Refined structure of the Hirudin-thrombin complex

J. Mol. Biol. (1991) 221, 583-601

Refined Structure of the Hirudin-Thrombin

Complex

Timothy J. Rydelt, Alexander Tulinskyl Department of Chemistry, Michigan East Lansing, MI 48824,

State University U.S.A.

Wolfram Bode and Robert Huber Max-Plan&-Institute fur Biochemie Da033 Martinsried, Germany (Received

11 February

1991; accepted 13 May

1991)

The structure of a recombinant hirudin (variant 2, Lys47) human a-thrombin complex has been refined using restrained least-squares methods to a crystallographic R-factor of 0.173. The hirudin structure consists of an N-terminal domain folded into a globular unit and a long 17.peptide C-terminal in an extended chain conformation. The N-terminal domain binds at the active-site of thrombin where Ilel’ to Tyr3’ penetrates to the catalytic triad. The x-amino group of He1 of hirudin makes a hydrogen bond with OG of Ser195 of t,hrombin, the side-chains of Ilel’ and Tyr3’ occupy the apolar site, Thr2’ is at the entrance to, but. does not enter, the Sl specificity site and Ilel’ to Tyr3’ form a parallel j-strand with Ser214 to Gly219. The latter interaction is antiparallel in all other serine proteinase-protein inhibitfor complexes. The extended C-terminal segment of hirudin, which is abundant, in acidic residues, makes many electrostatic interactions with the fibrinogen binding exosite while the last five residues are in a 3,, helical turn residing in a hydrophobic patch on the thrombin surface. The precision of the complementarity displayed by these two molecules produces numerous interactions, which although independently generally weak, together are responsible for the high degree of affinity and specificity. Although hirudin-thrombin and nPhe-Pro-Arg-chloromethyl ketone-thrombin differ in conformation in the autolysis loop (1~~~145 to Gly150), this is most likely due to different crystal packing interactions and changes in circular dichroism between the two are probably due to the inherent flexibility of the loop. An RGD sequence, which is generally known to be involved in cell surface receptor interactions, occurs in thrombin and is associated with a long solvent channel filled with water molecules leading to the surface from the end of the Sl site. However, the RGD triplet does not appear to be able to interact in concert in a surface binding mode. Ke2/words: hirudin:

thrombin;

blood

1. Introduction Thrombin (EC 3.4.21.5) is a glycoprotein that is representative of a sub-class of serine proteases that play a central role in thrombosis and hemostasis. It is generated in the final events of blood coagulation from prothrombin (Mann, 1987) where it converts fibrinogen into clottable fibrin in the

t Present address: Miami Valley Laboratories. The Procter & (iamble (Jo., PO Box 398707. Cincinnati, OH 45P39-8707, 1’.S.A. $ Author t,o whom all correspondence should be addressed.

coagulation;

inhibition;

exposite

formation of t#hrombi by exhibiting specificity largely attributed to an anion-binding recognition exosite that is distinct from the catalytic site (Fenton, 1981, 1986). Although this exosite is also implicated in thrombin interactions with other substances, heparin binds to a different exosite (Church et al., 1989). Thrombin also activates other coagulation factors such as V, VIII. XTII and protein C (Fenton, 1981) but the processes are modified when thrombin associates with thromhomodulin (Esmon et al., 1986). a-Thrombin consists of two polypeptide chains of 36 (A-chain) and 259 (Bchain) residues linked by a disulfide bridge (Table 1: Butkowski et al., 1977; Thompson et al., 1977; Degen et al., 1983). The crystallographic structure of

584

1’. J. Rydel et al.

Table 1 Summary

of final restrained least-squares paran~eter.~ldeviatio~Ls a?Ld R-factor

statistics

Distances (A): Bond lengths Bond angles Planar l-4 Planes (8): Peptides Aromatic groups (Ihiral volumes (A3): Non-bonded contracts (A): Single torsion Multiple torsion Possible H-bond Thermal parameters (A2) Main-chain bond Main-chain angle Side-chain bond Side-chain angle Torsion angles (deg) Planar Staggered Orthonormal Diffraction pattern a(lF,I) = A+U(sinfI/1-l/6) < llF,l-lE’,ll > = 54 dmin (A) 413 3.39 3.00 2.75 2.56 2.42 2.30

Reflection number

Wol)

< IIE’,F,II ’

K-factor shell

R-factor sphere

3043 3380 3335 3160 3112 2826 2200

34 30 27 25 23 22 21

89 64 56 47 43 39 38

0~150 0136 @I76 0.190 0212 0235 0265

ti150 0143 @151 0157 0163 0168 0,173

human a-thrombin inactivated with PPACK(t) has been determined at 1.9 A (18 = 0.1 nm) resolution (Bode et al., 1989a), where it was shown to possess structural similarity to other trypsin-like serine proteases but with insertion loops centered around Asp60E. Va1149C and Gly186C (Table 1); the 60A-I insertion loop protrudes into the active-site vicinity and, among other things, narrows the substrate cleavage binding cleft. a-Thrombin is susceptible to proteolytic cleavage by trypsin and autolysis at Arg77A (Fenton et al.. 1977). This form has been designated B-thrombin and can undergo further cleavage at Arg67 with the t Abbreviations used: PPACK, n-Phe-Pro-Argchloromethyl ketone; rHV2-K47. recombinant hirudin variant 2-Lys47; Cysls’. prime trailing residue number designates hirudin; PEG, polyethylene glycol; NAG, X-acetylglucosamine: c.d., circular dichroism; BPTI, bovine pancreatic trypsin inhibitor; n.m.r., nuclear magnetic resonance; r.m.s.. root-mean-square.

concomitant loss of the undecapeptide (Boissel et al., 1984). The loop from Lysl45 to Lysl49E (Fig. 1) is also highly sensit’ive to proteolytic cleavages, including a,utolysis, and has often been referred to as the autolysis loop. Thus, in addition to the p-cleavage, y-thrombin has a break at Lys149E. Other forms of thrombin are &-thrombin, resulting from limited proteolysis with elastase and characterized by a single cleavage between Ala149A-Asn149B (Kawabata et al., 1985) and [thrombin. produced by cathepsin G or chymotrypsin with a cleavage at Trp148 (Brezniak et al., 1990). All of the cleaved forms of thrombin exhibit reduced clotting activity. The principal inhibitor of thrombin in blood circulation is antithrombin III (Travis & Salvesen, 1983), however, the most potent natural inhibitor of thrombin is hirudin, a g&residue protein from the medicinal leech Hirudo ,medicinalis European (Markwardt, 1970). By virtue of disulfide links, hirudin consists of a compact N-terminal domain

Refined Structure

NN2-T

qf the Hirudin-Thrombin

CCLRPLTKKKSLED D

I G

R-CODE G D I Y s

K

A s

Complex

T K

E

R

G

K E

L L B-Chain

D

w

I[

P

P

P

N YT

S

p2-IVKGSDAKIGYSPWQVHLTRKPQKLLCGASLISDRWVLTAABCLLKNDLL 16 25 35

45

55

1

60

65

L

R

VRIGKBSRTRYLNIKKIS~LKKIYIBPRYNWRNLDRDIALNKLKKPVAF 85 75

95

105 V NG

------I ASL SDYIEPVCLPl~5RETALQAGYKGRVTGWGNLKKTWTGQPSVLQVVNLPIVE 135 115

AK

145

KG Y DK RPVCKDSTRIRITDNnlCAGKPRGDACKGDSGGPFVMKSPNRWYQNGIVS 185 175 165

195

R WGE-GCDDGKYGIYTEVCRLKKUIQKVIDQFGE-Cm6 225 215

245

235

155

TN 205

Figure 1. Sequence of a-thrombin. Insertions with respect to chymotrypsin indicated as protrusions from linear chain and designated with letters in text; as described in Bode it al.. 1989a).

possessing a two-disulfide, double-loop structure (B, C, D, Fig. 2) preceded by an ordinary disulfide loop (A) and followed by a long 26-residue C-terminal chain. Unlike antithrombin III, however, hirudin is specific for thrombin forms with fibrinogen activity (Fenton et al., 1979) with which, in the case of a-thrombin, it forms a remarkably stable non-covapossessing a lent, stoichiometric, 1 : 1 complex binding constant reportedly as low as 2 x lo-l4 M (Stone & Hofsteenge, 1986)) although catalytically active thrombin is not required for complexation (Stone et al.: 1987). Kinetic and equilibrium studies indicate that hirudin interacts simultaneously with the catalytic site and the fibrogen binding exosite of thrombin (Fenton, 1981; Chang, 1983). Studies with synthetic peptides have shown that the exosite also binds with the C-terminal decapeptide and related fragments of hirudin (Krstenansky & Mao, 1987; Ni et al., 1990) and that it contains a number of lysyl residues of thrombin (Chang, 1989). Whereas natural hirudins are mixtures of variants, recombinant techniques produce homogeneous preparations. The recombinant proteins lack a sulfated Tyr63; however, they only have about tenfold reduced affinity (Dodt et al., 1988; Braun et al., 1988) and possess Ki values in the pica-molar range (Degryse et al., 1989). The hirudin used in the crystallographic structure determination described here of the hirudin-thrombin complex was a recom-

binant form, variant 2 with Lys47 (Harvey et al., 1986). It differs from the most abundant natural iso-inhibitor in eight residues and by the absence of a sulfated tyrosine residue at position 63 (Fig. 2).

Figure 2. The sequence of recombinant hirudin variant 2-lysine 47 (rHV2-K47). Residues indicated outside circles are those of the most abundant natural iso-inhibitor.

T. J. Rydel et al.

586

Two solution structures of recombinant hirudins have been detemined by n.m.r. (Folkers et al., 1989; Haruyama & Wuthrich, 1989). Except for residues Gly31’ to Gly36’, the structure of the N-terminal domain of hirudin (residues 3’ to 48’) was fixed by n.m.r. while the remainder of the C-terminal was completely disordered (16 residues). We report here the highly refined X-ray crystallographic structure of the rHVZK47 human a-thrombin complex at 2.3 A resolution. A preliminary report of the structure determination has already appeared elsewhere (Rydel et al., 1990) as was a lower resolution structure of a variant 1 complex (Grutter et al.. 1990). A comprehensive report of the thrombin structure has been given by Rode et al. (1991). Unlike the n.m.r. solution struttures, the C-terminal of hirudin is structurally ordered in the complex. Tn contrast to predictions (Fenton & Bing, 1986; Johnson et nl., 1989). the N-terminal tripeptide of hirudin interacts with t’hr catalytic site of thrombin and Lys47’ does not occupy the specificity pocket while the interaction of the [:-terminal chain with the fibrinogen exosite of thrombin is confirmed.

2. Materials

and Methods

(‘rystals of t,he rHVSK47 human cc-thrombin complex were grown by vapor diffusion using PEG 4000. MgCl, and acetate buffer at pH 4.5 (Rydel et al.. 1990). The crystals are tetragonal. n = b = 9@39 a, c = 132.97 ,A. space group P-I,,,*) 2 l with 8 molecules per unit cell (protein fraction approx. 39yd). S-ray diffraction intensit) data were collected at, 6°C’ to 2.3 a resolution with an Enraf-Xonius FAST television area drtect,or using a Rigaku rotating anode S-ray source operat’ing at 5% kI%‘. Pertinent statistics regarding the diflraction data are summarized elsewhere (Rydel rt al.. 1990). The measurrments are fairl,v romplete. show excellent internal consistency and are observable out, to the diffraction limit. Patterson methods were used to d&ermine the orietrtation of the thrombin molecule in crystals of t.he nomples (Rossman & Blow. 1962: Huber. 196.5). The rotation search was performed with the thrombin structure of PPACK-thrombin (Bode rt al.. 1989a). The position of the t,hrombin molecule in the unit, cell was fixed by a translation search with programs written by Lattman (1985), modified by J. Deisenhofer & R,. Huber, using intensity data from 8.0 t,o 3.0 A resolution. These calculations established the position of the molecule and the correct enantiomorphic space group as well. The crystallographic R-value (R = XIIF,[-IF,ll/ClF,I) of this solution was 039. The rotational and the translational paramet’ers were refined further with t,he rigid body refinement program TRAREF (Huber & Schneider. 1985), reducing K to 031. The resulting model of the complex was refined using the energy-restraint crystallographic refinement program EREF (Jack & T,evit,t. 1978) with interac+ivr 1978). The graphics interventions using FRO110 (Jonrx cyclic procedure was repeated 5 times and the R-value (*onverged at @193. The refinement was c.ontinued using restrained leastthe program PROFFT squares procedures with (Hendrickson & Konnert, 1980: Finzel. 1987). The latter takes into account certain shortcomings associat’ed with EREF refinement. such as ability t’o restrain thermal

-90

-0

-90 L______.--___-

F

_1__________, I

-180

I

*

I

,

I

I

-90

I

0

I

,

,

90

I

I

1-180

180

PHI

Figure thrombin.

3. Ramachandran (ilycine

residues

4. rl/ angles not, displayed.

of hirudin

factors between at,oms. I)rI)tidr planarity and intrrmolecular contacts, along wit,h an option to refine orcupanties. In addition. since there was sufficient electron density in the last map near NIX of AsnCiOC+to model the 1st NA(: moiety of the oligosaccharide attached to thrombin. it was included into subsequent calrulations. The PROFFT refinement began at) 2.5 a resolut,ion with water st’ructure in the calculation whirh l)rovrd to he illconditioned showing many large positional and OIYUpancy shifts. Therefore. all the solvent was removrcl (R = 0.27): the strurture then refined quirkly and smoothly to K = 0.23. At this stage. water molecules were included again. These were chosen if they appeared in the (2lF,l-lF,l) density and in both the X.0 t,o 2.5 ip and 7.0 to 2.5 A resolut,ion (IF,/-lF,l) difference maps (>25 0). and it they were in the proximity of a hydrogen donor OI acceptor. The inclusion of 76 wat,er molrcules followed b?; 30 cycles of refinement rrduc>ed R tjo 0.20. The resolution was then including

increased to 2.3 A and the refinement c-ontinued. progressively more solvent along with graphical

interventions for model rehuilding purposes, The strut.ture ultimately converged at, K = 0.173 with an average thermal parameter of 35 a2 using 265 wat,er molecules and 21,056 reflertions from 7.0 t,o 2.3 14 resolut,ion. The final target parameters of the refinement and t,heir r.m.s. values are listed in Table 1. These values correspond to 2336 atoms (14 for the NAG of carhohydrak) of thrombin (96?6); 447 of hirudin (89’+,) and the water molerules all of which were defined in terms of 12,459 variahlrs (I.7

observations/parameter). The cl,-angles of 91 O<, of the peptide honds of t,he c~omplru are within +6” of’planarit>. and only 3 residues are not in conformationally allowrtl regions (Fig. 3): all of t*hesr occur in the A-chain with Serl E and AspI-CL being adjacent to the t,ermini of t,hca chain. The fit of the 1st hrxose of the carbohydrate c.haitl of thromhin to the electron densit’y was good but additional density for t,he remainder of t,hr chain tlicl not develop during the course of the refinement. inclicaating that it, was disordered in the interstit,ial solvent region between molecules in the crystal. From an examination of the R-factor as a function nf scattering angle (Tahlr I) a

Refined Structure of the Him&n-Throw&in

Complex

587

Figure 4. Stereoview of the CA structure of the hirudin-thrombin complex. Hirudin is shown as a bold line; disordered residues are shown as broken lines; N and C-terminal of thrombin and C-terminal of hirudin designated: N-terminal of hirudin in active site A; B and C are fibrinogen binding exosite and autolysis loop, respectively.

co-ordinate error (Luzzati. 1952) of about @20 to 025 L% has been estimated for the structure of the complex?.

3. Results (a) Structure of hirudin of hirudin in the The first 48 residues hirudin-thrombin complex are organized into a compactly folded domain similar to that observed in solution by n.m.r. (Folkers et al., 1989; Haruyama &

Wuthrich, 1989). However, the C-terminal chain of hirudin, completely disordered in solution, is welldefined in the complex and in an extended conformation (Fig. 4: Rydel et al, 1990). The first stretch (Glu49’ to Gly54’) makes numerous electrostatic and polar interactions with thrombin while the second (Asp55 to Gln56’) begins likewise but finishes in a distorted 3,c helical turn that resides in a hydrophobic patch on the thrombin surface. The conformation of the N-terminal domain of hirudin is related to close intramolecular contacts of a three-disulfide core (Fig. 5). The disulfide bonds of CysS’-Cysl4’ and Cysl&Cys28 are nearly perpendicular (distance between disulfide midpoints of 497 A) while Cys16’-Cys28’ and Cys22’-Cys39’ are nearly parallel (disulfide midpoint separation of 5.35 A). Similar close disulfide interactions have also been observed in C3a anaphylatoxin (Huber et al., 1980), in kringle structures of blood coagulation/ fibrinolysis (Tulinsky et al., 1988; Mulichak & Tulinsky, 1990) and in squash-seed trypsin inhibitor (Bode et d., 19893). A list of the sulfur-sulfur distances in the hirudin molecule is presented in Table 2. The net result is that the loop segments B, C and I), which comprise a double loop in hirudin (Fig. 2), fold into three unique loops. This, t The coordinates of the hirudin-thrombin have been desposited in the Protein numbers 1HTC and SHTC!).

Data

complex

Bank

(entry

combined with the close stacking of the other disulfide bond (Cys6’-Cysl4’). produces four threedimensional loops (Fig. 5). A solvent-accessibility calculation (Lee & Richards, 1971) indicates essentially zero accessibility for all the disulfide bonds of hirudin except possibly Cys39’, which, in any case, is very small (18% side-chain, 33% totad). The N-terminal domain possesses short stretches of antiparallel B-structure (Table 3). The j?l strands have two hydrogen bonds while another hydrogen bond precedes the strand (Table 4). The individual ribbons of this element are connected together by a type II’ reverse turn (Crawford et al., 1973; Tl of Table 3). A type II turn (T2) is present in loop C and a longer antiparallel p-finger (/IS of Table 3) is generated in loop D (Figs 4 and 5), which contains four hydrogen bonds (Table 4). Although a reverse turn must join the strands of this finger together (T3 of Table 3), this turn is also disordered in the crystal structure (Figs 4 and 5: Rydel et al.,1990), as it is in solution (Folkers et al., 1989; Haruyama &

Wuthrich, 1989). In addition to the disulfide core, many intramolecular hydrogen bonds also stabilize the N-terminal domain of hirudin (Table 4). These were chosen using distances less than 3.1 A and donorhydrogen-acceptor angles greater than 125” as criteria. In all, 22 hydrogen bonds occur in the domain, of which eight are in turns1 and p-structure. The hydrogen-bond interactions involving Lys47’ appear to be important in extending and terminating this globular domain out to and at residue Pro48’, respectively. Although there are a number of main-chain hydrogen bonds between Cys39’ and Pro48’, which is additionally a conserved stretch of sequence, and the remainder of the

1 Although the hydrogen bond distance is 3.3 A in Tl and T2, these turns are very clear and imply the hydrogen bond.

588

T. J. Rydel et al.

1’

) 6

\ 48’

8’

Figure 5. Stereoview of the folding of the hirudin K-terminal domain. The A to I) loops correspond to those of Fig. I: disulfide bridges and side-chains of Thr4’, Asp5’ and Lys47’ are shown as bold lines; hydrogen bonds and ill-defined residues 32’ to 35’ are shown as broken lines.

domain, the a-amino group of Lys47’ is involved in two apparently crucial hydrogen bonds with residues of the N-terminal pentapeptide of hirudin (Thr4’ and Asp5’: Fig. 5); moreover, Lys47’ N also hydrogen bonds to Asnl2’ 0 (Table 4). Such interactions bring the N and C-termini of the domain in relatively close proximity, increasing the size but maintaining the compactness of the domain. The two proline residues that flank Lys47’ on either side might well help maintain its position. These three residues also initiate a polyproline II helix (Hl: Table 3) like that of collagen (Yonath & Traub, 1969) which terminates at His51’. The folding of the N-terminal domain in the complex is similar to that observed by n.m.r. (Folkers et aE., 1989; Haruyama & Wuthrich, 1989). An optimal superposition of 38 CA atoms with the average of 32 n.m.r. structures? gave an r.m.s. difference of 0.86 A. Although the r.m.s. difference in the side-chain positions increases to about 1.95 A, it is comparable with the r.m.s. deviations from the average n.m.r. structure (Folkers et aE., 1989). The most serious discrepancy with respect to the crystallographic structure occurs in the disulfide positions (r.m.s. = 2.4 A) that are fairly well determined in the complex. Two of the disulfide bonds are displaced by about a half bond length and one bond t We thank Dr G. Marius Clore for providing us with the n.m.r. coordinates of the average solution structure of hirudin prior to their distribution by the Protein Data Bank.

length from the n.m.r. structure, respectively, while Cys16’-Cys28’ has a very different orientation in the complex: x . . x’ = -165, 81, 85. 76. -175” (complex); -60, -169, 75, -168, -40” (n.m.r.). The otherwise close correspondence between the n.m.r. and the crystal structures indicates that the N-terminal domain changes little when it is complexed with thrombin. The C-terminal of hirudin consists of t.wo extended stretches of chain with a bend at Asp55’ (Fig. 4). The first segment is approximately 18 A long. The initial residues are the last three of the polyproline helix while the last three residues have poorly defined electron density. The second segment of the C-terminal is approximately 19 A long and consists of 15 A in extended conformation (Asp55 to Pro60’) followed by a type III 3,, reverse turn (Crawford et al., 1973; Table 4). The extended

Table 2 h’u@~r-sulfur distances (if) Atom 6’ 14 16’ 22’ 28’ 39

6’

in hirudin

14’

lti’

p’

2411 *

4.72 6.43

6-11 856 4.70

* Indicates a disulfide

hand.

28’ 3.74 543 UK* 548

39’ 7.65 8.32 .522 205* 65H

Refined Structure of the Hirudin-Thrombin

589

Complex

Table 3 Secondary Element

structural elements of hirudin Residues

Type

~-strurturr Pl B2

Antiparallel Antiparallel

Helix Hi

Polyproline

Rrvrrsr Tl T2

Cys14’ to (‘~~16’: Asn20’ to Cys22’ Lys27’ t>o Gly31’; Gly36’ to Va140’

II

turns

Type 11

(:lu17’. (:ly18’, SerlS’, AsnXO Glp23’, Lys24’, Gly25’, AsnAG

1

Ser32’. Ssn33’.

Type II’

T3 T4

Type

(In) Hirudin-thrombin

i,nteraction

of the hirudin-thrombin The CA-structure complex is shown in Figure 4 where principal subsites and loops are designated. The docking of the N-terminal domain with thrombin is similar globally to the manner in which other natural serine prot)einase protein inhibitors interface with protease enzymes. Additionally, an important component to the binding in both is in or near the active site region. However, whereas the C-terminal peptide of hirudin is disordered in solution, it is well-defined in the complex where it extends across the surface of the t.hrombin molecule for 40 A terminating

Table 4 bonds of the X-terminal

Donor

(‘ys6’ Thr7’ c:1ux (:Iyio’ (iIn I’ Asnl:’ , lJPU13 11PU I I5’ (+:i,*)’ ._-

N N N N SE” s r h N s

Lysr’i (‘,vs”x’ 1Ie”!l’ l,ruXY (:I1138 c*ys:w V&O’ (:lp42’ (:Iy44’ ‘l’hr4.5’ I&7’ Lys47’ Lvs47’

s s N N N N N N N S s 2;71 S%

Acceptor Lru1.5 (+lnll’ (:lnl I’

0 OEl OEI

(‘vs48’ 0

(h’

Thr45’ (‘ys22’ Thr4’ (‘ysl.?’ Yal40’ (ilnll’ (+ln38’ Sri-9 Ilr”9’ (‘luli’ 1:ys?i ( :1,v2<5 Asn28’ (:lylo’ Asnll’ Thrl’ Asp5

OEl 0 0 0 0 0 0 0 OG 0 OEl 0 0 0 0 0 OGl 0

domain of hirudin Distance 2.75 2.48 3.0 1 238 2.63 2.24 2.60 283 283 2.39 3.07 2.53 2.66 2.72 2.58 2.42 307 298 2.78 2.70 2.64 290

(A)

Gly34’. Lys35’

Glu61’. C&62’. Tyr63’.

III

nature of the C-terminal of hirudin with no stabilizing back-interactions to the N-terminal domain is most likely the reason for the disorder observed in solution for this part of the molecule.

llydrogrn

Pro46’ to His51’

Element

Bl P2 s 82 p2

Leu64’

approximately diametrically opposit)e to the activesite (Fig. 4). The first ten residues of the terminal chain lie against a cliff-like wall on the surface of thrombin formed by the TrpSOD and Lys70 to Glu80 loops (Bode et al., 1989a, 1991) which probconstitutes the fibrinogen anion-binding ably exosite (Fent’on, 1981, 1986). The 3,,, helical turn at the C terminus fits in between the latter loop and the Phe34 to Leu41 loop. Tn all. 12 of 17 C-terminal residues of hirudin (70%) are involved in interactions with thrombin. Surprising and unexpected aspects of the N-terminal domain interaction with thrombin are: (1) the N-terminal tripeptide of hirudin penetrates into the active-site region, with the amino terminus of Ilel’ forming a hydrogen bond with O($ of Ser195 of the catalytic site and the carbonyl oxygen atom of Ser214, (2) Thr2’ is at the entrance of the Ml specificity site of thrombin hut is too small to occupy it extensively or interact with Asp189 at the end of the pocket in the manner observed for arginine of PPACK (Bode et al.. 1989a, 1991) or substrate, (3) Ilel’ and Tyr3’ are in an apolar dome consistent with the P2 position of thrombin substrates generally being non-polar and (4) llel’ t’o Tyr3’ form a parallel /3-.rtrandwith Ser214 t’o Gly219 of thrombin (Fig. 6: Rydel et al., 19930: (irutter et al., 1990). The lat’ter interaction is cwtipnrallel in PPACK-thrombin (Bode et al.. 1989a. 1991) and in all natural serine proteinase protein inhibitor complexes (Huber & Bode, 1978: Read & James. 1986). From Figure 6, it is clear that the binding of hirudin through the Sl site is not obligatory. The side-chains of Ilel’ and Tyr3’ basically occupy a contiguous spatial region of the acative-site very similar in position to Pro and n-Phe. respectively. of PPACK. The isoleucyl group is situat’rd in what appears to be the S2 subsite of t#hrombin and makes numerous hydrophobic contacts wit#h His57. Tyr60A. TrpSOD, Leu99 and Trplld (Table 5). The apolar site is complicated by the fact that it is expanded to an even larger region by t’he presence of Ile74 which, with Leu99 and Trp215, harbors Tyr3’ of hirudin and n-Phe of PPACK in their

T. J. Rvdel et al

590

Figure 6. Interaction of N-terminal tripeptide with the active-site pairs shown by _t; hydrogen bonds shown as broken lines.

respective complexes. This overall hydrophobic region is most likely the apolar binding-site originally reported for indole (Berliner & Shen, 1977) and elaborated upon by others (Sonder & Fenton, 1984; Fenton, 1988a) and first delineated by Bode et aE. (1989a) in the PPACK-thrombin structure. The hydrogen bonds from Lys47’ to Thr4’-Asp5 probably aid in effecting the N-terminal active-site interaction by reducing the degrees of freedom from that of a penta- to that of a tripeptide. Lastly, after the N-terminal tripeptide of hirudin penetrates the active-site of thrombin, it is locked into position with a salt-bridge between Asp5’ and Arg221A. Although most of the N-terminal domain of hirudin is not in contact with the thrombin surface (Fig. 4), many polar interactions exist at the interface of the two molecules. For instance, two ion pairs and two hydrogen bonds are in close proximity in the interface. The salt-bridges are Asp5’-Arg22 1A and Glul7’-Arg 173 while hydrogen bonds occur between Serl9’ OG-Lys224 NZ and

of thrombin.

Hirudin

is shown

as a bold line; ion

Va121’ N-Glu217 OEl. In addition, three residues make hydrophobic contacts; Leul3’ and Pro46 are close to Pro60C and Va121’ has two contacts with Ile174. In all, of the 48 residues of the N-terminal domain, 15 (31%) are directly involved in 103 interactions <4*0 A with thrombin (Table 5). The hirudin N-terminal tripeptide makes about half of these contacts (41) most of which are of a hydrophobic or neutral (involving Gly) nature. The C-terminal 17 residues of hirudin, by virtue of an unusually long extended conformation (Fig. 4), interact with a multitude of residues on the thrombin surface. This cliff-like binding exosite of thrombin is an extension of the active-site cleft and it is particularly abundant in positively charged side-chains of the Phe34 to Leu41 and Lys70 to Glu80 loops of thrombin (Bode et aE., 1989a: Fig. 7). Another extensive and highly electropositive surface region exists on the opposite side of the anion-binding exosite (Rydel et al., 1990) which might well be the site of heparin binding (Bode et

245

Figure 7. CA structure orientation

of thrombin and side-chains of positively to Fig. 4; side-chains are shown as bold lines.

charged

residues of anion binding

exosite. In similar

3 A, acceptor

t D, donor.

K36(33)

E39(3.0) R73(2.9. 32) K149E(49)

K60F(3.2)

R173(50)

R221A(3.6)

Contacts < 4.0 A (number in parentheses)

interations

L65(1), R67(3) Y76(5) K36(3), L65(4), 182(2) K36(3)

Y76(5), R77A(8)

F34(3), R73(2). T74(7) T74(4), R75(10), Y76(5)

W60D( 19). K60F(4) E192(3) E39(7) R73(7), T74(8). K149E(l)

E146(1), E192(1) R221A(4) P6OC(l) E217(2), G219(2). R221A(4) R173(1) R221A(l) E217(5), R221A(4), K224(3) R173(14), E217(2) 1174(2). E217(6) P6OC(3), W96(3) P6wl) W6OD(2)

C191(2), E192(2), G216(2). G219(3), C220(1) G216(5), E217(2). G219(1)

H57(7). Y60A(4), WsOD(2). L99(2) Sl95(1), S214(1). W215(3), G216(2)

intermolecular

Table 5

Ion pairs D(A)

159 P60 Y63’ Q65

2.9 %9

3.0 2.9 32

3.2

3.0

31

30 2.8

3.1 3.0 3.2

d(A)

R77A(3.3)

T740 E57’0E2

E390E2 D55’OD 1 D55’0D2

HBl’NDl R73NH2 R73NHl

E57’N Y76N

E49’OE 1

E2170El

V21’N

KGOFNZ

519’0

K224NZ

G2160 Y3’0

S1950G S2140 11’0

Hydrogen bonds Af

E58’

F56 E57

E49 s50 H51’ D55’

T4’ D5’ L13’ L15 E17’ G18 s19 N20’ V21’ K24’ P46 K47’

Y3’N G219N

Il’N Il’N G216N

11’

T2’ Y3’

w

Residues

Hirudin-thrombin

13

I2 19

23 3 7 16

2

2 4 1 8 1 1 12 16 8 6

10 9

22

Number of contacts

1

t 21

I

I 93

I

62

Tot>als

592

T. J. Rude1 et al.

Figure 8. Side-chain interactions brtwern Asp%’ to Cln6.i’ and thrumbin. Hirudin is shown as a bold shown by k; hydrogen bonds shown as broken lines: water molec~ules designated with an asterisk: # neighhoring molecule: Phe.56 omitted

al.. 1991) since the latter binds at an exosite different from that of fibrinogen (Church et al.. 1989). The first three residues of the C-terminal of hirudin, Glu49’. Ser50’ and His51’, participate exclusively in salt-bridge and hydrogen-bond interactions with thrombin. Residue Glu49’ forms an ion pair with Lys6OF and interacts indirectly with Arg35 through a mediating water molecule (W.508). Moreover. the imidazolium ion of His51’ makes an ion pair with Glu39 while OG of Ser50’ makes a bifurcated polar contact with the carboxylate oxygen atoms of Glu192. Thus, three positive and three negative charges are concentrated into a relatively small region of space producing a highly polar micro-environment. The next three residues of this e?gment’ are poorly defined in the electron density maps even though they were included in structure factor calculations. In all. Glu49’ to His 51’ have 33 contacts <4.0 a with thrombin including two ion pairs and one hydrogen bond (Table 5). The comp1ementarit.v of the anion-binding exosite and hirudin continues in the second segment of the (‘-terminal (Asp55’ to Gln65’), with t’hree to four more ion pairs. where eight of the 11 residues have 81 thrombin contacts <4.0 A (Table 5) with three-quarters of the electrostat’ic interactions concentrated in the first four residues (Fig. 8). The carhoxylate group of Asp5.5’ is hydrogen-bonded and salt-bridged with Arg73 and even makes a lesser salt-bridge contact with Lys149E. The y-cleavage site of g-thrombin is located at Lys149E. The charged interactions of the (‘-t,erminal are inter,rupted at this point when Phe56’ penetrates into a hydrophobic depression in the thrombin surface. The most notable cont’act here is an edge-on aromatic stacking interaction hetween Phe56’ and Phe34’ of thrombin. wit,h Trpl41 in the background. A total of ten hydrophobic contacts <4.0 A art’

made between Phe34 and Thr74 by l’he56’ (‘l’abl~~ 5). The Jjolar int’eractions pick up again after Phe56’ (Fig. 8) with Glu57’ which is engaged in t,wo hydrogen bonds. one with Thr74 and another with Tyr76: moreover. the carbonyl grouJ) of (~1~57’ hydrogen bonds to water W4Ol which in turn hydrogen bonds to NH1 and SH2 of &4rg67 of thrombin (Table 6). This glutamate residue also makes ten van der Waals csontacts < 4.0 ,& with Arg75 but even though Arg75 is nearby. Glu57’ makes an intermolwulur hydroyen-bond& ion pni,r with Arg75* of a neighboring thrombin molecule (Arg75*N~~(~lu57’OJ”,l. 23 A: Arg75*SH2--Glufji’OE2, 2.9 -4). However. the ~10s~~ proximity of Arg75 to C:lu57’ in the hirudin-thrombin com~)lrx suggests that these t,wc) residues Probably form a salt-bridge in the c~~mJ)lew in solution. Although the densit’\: of (‘13 and (Xi of’ (ilu58’ is Jjoor. nonetheless. it aJ)pears t,hat it formh a direct ion pair with Arg77A while (ilu.57 interacts more distantly wit’h this arginyl grouJ) through it mediating wat,er molecule (w467) (Table 6. Fig. 8). Considering the numher and diversity of the intrractions of Glu57’ and (:lufiX’ (Table 5). it is noi surprising t#hat t,hey are critical for efficient binding of the (!-terminal to thrombin and ant,it,hrombin act#ivit,v (Krstenansky it nl.. 19X7). Furthermortb. it should be noted that, A\rp77A is at the P-cleavage, sit,e of t’hrombin. Thus. t,he (‘-terminal of hirudirr hinders access of the two principal auto-cleavagr~ sites of’ cc-thrombin by interacting with t ht~ Pl residues (Lpsl49E and Arg7iA). Thtb first four residues of the second extended stretch of thcl C’-t]erminal producca t,he most int’imat,r c*ont)ac.t region with 60 thrombin contacts ~4.0 a. which includes three ion Jjairs and four hydrogen bonds (Table 5). An unexpectedly large number of’ hydrophobic* interactions follow the basically electrostatic nature of the Jjreceding contacts of the hirudin (l-terminal.

ReJined Structure of the Hirudin-Thrombin

Complex

593

Table 6 Water-mediated interactions Hirudin (H)

water (W)

Occupancy

A. Hirudin-thromhin Tyr3’ OH ASP5’ OD2 Gly18’ 0 AsndO ODI Asn20’ ODl

W606 W622 W622 w573 W723

052 079 0.79 l+JO 0.52

AsnZO’

ND”

W723

0.52

Mu49

Olcl 0 0 OE2 OE2

W508 w401 W401 W461 W467

097

H. Hirudin Thr4’ N Asp5’ ODP LeulY’ 0 (‘IulS’ N c&I62 s

W469 W622 W672 w502 W472

c:lu57’ Glu57’ Glu57’ MU.58

1.oo 1m 1.00 1.00

@79 0.65

These are concentrated in the vicinitv of the C-terminus 3,, helical turn (Fig. 8). The side-chains of Ile59’-Pro60’ and Tyr63’-Leu64’ are all on one side of the turn and form a non-polar face which fits into a hydrophobic cavity on the thrombin surface created by Leu65, Tyr76 and Ile82. In addition, Ile59’ makes three close contacts with Leu64’. The most abundant intermolecular contacts (Table 5) occur between Pro60’/Tyr76(5) and Tyr63’/Leu65, Ile82(6). Whether this obviously important hydrophobic interactSion of hirudin is also an integral part of the anion binding site of fibrinogen is a question which yet remains to he determined. The C terminus of Gln65’ concludes the binding of hirudin with an ion pair t’o Lys36. Lastly, whereas Glu57’ and Glu58’ are important’ in the binding of hirudin, the glutamate residues at position 61’ and 62’, the latter being ill-defined. extend out in solvent and display no interactions with thrombin. It has already been mentioned that recombinant hirudins lack a sulfated Tyr63’. This leads to about a tenfold reduction in the affinity of hirudin binding to thrombin (Dodt et al., 1988; Braun et al., 1988) but still leaves the binding constant in the picomolar range (Degryse et aZ., 1989). The rHV2-K47 used in the present work lacks a sulfated Tyr63’ but it, was pointed out elsewhere (Rydel et al., 1990) that Lys81 and Lys109 to LysllO are nearby and could conceivably form ion pairs with a sulfated tyrosine residue by free bond rotations. However, we have since solved the structure of the hirugen-thrombin complex at 2.2 A resolution where hirugen is the dodecapetide Asn53’ to Leu64’ C-terminal of hirudin but which additionally possesses a sulfated Try63’ residue (Skrzypczak-Jankun et al., 1991). The hirugen binds to thrombin in a similar manner to hirudin but with the sulfat’e oxygen atoms of Tyr63’

Thrombin (T)

H’..W(A)

Tyr6OA Arg221A Arg221A Arg173 &xl71 Lys224 Serl71 Lys224 Aig35 Ax67 Arg67 Arg77A Arg77A

OH NH2 NH2 x 0 NZ 0 NZ NH1 NH1 NH2 NE NE

3.0 2.6 2.9 2.3 3.3

Leul3 Gly18 Lys47’ Am20 Glu62’

0 0 0 0 OF“

2.7 2.6 3.1 3.0 2.9

A_

W...T(A)

3.3 3.2 2.4 2.4 3.7 2.7

2.8 2.9

2% 2.9 2.9

hydrogen bonding to Try76 OH, Ile82 h’ with a nearby water molecule. Thus, Tyr63’ does not appear to participate in ion pair interactions and the increase in binding affinity due to posttranscriptional sulfation results from a precisely organized hydrogen-bonding network with thrombin. The importance of hydrophobic int’eractions in the anticoagulant activity of the Ct,erminal of hirudin has been suggested from studies with synthetic polypeptides (Krstenansky et al., 1987). These indicate that the minimal peptidr necessary for detection of activity is Phe56’ to Gln65’ and that it is sensitive to modification of residues Phe56’. Glu57’, Ile59’. pro60 and Leu64’. The role of the first two of these has already been discussed: that of the remaining three is most likely associated with their ability to form a 3,c helical turn with a hydrophobic face along with the additional interactions accompanying Tyr63’ sulfation. Disruption of t)he 3,, turn leads to a diminution of the binding at a complementary patch on the thromhin surface (Table 5). A number of water-mediated int,eractions occur not only between the N-terminal domain and thrombin but with the C-terminal pept#ide as well, and such contacts are another important component of the hirudin binding interaction. These are listed in Table 6 and are characterized by highoccupancy factors. In particular, while Asp5’ and Arg221A form a salt-bridge, W622 also bridges Gly18’ with the interaction: this water molecule makes three hydrogen bonds involving Asp5’ OD2, Gly18’ 0 and Arg221A NH2. It should also he noted that practically all of the hirudin-thrombin wat,ermediated interactions involve a positively charged arginine residue of thrombin. Five solvent-bridged

T. J. Rydel et al.

594

Table 7 Loss of solvent-accessibility

hirudin-thrombin :fOLoss Hirudin 1 4 49 55 59

to to to to to

3 48 51 58 65

(total) 91 20 47 56 36

Table 8

surface of hirudin in the complex % Loss (main-chain) 85 7 30 32 35

Root-mean-square the autolysis

O/o Loss (side-chain) 93 25 51 62 36

intramolecular hydrogen bonds occur within the hirudin molecule providing additional stability t,o the inhibitor (Table 6). The total number of intermolecular contacts of <4.0 i% between hirudin and thrombin is 217 which involve 26 or 65 or 40% of the hirudin residues (Table 5). These interactions comprise eight ion pairs (possibly nine with an intramolecular Glu57’-Arg75*), 13 hydrogen bonds and numerous hydrophobic contacts at the N and C terminus regions of hirudin. The close complementarity displayed by the hirudin and thrombin molecules that produces such an abundance of stabilizing although independently generally interactions, weak, together are responsible for the high degree of affinity and specificity which is displayed between these two molecules. The extensive hirudin-thrombin interactions lead to noteworthy aspects in the solvent-accessibility of the complex. The accessible surface area has been calculated for u-thrombin, hirudin and the hirudin-thrombin complex using a probe of 1.4 a radius (Lee & Richards, 1971). In the complex, hirudin blocks 12% of the thrombin-accessible surface masking 10 y0 of the main-chain and 12 y. of the side-chains. The coverage is relatively small because hirudin binds to only a small fraction of a Conversely, much larger thrombin surface. thrombin masks about 38% of the solvent-accessibility of hirudin where the loss is 24% for mainchain and 42% for side-chains. Moreover, the bulk of the loss is concentrated in small regions (Table 7) which are also the stretches of hirudin which make a large number of contacts with thrombin (Table 5). The N-terminal tripeptide is practically buried with a total loss of 91% accessibility upon complexation and the folded N-terminal domain, which only interfaces thrombin, shows the least amount of loss (20%), most of it residing in the side-chains (Table 7). The loss in the anion-binding exosite region totals about half but most of it is in side-chains consistent with them being buried by the The relatively hirudin-thrombin interaction. exposed main-chain of hirudin in this region agrees with its general lack of interactions with thrombin (Table 5). Another notable aspect of the hirudin-thrombin interaction is the number of insertions with respect to the chymotrypsin sequence that appear to be of

Residue Lys145 Blu146 Thr147 Trp148 Thrl49 Alltl49A Asn149H Val149C GIy159D Lysl49E Gly150

diflerences

between the residues vj

loop of hirudin and PPACK-thrombin. Main--chain

(A)

0.8 1.5 44 7.2 i.5 7.5 X.8 6.6 51 2.8 0.9

Side-chains

(A)

3.2 1.1 5.2 X.0 95 I.4 5+ 2.3 1.5

pertinent utility in thrombin (Fig. 1). The 6OA-I insertion loop narrows access to the active-site with Tyr60A-Pro60B-Pro60C-Trp6OD also being a major component of the S2 apolar site. Furthermore, 60C and 60D interact with Pro46’ and Pro48’ at the beginning of the polyproline helix. Other important insertions participating in interactions already mentioned are Lys60F, 4rg77A, Lys149E and Arg221A. The autolysis loop of thrombin is topographically located in the vicinity of the B-C chain cleavage-site of chymotrypsinogen that leads to the formation of cr-chymotrypsin; the insertions in the loop add diversity in the case of thrombin to make it susceptible to cleavage at several positions by proteases exhibiting different specificities leading to the several different forms of thrombin. The only major insertion unaccounted for is that of the Gly186C loop which will be discussed below.

(c) Conformational

chanye

When hirudin binds to thrombin, the latter undergoes a change in c.d. that is not exhibited in PPACK inhibition (Konno et al., 1988). Furthermore, a change in c.d. is also observed when hirugen-like peptides bind to thrombin (Mao et al., 1988), although this change need not necessarily correspond to the same conformational transition. Consequently, an optimal superposition of t,he thrombin structure of the hirudin complex and PPACK-thrombin was carried out to ascertain structural differences by determining a rotation matrix and a translation vector that minimizes the sum of the squares of the differences between the atomic positions in the two. The r.m.s. difference for the CA, C and N atoms of 818 atom pairs is 0.45 A. which reduces to 0.27 A (609 pairs) when deviations greater than 1 (T are not considered while the r.m.s. difference for side-chain atoms is 1.4 A (1103 pairs). The differences of the main-chain are within the expected error of the two determinations while that of the side groups is about twice that expected. Although the structure of thrombin is essentially

ReJined Structure of the Hirudin-Thrombin

F

102

-c 195

595

102

c 57 1'

Complex

P6 57 1' I?@

-c 195

K

Figure 9. Stereoview of the conformational difference between hirudin-thrombin and PPACK-thrombin (GM46 to Gly150). Hirudin-thrombin is shown as a bold line; hirudin X-terminal pentapeptide and catalytic triad of the thrombin complex also included.

Figure 10. Stereoview of crystal packing interactions involving the autolysis loop shown as a bold line; hydrogen bonds are shown as broken lines; neighboring (b) PPACK-thrombin; in different orientation from hirudin-thrombin. Autolysis shown as bold lines; neighboring molecules are indicated with asterisks; aromatic F245*.

loop. (a) Hirudin-thrombin; autolysis molecules are indicated with asterisks. loop and G219C220 to C191E192 are residues are W51*, Y89*, W23’7* and

596

T. J. Rydel et al.

the same in PPACK-thrombin and the hirudin nonetheless, complexes, a large conformational difference occurs in the autolysis loop region between Lysl45 to (21~150 of the hirudin complex (Table 8) that could correspond to the conformational transition producing the change in c.d. The average r.m.s. difference of the main-chain in this region is 4.9 A and that of side-chains is 4.6 A. The conformational difference of the autolysis loop is shown in Figure 9 from which it will be seen that the position of Trp148 in PPACK-thrombin is sterically incompatible with the position of the N-terminal of hirudin in the hirudin-thrombin complex. In the case of the latter, Trp148 is encircled by the autolysis loop. Thus, such a steric collision in the formation of the hirudin complex could be the trigger for the onset of a conformational change. However. since hirugen-like peptides also induce a c.d. change on thrombin binding, it would seem that’ binding in the anion exosite is responsible for the conformational change. This would also be in agreement with kinetic studies of hirudin inhibition that indicate that: (1) C-terminal binding precedes the docking of the N-terminal domain since the rate of t,he first step of complex formation decreases with increasing ionic strength and (2) it is unaffected by binding at t,he active site (Stone & Hofsteenge, 1986; Braun et nl., 1988). However, examining the electron density complex in the vicinity of of the hirugen-thrombin the aut’olysis loop (Skrzypczak-Jankun et rcl.. 1991) shows that there is little or no electron density for the Thr147 t,o Lys149E octapeptide so that, it is disordered in the crystal structure. This observation is consistent with the flexibility that the autolysis between structures of the loop displays PPACK-thrombin and the hirudin complex. The fact that the autolysis loop is sometimes ordered (hirudin and PPACK-thrombin) and sometimes not (hirugen-thrombin; hirulog-thrombin: Skrzypczak-Jankun rt al.. 1991) led us to examine it’s intermolecular crystal packing contacts in the four structures. Whrle there are no major intermolecular contacts in the region of the isomorphous hirugenthrom bin, hirulog-throm bin struct,ures. in the case of the hirudin-thrombin complex, N and (‘-terminal residues from another molecule make eight hydrogen bonds with the autolysis loop (Fig. 10). The amide nitrogen atom of AlalB and the carbonyl group of GlylD form hydrogen bonds with the peptide of Trp148. Furt#hermore. Gln24-4. tjhe residue of ordered the next-to-last C-terminal. participates in four hydrogen honds and t,he aromatic rings of Trp148 and Phe2-15 are in the stacking edge-on aromatic highly stabilized The sit,uation is analogous in interaction. PPAC:K-thrombin, except here, Trp148 is involved in stabilizing intermolecular contacts consisting of a large intermolecular hydrophobic aromat,ic clust.er (Fig. 10). Thus, the evidence points fairly conclusively to flexibility in the autolysis loop which is constrained in different ordered conformations in huirudin-thrombin and PPACK-thrombin by interactions. The cd. packing intermolecular

changes displayed by the hirudin and hirugen-like complexes must therefore be related t’o different conformations of the autolysis loop and in t,he flexibility inherent’ thereof. The source or trigger of t,he conformational changes remains t,o he determined, 4. Discussion The average H-values of thrombin (31.5 -4’) and hirudin (48.3 A) reveal a noteworthy aspect of the hirudin binding interaction: hirudin is more disordered than thrombin in the crystal structure. The R-values of hirudin are about ,509, greater than those of thrombin and since a difference of this magnitude cannot be solely due to thermal vibrations. it, suggests that the hirudin molecule does not bind to thrombin with the highest, degree of fidelity. even though its binding constant is in the femtomolar range. This is not surprising for the Thr4’ to Pro48’ domain interaction that only has ‘1 relatively small number of cornacts in the interface region (Table 5) for its tot’al bulk (62<4.0 I! for 43 residues). However. the same does not apply for Asp55’ t,o ~~1~58’. which participates in 60 contacts has average B-values (55 I”, <4.0 .a, hut significantly; greater than the N-terminal domain. A better fidehty of binding is displayed b): the 3,, helical turn wit.h an average H-value of approximately 42 A’. Nonetheless. the electron density of bot,h of these regions is well-defined and nnarnbiguous and its interpretation is as reliable as for tht thrombin part’ (Fig. 11). Thus. it appears that) the binding of hirudin, although tenacious, most likely possesses a distribution over a number of different positionings. A similar observation was also made in the structure determination of the hiruyrn thrombin comples (Skrzypcz~~k-,JankuIl rf (II.. 1991). ?;ot surprisingly. the best hinding is displayrtl by the X-terminal pentapeptide that is cornpletel>~ buried in the active-site (Table 7) ant1 has ittt H-value (1% _A2) similar to that ot’ the average thrombin molecule. From Figure 1. it is clear that the binding of hirudin to t,hrombin through the Sl specificity site is not required. which is consist,ent with hirudin binding to inact,ivated forms of thrombin (St,one it nl.. 1987). However. chemical moditications of the X terminus of hirudin have shown its critical role in complexation. In particular. extension of the ?; terminus affects complex formation adverseI> (Bergmann et ccl., 1986: Loison it 01.. 19X8) and a positively charged N terminus appears to stabilize the complex (Wallace ut al.. 1989). The crystals were grown and stored at pH 4.5. which implies a prot,onated His?57 as well as a charged Ilel’. These charges are buried in the complex. requiring dispersion which is accomplished t,hrough the His57 link IO Asp102 and the many other polar and hydrogen bonding interactions of these groups. An unexpected aspect of the hirudin binding is the role that Lys47’ appears to assume. The last three residues of the N-terminal domain. Pro46’. Lys4T’ mtl Pro/H’. are only inclire+involved in

Refined Structure of the Hirudin-Thrombin

Complex

597

(D)

Figure 11. Stereoview of the electron density of Asp55’ to Glu65’ in hirudin-thrombin ProGO’to Glu65’: contoured at 1.5 0.

the N-terminal active-site interaction (Fig 5 and 6). However, this is not because this stretch resembles an a-thrombin substrate-like sequence with Lys47’ at the specificity position and a proline residue at the apolar P2 position (Chang, 1985) nor because Cys39’ to Pro48’ displays 50% homology to Arg148 to Ser157 of the thrombin cleavage-site of prothrombin (Dodt et al., 1988), although it has often been viewed that Lys47’ is the Pl residue of substrate in the hirudin-thrombin complex (Chang, 1983; Braun et aZ., 1988; Johnson et al., 1989). The lysyl side-chain does not occupy the specijkity pocket of thrombin and is located approximately 11 A away from it (Fig. 6). This is in agreement with sitedirected mutagenesis modifications that failed to detect a critical role for Lys47’ (Dodt et al., 1988; Braun et aE., 1988; Degryse et al., 1989). The hydrogen bonds of the a-amino group of Lys47’ to

complex. (a) Asp55’ to Ile59’; (b)

Thr4’0G and Asp5’0 and the water-mediated interaction of Asp5’ with Arg221A (Figs 5 and 6) could conceivably help maintain the N-terminal tripeptide in an extended conformation by reducing the conformational freedom from that of a pentapeptide in order for the former to penetrate the active-site and form a hydrogen bond with Ser195. The Asp5’-Arg221A salt-bridge then anchors the N-terminal peptide durably to thrombin when The contacts between docking is completed. Pro46’-Lys47’ and ProGOC-Trp6OD probably also assist in the anchoring. The Sl specificity pocket of thrombin, which displays a preference for arginyl and lysyl sidechains, is only marginally occupied by Thr2’ in the hirudin-thrombin complex (Figs 6 and 12). Therefore, its structure should be a good approximation of the site in the native state of u-thrombin.

598

T. J. Rude1 et al.

Figure 12. Specificity site channel. Ilel’ to Tyr3’ of hirudin and Asp189 of thrombin molecules are indicated by crosses; Gly186C insertion loop at one end of channel.

The Sl site is formed by two large loops comprising Cys182 to Ser195 and Va1213 to Tyr228. The residues of these loops are characterized by a high content of polar side-chains (5 Arg/Lys, 8 Asp/Glu) or essentially no side-chains (7 Gly/Ala) that line an elongated channel extending from the active-site all the way to the surface of the molecule. One wall of the channel is abundant in aromatic residues while the entrances are from the active-site and from the thrombin surface, where, in the case of the latter, it is also lined with the hydrophilic residues of the Gly186C insertion loop (Fig. 1) accounting for the last major insertion in thrombin with respect to chymotrypsin. The arginyl group of PPACK in the PPACK-thrombin structure fits into this channel and forms an ion pair with Asp189 located about half way down the channel (Bode et al., 1989a), while in the hiruden-thrombin complex, the 61 site is not penetrated (Fig. 12) and very significantly, the channel is occupied by 25 water molecules (11 with occupancy factors of 1.0, averaging 0.92). The distribution of the water molecules in this network is shown in Figure 12 from which it should also be clear that at least some of the water must be displaced and/or expelled from the specificity site upon binding an arginyl or lysyl residue. The channel is about 8 A in diameter and about 18 to 19 A in length and is marked by the hydrophobic aromatic wall contrasting its counterpart which is lined with hydrophilics. Moreover, Asp189 of the Sl site is also part of an RGD sequence on the hydrophilic wall of the channel with Arg187 about 7 to 8 A from the Gly186C loop entrance (Fig. 12). The RGD sequence, known to be generally involved in cell-surface receptor interactions (Ruoslahti $ Pierschbacher, 1987) has been implicated as a possible candidate in platelet binding of thrombin (Glenn et aE., 1980; Charo et al.. 1987; Fenton,

are shown

as bold lines: water

19886). However, although Argl87 is exposed to solvent’ and makes a salt-bridge with Asp222, it, is difficult to envision how it might function in concert with Asp189 in a binding mode since the latter is located in the interior of the channel. Prior to the X-ray crystal structure determination of the hirudin-thrombin complex, Chang (1989) identified inaccessible lysyl residues in the hirudinblocked complex (Lys36, 60F, 70, 109. 110 and 149E) and additionally implied the participation of Arg67, 73, 75 and 77A in the anion-binding recognit#ion exosite and more recently showed that hirugen also protects the same residues from modification (Chang Pt al., 1990). The involvement of Lys36, 60F. 110 and 149E. and Arg67, 73, 75 and 77A in the hirudin-t’hrombin interface have already been discussed, and all appear to he sterically protected by hirudin whereas Lys70 and 109 do not appear to be significantly blocked by hirudin; however, Lys70 forms internal salt-bridges with Glu77 and Glu80 and hydrogen bonds with the carbonyl oxygen atoms of Ser72 and Arg75. Similar lysyl accessibility studies have also been performed by reductive methylation which imply hirudin protect’ion of Lys36, 6OF. 70, 81, 145 and 224 (Zuck & Owen, personal caommunication). Of the newly implicated lysine residues, Lys81 could conceivably interact with a sulfated Tyr63’ (Skrzypczak-Jankun et n.l., 1991) and Lys224 makes an internal ion pair with Glu217; Lys224 also hvdrogen bonds with both Ser1710 and Asn20’ z& mediating water W723 (Table 6). The final lysine residue (Lys145) resides in the autolysis loop and appears to be completely exposed and unprotected by hirudin in the crystal structure but interacts with Gh118 via mediating water molecule W70.5. However, its position might, be influenced by close crystal intermolecular contacts (Fig. 10). Most of the foregoing residues of

Refined Structure of the Hirudin-Thrombin thrombin are located in the anion-binding exosite comprised of loops Phe34 to Leu41 and Lys70 to Glu80. In summary, the coincidences between the chemical protection studies and the crystal structure of the complex are excellent. The few exceptions may be the result of interactions or differences between solution and crystal structures. BPTI forms a complex with trypsin comparable to that of hirudin-thrombin (Huber & Bode, 1978), which possesses a Ka of about 1013 M-I (Lazdunski interaction is et al., 1974). The BPTI-trypsin exemplary in that: (1) it engages trypsin as a substrate presenting the carbonyl group of the scissile peptide bond within 2.6 a of Ser1950G of the catalytic site in an antiparallel B-strand interaction involving Prol3’ to Lysl5’ of BPTI and Ser214 to Gly216 of trypsin, (2) it utilizes the Sl site by means of Lysl5’ at the Pl subsite which forms an ion pair with the carboxylate group of Asp189, (3) its interactions with trypsin are generally concentrated in and around the active site region (Read & James, 1986). Hirudin, by contrast, makes a parallel p-strand in the active-site and hydrogen bonds with Ser1950G, does not use the Sl specificity site and additionally interacts extensively with regions distantly removed from the active-site. Moreover, the BPTT-trypsin interaction is essentially a complementary one between rigid bodies in which the t’wo components exhibit practically perfectly fitting surfaces requiring little entropy loss, whereas the hirudin-thrombin interaction is better thought of as an induced-fit complementary coupling in which a flexible hirudin molecule suffers a considerable entropy loss in adapting to interact extensively with the thrombin molecule. In both complexes, t,he inhibitor loses about 35% of its solvent-accessible surface upon formation, leading to 217 contacts less than 40 a in hirudin-thrombin and 163 in BPTI-trypsin; the number of hydrogen bonds formed also occurs in a similar ratio (13 and 9, respectively). The crucial p-strand interaction in HPTI-trypsin involves five of the hydrogen bonds while five occur with the N-terminal tripeptide of hirudin. However, whereas the hydrogen bonds of BPTI-trypsin are confined to the enzyme-substrate interaction from the P3 to the P2’ subsites, those of hirudin-thrombin are associated mostly with the bindin exosit,e (Table 5). Another highly significant difference between the two complexes is in the ion pairs that are number of formed; hirudin-thrombin has eight, while BPTI-trypsin has only one. In the case of hirudin, two occur in the N-terminal domain while the remainder are found in the C-terminal peptide (Table 5) which wraps halfway around the thrombin molecule. The high affinity that both the inhibitors display for their host is clearly the net result of precise complementarity achieved in different ways. The high affinity of hirudin, which lacks an enzyme substrate-like interaction, is most likely the result of its divalent nature producing many interactions far beyond the active-site region lt is likely that naturally occurring hirudins

Complex

599

interact with the active-site of thrombin as does recombinant hirudin rHV2-K47, since they possess Val-Val-Tyr or Ile-Thr-Tyr N-terminal sequences (Scharf et al., 1989). The importance of maintaining hydrophobic character in the first two positions has been demonstrated in binding studies with specific l’-2’ position site-directed mutants (Wallace et al., 1989) where the experiments showed that replacing the Val-Val residues with polar side-chains resulted in significant increases in inhibition constant, while replacement of the residues with hydrophobic amino acids had little affect on the binding constant. The fact that hirudin interacts in such a novel fashion with the active site region of thrombin coupled with the fact that it displays no close sequence or topological similarity to the ten existing classes of serine inhibitors (Huber & Bode, 1978: proteinase Laskowski & Kato, 1980) suggests that hirudin unknown family of represents a heretofore inhibitors (Dodt et al., 1985). We thank Dr K. G. Ravichandran for his help during the earlier stages of the work and Drs Carolyn Roitsch and John W. Fenton II for providing us with rHVZ-K47 and human a-thrombin, respectively, and for their continued interest and many helpful discussions. This work was supported by NIH grant HL43229 (A.T.) and by the Deutsche Forschungsg
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