Interaction of the N-terminal region of hirudin with the active-site cleft of thrombin.

Site-specific substitutions of the first five amino acids of the thrombin inhibitor hirudin have been made and the effects of these substitutions on the kinetics of formation of the thrombin-hirudin complex evaluated. The effects of different substitutions of Val1 indicate that nonpolar interactions play a major role in the binding of this residue. In the second position (Val2), polar amino acids were better accommodated than in the first. The mutant with arginine in the second position bound particularly well to thrombin; its dissociation constant was 9-fold lower than that of wild-type recombinant hirudin. Comparison of the effects of single and double mutations involving Val1 and Val2 indicates that there was no cooperativity in the binding of these two residues. Elimination of the hydrophobic interactions made by the aromatic ring of Tyr3 of hirudin resulted in a large loss of binding energy (12.7 kJ mol-1). Replacement of Thr4 of hirudin by serine and alanine suggested that both the gamma-methyl and the hydroxyl group of the threonine were important in the stabilization of the thrombin-hirudin complex. Replacement of Asp5 of hirudin by alanine and glutamate caused about the same loss in binding energy (5 kJ mol-1). The effects of site-specific substitutions are discussed in terms of the crystal structure of the thrombin-hirudin complex. Molecular modeling provided plausible explanations for many of the observed effects. For instance, such studies suggested that the improved binding of the mutant with arginine in the second position could be due to an interaction of the arginine with the primary specificity pocket.     

Molecular dissection of Na+ binding to thrombin

Na(+) binding near the primary specificity pocket of thrombin promotes the procoagulant, prothrombotic, and signaling functions of the enzyme. The effect is mediated allosterically by a communication between the Na(+) site and regions involved in substrate recognition. Using a panel of 78 Ala mutants of thrombin, we have mapped the allosteric core of residues that are energetically linked to Na(+) binding. These residues are Asp-189, Glu-217, Asp-222, and Tyr-225, all in close proximity to the bound Na(+). Among these residues, Asp-189 shares with Asp-221 the important function of transducing Na(+) binding into enhanced catalytic activity. None of the residues of exosite I, exosite II, or the 60-loop plays a significant role in Na(+) binding and allosteric transduction. X-ray crystal structures of the Na(+)-free (slow) and Na(+)-bound (fast) forms of thrombin, free or bound to the active site inhibitor H-d-Phe-Pro-Arg-chloromethyl-ketone, document the conformational changes induced by Na(+) binding. The slow --> fast transition results in formation of the Arg-187:Asp-222 ion pair, optimal orientation of Asp-189 and Ser-195 for substrate binding, and a significant shift of the side chain of Glu-192 linked to a rearrangement of the network of water molecules that connect the bound Na(+) to Ser-195 in the active site. The changes in the water network and the allosteric core explain the thermodynamic signatures linked to Na(+) binding and the mechanism of thrombin activation by Na(+). The role of the water network uncovered in this study establishes a new paradigm for the allosteric regulation of thrombin and other Na(+)-activated enzymes involved in blood coagulation and the immune response.     

Electrostatic interactions in the association of proteins: an analysis of the thrombin-hirudin complex.

The role of electrostatic interactions in stabilization of the thrombin-hirudin complex has been investigated by means of two macroscopic approaches: the modified Tanford-Kirkwood model and the finite-difference method for numerical solution of the Poisson-Boltzmann equations. The electrostatic potentials around the thrombin and hirudin molecules were asymmetric and complementary, and it is suggested that these fields influence the initial orientation in the process of the complex formation. The change of the electrostatic binding energy due to mutation of acidic residues in hirudin has been calculated and compared with experimentally determined changes in binding energy. In general, the change in electrostatic binding energy for a particular mutation calculated by the modified Tanford-Kirkwood approach agreed well with the experimentally observed change. The finite-difference approach tended to overestimate changes in binding energy when the mutated residues were involved in short-range electrostatic interactions. Decreases in binding energy caused by mutations of amino acids that do not make any direct ionic interactions (e.g., Glu 61 and Glu 62 of hirudin) can be explained in terms of the interaction of these charges with the positive electrostatic potential of thrombin. Differences between the calculated and observed changes in binding energy are discussed in terms of the crystal structure of the thrombin-hirudin complex.     

Role of interactions involving C-terminal nonpolar residues of hirudin in the formation of the thrombin-hirudin complex

The role of interactions involving C-terminal nonpolar residues of hirudin in the formation of the thrombin-hirudin complex has been investigated by site-directed mutagenesis. The residues Phe56, Pro60, and Tyr63 of hirudin were replaced by a number of different amino acids, and the kinetics of the inhibition of thrombin by the mutant proteins were determined. Phe56 could be replaced by aromatic amino acids without significant loss in binding energy. While substitution of Phe56 by alanine decreased the binding energy (delta G degrees b by only 1.9 kJ mol-1, replacement of this residue by amino acids with branched side chains caused larger decreases in delta G degrees b. For example, the mutant Phe56----Val displayed a decrease in delta G degrees b of 10.5 kJ mol-1. Substitution of Pro60 by alanine or glycine resulted in a decrease in delta G degrees b of about 6 kJ mol-1. Tyr63 could be replaced by phenylalanine without any loss in binding energy, and replacement of this residue by alanine caused a decrease of 2.2 kJ mol-1 in delta G degrees b. Substitution of Tyr63 by residues with branched side chains resulted in smaller decreases in delta G degrees b than those seen with the corresponding substitutions of Phe56; for example, the mutant Tyr63----Val showed a decrease in binding energy of 5.1 kJ mol-1. The effects of the mutations are discussed in terms of the crystal structure of the thrombin-hirudin complex.     

Characterization of the interactions of a bifunctional inhibitor with alpha-thrombin by molecular modelling and peptide synthesis.

A potent thrombin inhibitor, [D-Phe45, Arg47] hirudin 45-65, that contains an active site-directed sequence D-Phe-Pro-Arg-Pro, an exosite specific fragment hirudin 55-65 (H55-65) and a linker portion hirudin 49-54, was designed based on the hirudin sequence [DiMaio et al. (1990) J. Biol. Chem., 265, 21698-21798]. A three-dimensional model of the complex between the B-chain of human thrombin and the inhibitor [D-Phe45, Arg47] hirudin 45-65 was constructed using molecular modelling starting from the X-ray C alpha coordinates of the thrombin-hirudin complex and the NMR-derived structure of the thrombin-bound hirudin 55-65. The contribution of the H49-54 fragment to the thrombin-inhibitor interaction was deduced by examining a series of analogs containing single glycine substitution and analogs with reduced number of residues within the linker. The results were consistent with the molecular modelling observations i.e. the H49-54 fragment serves the role of a spacer in the binding interaction and could be replaced by four glycine residues. The studies on the interaction of the exosite-directed portion of the inhibitor with thrombin using a series of synthetic H55-65 analogs demonstrated that residues AspH55 to ProH60 play a major role in binding to human thrombin where the side chains of PheH56, IleH59 and GluH57 showed critical contributions. Molecular modelling suggested that these side chains may contribute to inter- and intramolecular hydrophobic and electrostatic interactions, respectively.     

The co-crystal structure of unliganded bovine α-thrombin and prethrombin-2: Movement of the Tyr-Pro-Pro-Trp segment and active site residues upon ligand binding

Unliganded bovine α-thrombin and prethrombin-2 have been co-crystallized, in space group P2₁2₁2, using either ammonium sulfate or polyethylene glycol 2000 (PEG2K), and their structures determined at 2.2 Å and 2.3 Å, respectively. Initial phases were determined by molecular replacement and refined using XPLOR to final R factors of 0.187 (R_free = 0.255) and 0.190 (R_free = 0.282) for the salt and PEG2K models, respectively. The apo-enzyme form of bovine α-thrombin shows dramatic shifts in placement for the Tyr-Pro-Pro-Trp segment, for Glu-192, and for the catalytic residues His-57 and Ser-195, when compared to 4 thrombin complexes representing different states of catalysis, namely (1) the Michaelis complex (residues 7-19 of fibrinogen Aa with a non-cleavable scissile bond), (2) enzyme-inhibitor complex (D-Phe-Pro-Arg chloromethylketone), (3) enzyme product complex (residues 7-16 of fibrinopeptide A), and (4) the exosite complex (residues 53-64 of hirudin). The structures of bovine and human prethrombin-2 are generally similar to one another (RMS deviation of 0.68 8,) but differ significantly in the Arg-15/Ile-16 cleavage region and in the three activation domains, which are disordered in bovine prethrombin-2, analogous to that seen for trypsinogen.     

Glucose phosphorylation. Site-directed mutations which impair the catalytic function of hexokinase

Recent studies from this and other laboratories have resulted in the cloning and sequencing of hexokinases from a variety of tissues including yeast, human kidney, rat brain, rat liver, and mouse hepatoma. Significantly, studies on the hepatoma enzyme conducted in this laboratory (Arora, K.K., Fanciulli, M., and Pedersen, P.L. (1990) J. Biol. Chem. 265, 6481-6488) resulted also in its overexpression in Escherichia coli in active form. We have now used site-directed mutagenesis for the first time in studies of hexokinase to evaluate the role of amino acid residues predicted to interact with either glucose or ATP. Four amino acid residues (Ser-603, Asp-657, Glu-708, and Glu-742) believed to interact with glucose were mutated to alanine or glycine, whereas a lysine residue (Lys-558) thought to be directly involved in binding ATP was mutated to either methionine or arginine. Of all the mutations in residues believed to interact with glucose, the Asp-657----Ala mutation is the most profound, reducing the hexokinase activity to a level less than 1% of the wild type. The relative Vmax values for Ser-603----Ala, Glu-708----Ala, and Glu-742----Ala enzymes are 6, 10, and 6.5%, respectively, of the wild-type enzyme. Glu-708 and Glu-742 mutations increase the apparent Km for glucose 50- and 14-fold, respectively, while the Ser-603----Ala mutation decreases the apparent Km for glucose 5-fold. At the putative ATP binding site, the relative Vmax for Lys-558----Arg and Lys-558----Met enzymes are 70 and 29%, respectively, of the wild-type enzyme with no changes in the apparent Km for glucose. No changes were observed in the apparent Km for ATP with any mutation. These results support the view that all 4 residues predicted to interact with glucose from earlier x-ray studies may play a role in binding and/or catalysis. The Asp-657 and Ser-603 residues may be involved in both, while Glu-708 and Glu-742 clearly contribute to binding but are not essential for catalysis. In contrast, Lys-558 appears to be essential neither for binding nor catalysis.     

The structure of residues 7-16 of the A alpha-chain of human fibrinogen bound to bovine thrombin at 2.3-A resolution.

The tetradecapeptide Ac-D-F-L-A-E-G-G-G-V-R-G-P-R-V-OMe, which mimics residues 7f-20f of the A alpha-chain of human fibrinogen, has been co-crystallized with bovine thrombin from ammonium sulfate solutions in space group P2(1) with unit cell dimensions of a = 83.0 A, b = 89.4 A, c = 99.3 A, and beta = 106.6 degrees. Three crystallographically independent complexes were located in the asymmetric unit by molecular replacement using the native bovine thrombin structure as a model. The standard crystallographic R-factor is 0.167 at 2.3-A resolution. Excellent electron density could be traced for the decapeptide, beginning with Asp-7f and ending with Arg-16f in the active site of thrombin; the remaining 4 residues, which have been cleaved from the tetradecapeptide at the Arg-16f/Gly-17f bond, are not seen. Residues 7f-11f at the NH2 terminus of the peptide form a single turn of alpha-helix that is connected by Gly-12f, which has a positive phi angle, to an extended chain containing residues 13f-16f. The major specific interactions between the peptide and thrombin are 1) a hydrophobic cage formed by residues Tyr-60A, Trp-60D, Leu-99, Ile-174, Trp-215, Leu-9f, Gly-13f, and Val-15f that surrounds Phe-8f; 2) a hydrogen bond linking Phe-8f NH to Lys-97 O;3) a salt link between Glu-11f and Arg-173; 4) two antiparallel beta-sheet hydrogen bonds between Gly-14f and Gly-216; and 5) the insertion of Arg-16f into the specificity pocket. Binding of the peptide is accompanied by a considerable shift in two of the loops near the active site relative to human D-phenyl-L-prolyl-L-arginyl chloromethyl ketone (PPACK)-thrombin.     

Quantitative evaluation of the contribution of ionic interactions to the formation of the thrombin-hirudin complex

The effect of ionic strength on the kinetics of inhibition of human alpha-thrombin has been examined by using genetically engineered forms of hirudin that differed only in the number of negatively charged residues in the carboxyl-terminal region of the molecule. Analysis of the data obtained allowed the binding energy for the thrombin-hirudin complex to be divided into contributions from ionic and nonionic interactions. The contribution of nonionic interactions to the binding energy was the same for each of the forms whereas the ionic contribution varied with the charge of the molecule. Each of the negatively charged residues made an approximately equal contribution of -4kJ mol-1 to the binding energy. For native hirudin, ionic interactions accounted for 32% of the binding energy at an ionic strength of zero.     

Interaction of hirudin with thrombin: identification of a minimal binding domain of hirudin that inhibits clotting activity

Hirudin, isolated from the European leech Hirudo medicinalis, is a potent inhibitor of thrombin, forming an almost irreversible thrombin-hirudin complex. Previously, we have shown that the carboxyl terminus of hirudin (residues 45-65) inhibits clotting activity and without binding to the catalytic site of thrombin. In the present study, a series of peptides corresponding to this carboxyl-terminal region of hirudin have been synthesized, and their anticoagulant activity and binding properties to thrombin were examined. Binding was assessed by their ability to displace 125I-hirudin 45-65 from Sepharose-immobilized thrombin and by isolation of peptide-thrombin complexes. We show that the carboxyl-terminal 10 amino acid residues 56-65 (Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln) are minimally required for binding to thrombin and inhibition of clotting. Phe-56 was critical for maintaining anticoagulant activity as demonstrated by the loss of activity when Phe-56 was substituted with D-Phe, Glu, or Leu. In addition, we found that the binding of the carboxyl-terminal peptide of hirudin with thrombin was associated with a significant conformational change of thrombin as judged by circular dichroism. This conformational change might be responsible for the loss of clotting activity of thrombin.     

Antithrombin properties of C-terminus of hirudin using synthetic unsulfated N alpha-acetyl-hirudin45-65

Unsulfated N alpha-acetyl-hirudin45-65 (MDL 27 589), which corresponds to the C-terminus of hirudin1-65, was synthesized by solid-phase methods. The synthetic peptide was able to inhibit fibrin formation and the release of fibrinopeptide A from fibrinogen by thrombin. The catalytic site of thrombin was not perturbed by the synthetic peptide as H-D-Phe-Pip-Arg-pNA hydrolysis (amidase activity) was not affected. The binding of synthetic peptide and thrombin was assessed by isolation of the complex on gel-filtration chromatography. A single binding site with a binding affinity (Ka) of approx. 1.0 X 10(5) M-1 was observed for thrombin-hirudin45-65 interaction. The data suggest that the C-terminal residues 45-65 of hirudin contain a binding domain which recognizes thrombin and yet does not bind to the catalytic site of the enzyme.     

Structural basis of Na+ activation mimicry in murine thrombin

Unlike human thrombin, murine thrombin lacks Na+ activation due to the charge reversal substitution D222K in the Na+ binding loop. However, the enzyme is functionally stabilized in a Na+-bound form and is highly active toward physiologic substrates. The structural basis of this peculiar property is unknown. Here, we present the 2.2 A resolution x-ray crystal structure of murine thrombin in the absence of inhibitors and salts. The enzyme assumes an active conformation, with Ser-195, Glu-192, and Asp-189 oriented as in the Na+-bound fast form of human thrombin. Lys-222 completely occludes the pore of entry to the Na+ binding site and positions its side chain inside the pore, with the Nzeta atom H-bonded to the backbone oxygen atoms of Lys-185, Asp-186b, and Lys-186d. The same architecture is observed in the 1.75 A resolution structure of a thrombin chimera in which the human enzyme carries all residues defining the Na+ pore in the murine enzyme. These findings demonstrate that Na+ activation in thrombin is linked to the architecture of the Na+ pore. The molecular strategy of Na+ activation mimicry unraveled for murine thrombin is relevant to serine proteases and enzymes activated by monovalent cations in general.     

Analysis of the secondary structure of hirudin and the mechanism of its interaction with thrombin

Highly purified hirudin with a specific activity of 13,950 antithrombin units/mg was isolated from a commercial preparation by reversed-phase chromatography. The circular dichroism (CD) spectrum of hirudin was investigated and it was found that the spectrum cannot be accounted for solely in terms of the traditional three components of peptide backbone. It was also found that the CD spectrum of the thrombin-hirudin complex was not additive with respect to the individual spectra of thrombin and hirudin. This deviation from additivity was significant between 210 and 225 nm, indicating alterations in the secondary structures of the proteins during complex formation. When thrombin was titrated with hirudin, the spectral deviation from additivity was sigmoidal, suggesting the cooperative nature of the binding process. Gel filtration of the thrombin-hirudin mixture showed no molecular species greater than a 1:1 complex (Mr 45,500), but gel filtration of free hirudin showed a multimeric form (Mr 51,300) under the same experimental conditions. It is concluded that the cooperative nature of the binding process is due to the binding of thrombin molecules to the multimeric form of hirudin. This initial binding occurs with little or no change in the CD spectrum. In the second step, the multiple complex dissociates to form 1:1 complexes, resulting in larger conformational changes and a considerable increase in binding affinity.     

Impact of protein-protein contacts on the conformation of thrombin-bound hirudin studied by comparison with the nuclear magnetic resonance solution structure of hirudin(1-51).

The impact of protein-protein interactions on the conformation of the N-terminal hirudin domain consisting of residues 1 to 51 in the X-ray crystal structure of a hirudin-thrombin complex was investigated through comparisons with the nuclear magnetic resonance solution structure of hirudin(1-51). The close overall similarity observed between these two structures contrasts with the behavior of the C-terminal 17-residue polypeptide segment of hirudin, which is flexibly disordered in solution but exhibits a defined conformation in the complex with thrombin. Localized structural differences in the N-terminal domain include that residues 1 to 3 of hirudin in the crystalline complex form a hydrogen-bonding network with thrombin that is reminiscent of a parallel beta-sheet. Moreover, the backbone conformation of residues 17 to 20 in the complex does not contain the characteristic hydrogen bond observed for the type II' reverse turn in the solution structure, and the side-chains of Ser19 and Val21 have significantly different orientations in the two structures. Most of these structural changes can be related directly to thrombin-hirudin contacts, which may also be an important factor in the mechanism of hirudin action. In this context, it is of special interest that other residues that also make numerous contacts with thrombin, e.g. Thr4, Asp5 and Asn20, have identical conformations in free hirudin and in the complex.     

pH dependence of the interaction of hirudin with thrombin.

The kinetics of the inhibition of human alpha-thrombin by recombinant hirudin have been studied over the pH range from 6 to 10. The association rate constant for hirudin did not vary significantly over this pH range. The dissociation constant of hirudin depended on the ionization state of groups with pKa values of about 7.1, 8.4, and 9.2. Optimal binding of hirudin to thrombin occurred when the groups with pKa values of 8.4 and 9.0 were protonated and the other group with a pKa of 7.1 was deprotonated. The pH kinetics of genetically engineered forms of hirudin were examined in an attempt to assign these pKa values to particular groups. By using this approach, it was possible to show that protonation His51 and ionization of acidic residues in the C-terminal region of hirudin were not responsible for the observed pKa values. In contrast, the pKa value of 8.4 was not observed when a form of hirudin with an acetylated alpha-amino group was examined, and, thus, this pKa value was assigned to the alpha-amino group of hirudin. The requirement for this group to be protonated for optimal binding to thrombin is discussed in terms of the crystal structure of the thrombin-hirudin complex. Examination of this structure allowed the other pKa values of 7.1 and 9.2 to be tentatively attributed to His57 and the alpha-amino group of Ile16 of thrombin.     

Determinants of the specificity of rotavirus interactions with the alpha2beta1 integrin

The human α2β1 integrin binds collagen and acts as a cellular receptor for rotaviruses and human echovirus 1. These ligands require the inserted (I) domain within the α2 subunit of α2β1 for binding. Previous studies have identified the binding sites for collagen and echovirus 1 in the α2 I domain. We used CHO cells expressing mutated α2β1 to identify amino acids involved in binding to human and animal rotaviruses. Residues where mutation affected rotavirus binding were located in several exposed loops and adjacent regions of the α2 I domain. Binding by all rotaviruses was eliminated by mutations in the activation-responsive αC-α6 and αF helices. This is a novel feature that distinguishes rotavirus from other α2β1 ligands. Mutation of residues that co-ordinate the metal ion (Ser-153, Thr-221, and Glu-256 in α2 and Asp-130 in β1) and nearby amino acids (Ser-154, Gln-215, and Asp-219) also inhibited rotavirus binding. The importance of most of these residues was greatest for binding by human rotaviruses. These mutations inhibit collagen binding to α2β1 (apart from Glu-256) but do not affect echovirus binding. Overall, residues where mutation affected both rotavirus and collagen recognition are located at one side of the metal ion-dependent adhesion site, whereas those important for collagen alone cluster nearby. Mutations eliminating rotavirus and echovirus binding are distinct, consistent with the respective preference of these viruses for activated or inactive α2β1. In contrast, rotavirus and collagen utilize activated α2β1 and show an overlap in α2β1 residues important for binding.     

Electrostatic steering and ionic tethering in the formation of thrombin-hirudin complexes: the role of the thrombin anion-binding exosite-I

Electrostatic interactions between the thrombin anion-binding exosite-I (ABE-I) and the hirudin C-terminal tail play an important role in the formation of the thrombin-hirudin inhibitor complex and serves as a model for the interactions of thrombin with its many other ligands. The role of each solvent exposed basic residue in ABE-I (Arg(35), Lys(36), Arg(67), Arg(73), Arg(75), Arg(77a), Lys(81), Lys(109), Lys(110), and Lys(149e)) in electrostatic steering and ionic tethering in the formation of thrombin-hirudin inhibitor complexes was explored by site directed mutagenesis. The contribution to the binding energy (deltaG(degrees)b) by each residue varied from 1.9 kJ mol(-)(1) (Lys(110)) to 15.3 kJ mol(-1) (Arg(73)) and were in general agreement to their observed interactions with hirudin residues in the thrombin-hirudin crystal structure [Rydel, T. J., Tulinsky, A., Bode, W., and Huber, R. (1991) J. Mol. Biol. 221, 583-601]. Coupling energies (delta deltaG(degrees) int) were calculated for the major ion-pair interactions involved in ionic tethering using complementary hirudin mutants (h-D55N, h-E57Q, and h-E58Q). Cooperativity was seen for the h-Asp(55)/Arg(73) ion pair (2.4 kJ mol(-1)); however, low coupling energies for h-Asp(55)/Lys(149e) (deltadeltaG(degrees)int 0.6 kJ mol(-1)) and h-Glu(58)/Arg(77a) (deltadeltaG(degrees)int 0.9 kJ mol(-1)) suggest these are not major interactions, as anticipated by the crystal structure. Interestingly, high coupling energies were seen for the intermolecular ion-pair h-Glu(57)/Arg(75) (deltadeltaG(degrees)int 2.3 kJ mol(-1)) and for the solvent bridge h-Glu(57)/Arg(77a) (deltadeltaG(degrees)int 2.7 kJ mol(-1)) indicating that h-Glu(57) interacts directly with both Arg(75) and Arg(77a) in the thrombin-hirudin inhibitor complex. The remaining ABE-I residues that do not form major contacts in tethering the C-terminal tail of hirudin make small but collectively important contributions to the overall positive electrostatic field generated by ABE-I important in electrostatic steering.     

Basis for the reduced affinity of beta T- and gamma T-thrombin for hirudin

Partial proteolysis of human alpha-thrombin by trypsin results in the formation of beta T-thrombin and gamma T-thrombin which have a reduced affinity for the inhibitor hirudin and the cell-surface cofactor thrombomodulin as well as reduced activity with fibrinogen. The basis of the reduction in affinity of these thrombin derivatives for hirudin has been investigated by examining their kinetics of interaction with a number of hirudin mutants differing in their C-terminal charge properties as well as with a truncated form of hirudin. The results indicate that the reduced affinity of beta T-thrombin for hirudin is most likely due to a decrease in the strength of nonionic interactions between thrombin and the C-terminal region of hirudin. No decrease in the strength of ionic interactions was observed with beta T-thrombin. In contrast, the reduced affinity of gamma T-thrombin was due to a decrease in the strength of both ionic and nonionic interactions. The N-terminal core region of hirudin, which interacts predominantly with the active-site cleft of thrombin, exhibited similar affinities for alpha-, beta T-, and gamma T-thrombin, indicating that thrombin-hirudin interactions within the active site are largely preserved in beta T- and gamma T-thrombin.     

Bifunctional thrombin inhibitors based on the sequence of hirudin45-65

The interaction of alpha-thrombin with the hirudin (HV1) fragment N alpha-acetyl desulfo hirudin45-65 (P51) was investigated. Kinetic analysis revealed that P51 inhibits the proteolysis of a tripeptidyl substrate with Ki = 0.72 +/- 0.13 and 0.11 +/- 0.03 microM for bovine and human alpha-thrombins, respectively. The inhibition was partially competitive, affecting substrate binding to the enzyme-inhibitor complex by a factor alpha = 2 (bovine) and alpha = 4 (human) characteristic of hyperbolic inhibitors. P51 also inhibited thrombin-induced fibrin clot formation with IC50 values of 0.94 +/- 0.20 and 0.058 +/- 0.006 microM for bovine and human alpha-thrombins, respectively. The enhanced antithrombin activity for human thrombin could be attributed to species variations in the putative auxiliary "anion" exosite since N alpha-acetyl desulfo hirudin55-65 displayed the same rank order of potency shift in a clotting assay without inhibiting the amidolytic activity of either enzyme. From these observations, a potent thrombin inhibitor was designed having modified residues corresponding to the P1 and P3 recognition sites. N alpha-Acetyl[D-Phe45, Arg47] hirudin45-65 (P53) emerged as a pure competitive inhibitor with a Ki = 2.8 +/- 0.9 nM and IC50 = 4.0 +/- 0.8 nM (human alpha-thrombin) and is designated as a "bifunctional" inhibitor. Its enhanced potency could be explained by a cooperative intramolecular interaction between the COOH-terminal domain of the inhibitor and the auxiliary exosite of thrombin on the one hand, and the modified NH2-terminal residues with the catalytic site on the other.     

Hirudin binding reveals key determinants of thrombin allostery

Thrombin exists in two allosteric forms, slow (S) and fast (F), that recognize natural substrates and inhibitors with significantly different affinities. Because under physiologic conditions the two forms are almost equally populated, investigation of thrombin function must address the contribution from the S and F forms and the molecular origin of their differential recognition of ligands. Using a panel of 79 Ala mutants, we have mapped for the first time the epitopes of thrombin recognizing a macromolecular ligand, hirudin, in the S and F forms. Hirudin binding is a relevant model for the interaction of thrombin with fibrinogen and PAR1 and is likewise influenced by the allosteric S-->F transition. The epitopes are nearly identical and encompass two hot spots, one in exosite I and the other in the Na+ site at the opposite end of the protein. The higher affinity of the F form is due to the preferential interaction of hirudin with Lys-36, Leu-65, Thr-74, and Arg-75 in exosite I; Gly-193 in the oxyanion hole; and Asp-221 and Asp-222 in the Na+ site. Remarkably, no correlation is found between the energetic and structural involvements of thrombin residues in hirudin recognition, which invites caution in the analysis of protein-protein interactions in general.