond betweenthe amide nitrogen of Glyl4 and the carbonyl group of Gly216(2.4 A) that appears to be important in positioning the bicyclicring and is different from that in PPACK-thrombin. The bicyclicring corresponding to a /3-bend, which was presumed to be atthe 12-13 position of FPA (Ni et al., 1989), has a (S,S)conformation according to the chirality at carbon atoms CH andCB (Figure 2) and is located in the region bordered by residuesof Tyr60A, Trp60D, Leu99, Trp215 and Glu217 (Figure 5). Thering, although not aromatic, forms an end to face contact withthe indole ring of Trp215 producing an aromatic-like interactionthat is common in proteins (Burley and Petsko, 1985). In additionto the stacking interaction, the bicyclic ring is also stabilized byother hydrophobic contacts with side chains of Tyr60A, Trp60Dand Leu99. The N-terminal of the FPAM concludes with twophenyl groups that are also located near the non-polar region ofthe S2 and S3 subsites. The Phal ring makes a good van der

    195

    Fig. 5. Stereoview of FPAM bound in active site of thrombin. FPAM in bold.

    16

    Fig. 6. Stereoview of the comparison of FPAM and FPA in their thrombin complexes. FPAM in bold; FPA, human thrombin but bovine in FPA complex(Martin et al., 1992).

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    Waals contact with the side chain of He 174 (2.6 A) and showsdensity for its phenyl ring, however, Ben2, on the other side,has no significant interaction with thrombin except for looserhydrophobic contacts with residues of Tyr60A and Pro60C andis most likely the reason why the observed density is not as well-defined as that of Phal.

    100

    10 20 30TIME (min)

    100

    90

    u

    10

    R0KIKA3E I-3.0X1OKALUKREN1-9.0 X ICT'U

    FACTOR

    TRYPSIN1-2.3 X 10 M

    10 20 30TIME (min)

    Fig. 7. Activity of FPAM (top) and PPACK (bottom) with respect tothrombin and several other trypsin-like proteinases.

    Comparison of FPA-related structuresThere are five FPA-derived structures that are relevant in anycomprehensive comparison of its bound state in thrombin. Theseare (i) the FPA bovine thrombin complex (Martin et al., 1992);(ii) the chloromethyl ketone of FPA alkylating His57 of the activesite (Stubbs et al., 1992); (iii) the solution NMR structure of FPAbound to thrombin (Ni etal., 1989); (iv) the presentFPAM-thrombin structure; and (v) the modeled structure of theFPAM-thrombin complex (Nakanishi etal., 1992). Thestructures of FPA in (i) and (ii) are practically the same(r.m.s.A = 0.6 A and only 0.3 A if side chains of Leu9 andGlull are omitted). The 4>, \p, conformational angles of thePI P2P3 residues of (i), (ii), (iv) and (v) are all in fairly closeagreement with the conformation of PPACK in PPACK-thrombin (except for i/^, which is negative in PPACK due tothe D-enantiomer) (Bode et al., 1992). This can be seen fromFigures 4 and 6 which compare three of the structures. Inaddition, the conformation is also similar to that ofProl3-Cysl4-Lysl5 of BPTI bound in the BPTI-trypsincomplex (Huber and Bode, 1978). Only the NMR structure ofthe P 1 - P 2 - P 3 residues (Ni et al., 1989) of FPA bound tothrombin is not in agreement (Martin et al., 1992), especiallywith respect to the \j/2 and 3, \p3 angles. This was first noticedin attempts to dock and model the NMR structure in the activesite of thrombin by placing Argl6 in the SI subsite, which faileduniformly because of massive collisions of the NMR FPAstructure with the enzyme (Nakanishi et al., 1992). When theBPTI trypsin-bound structure was used as a template to reorientthe P 1 - P 2 - P 3 residues of the NMR structure, an excellent fitof these residues in the active site of thrombin was achieved.In the same work, similar rationalizations were employed to alsomodel FPAM in the active site.

    The structure of FPAM bound to thrombin is compared withthat of FPA in the thrombin complex (Martin et al., 1992) inFigure 6 from which it will be seen that the first generation FPAmimetic does not correspond to FPA in two important aspects.The first is that a peptide insertion following the bicyclic systemwould place the /3-tum of the mimetic more optimally with respectto the turn of FPA. Initially, the positioning of the turn in FPAMwas based on the NMR position (between Glyll and Vail5).The other point of difference of FPAM in mimicking FPA isin the conformation of the Phal moiety: rather than reversingto interact with Vall5 as in FPA, the FPAM molecule assumesa more or less extended conformation in the thrombin complex(Figures 5 and 6). Both of these shortcomings can be easilyrectified with some additional design features and synthesis(insertion of a peptide, reconforming the bicyclic system).Another notable difference from the FPA complex is that Gly 14Nof FPAM makes a hydrogen bond with Gly216O thereby alter-ing the conformation somewhat between the P2 and P3 positions.

    Table m.

    Protease

    TrypsinThrombinKallikreinFactor XaPlasminUrokinase

    Inhibition of selected

    Concentrationof affinitylabel(M)

    5.0 x 10-"5.0 x 10" '2.5 x 10-31.0 x 10"31.0 x 10"32.5 x 10""

    trypsin-like

    'v>(min)

    28.048.548.5

    130.8141.511.8

    proteinases by FPAM and

    (M"1 x min"1)

    5.0 x 1032.9 x 10"5.7 x 1025.3 x 1024.9 x 1022.5 x 10

    PPACK

    Relativerate ofinactivation

    20 0001 160

    232220

    1

    Concentrationof affinitylabel(M)

    5.0 x 10"'5.0 x 10"95.0 x 10-'5.0 x 10"'3.8 x 10"'5.0 x 10"'

    'v,(min)

    12.98.9

    86.613.635.5

    161.2

    (M

    1.11.61.11.15.28.6

    - l

    XXXXXX

    X min ')

    10810'10"10310"103

    Relativerate ofinactivation

    12 7901 861

    11361

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  • Mimetic thrombin Inhibitor

    The most important aspect that emerges, however, is that theFPAM-thrombin complex displays yet another binding modein the active site of thrombin (Tulinksy and Qiu, 1992) bringingthe total to four. The other three are

    (i) FP A/substrate-like,(ii) N-terminal hirudin-like (Rydel etal., 1990, 1991) and(iii) argatroban-like (Banner and Hadvary, 1992; Brandstetter

    etal., 1992).Specificity of thrombin and related proteinasesKinetic studies to explore the specificity of inhibition of theFPAM chloromethyl ketone with thrombin and a series of relatedtrypsin-like serine proteinases (factor Xa, trypsin, urokinase,kallikrein and plasmin) were conducted. We anticipated that theFPAM chloromethyl ketone would exhibit a high degree ofselectivity for thrombin and it does so, in that it was designedto mimic the bound conformation of the natural thrombinsubstrate fibrinogen. For comparison, we also evaluated thehighly thrombin selective affinity label PPACK (kindly suppliedby Dr M.Greco, R.W.Johnson Pharmaceutical ResearchInstitute). The rates of inactivation of this family of trypsin-likeserine proteinases are shown in Figure 7 for a given concentrationof affinity label as a function of time while Table HI lists thekinetic parameters, as well as the relative rates of inactivationand specificities of both FPAM and PPACK. Even though thereis nearly two orders of magnitude decline in potency for FPAMin comparison with PPACK the selectivity of inactivation ofthrombin is satisfyingly qualitatively similar.

    DiscussionA hallmark of the enzyme thrombin is its remarkable specificity.This is due to the inherently deep and constricted binding siteof thrombin and a requirement of its substrates to adopt a specificconformation to productively bind in the active site. Limitedproteolysis by trypsin-like serine proteases plays an importantrole in coagulation/hemostasis (Davie and Fujikawa, 1975) andcomplements activation (Muller-Eberhard, 1975). The potentialto specifically intervene in these processes to ameliorate a numberof disease states is significant and well-recognized. However,controlled and selective interference is critical to the success ofthe strategy that is made considerably more difficult by the highdegree of sequence homology within this family of proteases,which generally contain a trypsin-like core with insertions whichmodify specificity and are responsible for interaction withadditional macromolecular components (Magnusson et al., 1976).

    Of the 181 Arg/Lys-Xaa sequences in fibrinogen (Blomback,1967), only two bonds are cleaved by thrombin. Experimentalrationalization of this was provided through NMR investigationsof the complex between FPA and bovine thrombin (Ni et al.,1989). A striking feature that emerged in this study is the clusterof non-polar residues (Phe8, Leu9 and Vall5) which wereapparently brought into close proximity by the reverse turn.Reverse turns have been implicated in enhancing the specificityof proteolytic processing of prohormones, zymogens and viralproteins (Rholam et al., 1986, 1990; Bek and Berry, 1990). Wehave been investigating the role of secondary structure elementsin proteolysis through the incorporation of peptidometic prostheticunits (Kahn et al., 1988, 1991; Shen et al., 1992) and recentlyproposed a model for the bound structure of FPA (Nakanishiet al., 1992). The most critical feature of the model involvedthe reorientation of the P1-P3 residues of the NMR-derivedFPA structure to coincide in alignment with the active siteconformation of BPTI, which is believed to represent a canonical

    loop proteolysis substrate mimic (Huber and Bode, 1978;Laskowski and Kato, 1980; Huber and Carrell, 1989). Thisproved to be very effective in that the reoriented FPA model thenfitted well into the thrombin active site and satisfied all of thepreviously reported NOE data (Ni et al., 1989). Hybrid mimetic(i.e. part peptide and part /3-turn prosthetic unit) substrates basedon the foregoing were found to have similar kinetic parametersto those of FPA!_52 and were thus believed to effectively mimicthe bound conformation of natural substrates.

    We anticipated that because the FPAM inhibitor was designedaround a specific natural substrate for thrombin, it would exhibita high degree of selectivity. This is indeed the case, in that FPAMexhibits a degree of specificity similar to that of PPACK, a well-known selective inhibitor of thrombin (Table HE). Utilization ofthe canonical loop motif (Laskowski and Kato, 1980) of a naturalproteinaceous inhibitor as a lead for designing reverse turnpeptidomimetic inhibitors may provide a general strategy forintroducing specificity into an inhibitor.

    Although our model for the bound structure of FPAM isconsistent with the observed crystallographic structure (Martinet al., 1992), particularly in the orientation of the P; - P 3 sites,not suprisingly there are some significant differences. Moststriking is the fact that the hydrophobic pocket, formed by the60 insertion loop thrombin and residues Leu 99, De 174 and Trp215, is not fully occupied by the N-benzyl group backtrackingto Vall5 as anticipated, but rather by the bicyclic /9-turn templatein a similar manner to the dansyl group of DAPA -thrombin(Tulinsky and Qiu, 1992) and related molecules (Banner andHadvary, 1992). This is due to the relatively extended structureof FPAM and is yet another example of the intriguing dichotomythat exists between the specificity and promiscuity of thrombin(Tulinksy and Qiu, 1992). A peptide insertion between Glyl4and Rng3 (Figure 2) and an alternate conformation orstereochemistry in the C-7 ring of the bicyclic /3-turn prostheticunit could conceivably match the bound FPA conformation withconsiderable fidelity (Figure 6).

    Information gathered from these investigations is being utilizedin the design and synthesis of novel non-peptidic thrombininhibitors. This stepwise process, starting with natural proteasesubstrates or inhibitors and culminating in truly non-peptideinhibitors will generate new structures that should maintain thespecificity that nature has so elegantly and carefully crafted.

    AcknowledgementsWe would like to thank Drs Phil Martin and Brian Edwards for providing uswith the coordinates of FPA bound to thrombin, Dr Wolfram Bode for the FPAchloromethyl ketone-thrombin coordinates and NIH grams HL43229 (A.T.) andGM3826O (M.K.) for supporting this work. Additionally, M.K. wishes to thankGlaxo for a Cardiovascular Discovery Grant and the American Heart Associationfor an Established Investigators Award.

    ReferencesBanner.D.W. and Hadvary,P. (1992) J. Biol. Chenu, 266, 20085-20093.Bek.E. and Berry,R. (1990) Biochemistry, 29, 178-183.Blomback.H. (1967) In Seegers,W.H. (ed.), Blood Clotting Enzymology.

    Academic Press, New York, pp. 143-215.Bode.W., Mayr,I., Baumann.U., Huber.R., Stone.S.R. and HofsteengeJ. (1989)

    EMBOJ., 8, 3467-3475.Bode.W., Turk.D. and Karshikov.A. (1992) Protein Sri., 1, 426-471.Brandstetter.H., Turk.D., Hoeffken.H.N., Grosse.D., Sturzebecher.J.,

    Martin.P.D., Edwards.B.F.P. and Bode.W. (1992) J. MoL Biol, 226,1085-1099.

    Burley.S.K. and Petsko.G.A. (1985) Science, 229, 23-28.DaleJ.A., DuU.D.L. and Mosher^.S. (1969)7. Org. Chem., 34, 2543-2549.Davie.E.W. and Fujikawa.K. (1975) Annu. Rev. Biochem., 44, 799-829.Evans.D.A., BartolU. and Shih.T.L. (1981) /. Am. Chem Soc., 103,

    2127-2129.

    477

    at Penn State University (Paterno Lib) on M

    ay 8, 2012http://peds.oxfordjournals.org/

    Dow

    nloaded from

  • T.-P.Wu et al.

    Heodrickson.W.A. and KonnerU.H. (1980) In Srinivasan.R. (ed.), BiomoiecularStructure, Function, Conformation and Evolution. Pergamon, Oxford, pp.43-57.

    Howard.A.J., Hilliland,G.L., Finzel.B.C, Poulous.T.L., Olhendorf.D.H. andSalemme.F.R. (1987) / Appl. CrystaUogr., 20, 383-387.

    Hubbard.S.J., Campbell.S.F. and ThorntonJ.M. (1991) J. MoL Biol., 220,507-530.

    Huber.R. and Bode.W. (1978) Ace. Chan. Res., 11, 114-122.Hubcr.R. and Carrell.R.W. (1989) Biochemistry, 28, 8951-8966.Jones.T.A. (1982) In Sayre.D. (ed.) Computational Crystallography. Clarendon

    Press, Oxford, pp. 303-317.Kahn.M., Wilke.S., Chea.B. and Fujita.K. (1988) J. Am. Chan. Soc., 110,

    1638-1639.Kahn.M., Nakanishi,H., Chruscid.R.A., Fdzpatrk,D. andJohnson.M.E. (1991)

    J. Med, Chem., 34, 3395-3399.Kettner,C. and Shaw.E. (1979) Thrombosis Res., 14.Laskowski.M. and Kato.I. (1980) Annu. Rev. Biochem., 49, 593-626.Magnusson.S.L., Sottrup-Jensen.T.E., Petersen, Wojciechowska.G.D. and

    daeys.H. (1976) In Ribbons.D.W. and Brew JC (eds), Miami Winter SymposiaVoL 11, Proteotysis and Physiological Regulation. Academic Press, New York,pp. 203-239.

    Mann.K.G. (1987) Trends Biochem. Sri., 12, 229-233.Mardn.P.D., Robertson.W., Turk.D., Huber.R., Bode.W. and Edwards.B.F.P.

    (1992) J. Biol. Chem., 267, 7911-7930.Muller-Eberhard.H.J. (1975) Annu. Rev. Biochem., 44, 697-724.Nakanishi.H., Chrusciel.R.A., Shen.R., Bertenshaw.S., Johnson.M.E.,

    Rydel.T.J., Tulinsky.A. and Kahn.M. (1992) Proc. NatlAcad. Sci. USA, 89,1705-1709.

    Ni,F., Meinwald.Y.C, Vasquez.M. and Scheraga.H.A. (1989) Biochemistry,28, 3094-3105.

    Priebe,C.L. (1991) PhD Thesis, University of Illinois at Chicago.Qiit.X., Carperos,V.E., Tulinsky.A., Kline.T., Maraganore.J.M. and

    FentonJ.W.,n (1992) Biochemistry, in press.Rholam.M., Nicolas.P. and Cohen,P. (1986) FEBSLett., 207, 1-6.Rnc4flm,M., CohenJ>., Brakch.N., PaohTlo.L., Scatturin^. and DiBello.C. (1990)

    Biochem. Biophys. Res. Commun., 168, 1066-1073.Rydel.T.J., Ravkhandran.K.G., Tulinsky.A., Bode.W., Huber.R., Roitsch.C.

    and Fenton,J.W.,n (1990) Science, 249, 277-280.Rydel.T.J., Tulinsky.A., Bode.W. and Huber.R. (1991) J. Mol. Biol, 221,

    583-601.Shen.R., Priebe.C, Patel.C, Rubo.L., Su,T., Kahn.M. and Sugasawara.R.

    (1992) Tetrahedron Lett., 33, 3417-3420.Skrzypczak-Jankun.E., Carperos.V., Ravichandran.K.G., Tulinsky.A.,

    Westbrook,M. and Maraganore.J.M. (1991)7. Mol. Biol., HI, 1379-1393.Stubbs.M.T., Oschkinat,H., Mayr.I., Huber.R., Angliker.H., Stone.S.R. and

    Bode.W. (1992) Eur. J. Biochem., 206, 187-195.Tulinsky,A. and Qhi.X. (1992) Sem. Thrombosis Hemost., in press.

    Received on November 19, 1992; revised on February 10, 1993; accepted onMarch 30, 1993

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