Biomedical subjects

A Tulinsky

Publications and source records attributed to A Tulinsky.

At least 37 records · Page 2Linked to original sources

PubMed1996

Synthesis, structure, and structure-activity relationships of divalent thrombin inhibitors containing an alpha-keto-amide transition-state mimetic.

A new class of divalent thrombin inhibitors is described that contains an alpha-keto-amide transition-state mimetic linking an active site binding group and a group that binds to the fibrinogen-binding exosite. The X-ray crystallographic structure of the most potent member of this new class, CVS995, shows many features in common with other divalent thrombin inhibitors and clearly defines the transition-state-like binding of the alpha-keto-amide group. The structure of the active site part of the inhibitor shows a network of water molecules connecting both the side-chain and backbone atoms of thrombin and the inhibitor. Direct peptide analogues of the new transition-state-containing divalent thrombin inhibitors were compared using in vitro assays of thrombin inhibition. There was no direct correlation between the binding constants of the peptides and their alpha-keto-amide counterparts. The most potent alpha-keto-amide inhibitor, CVS995, with a Ki = 1 pM, did not correspond to the most potent divalent peptide and contained a single amino acid deletion in the exosite binding region with respect to the equivalent region of the natural thrombin inhibitor hirudin. The interaction energies of the active site, transition state, and exosite binding regions of these new divalent thrombin inhibitors are not additive.

Amides↗

PubMed1996

Comparison of the structures of the cyclotheonamide A complexes of human alpha-thrombin and bovine beta-trypsin.

Thrombin, a trypsin-like serine protease present in blood, plays a central role in the regulation of thrombosis and hemostasis. A cyclic pentapeptide, cyclotheonamide A (CtA), isolated from sponges of the genus Theonella, inhibits thrombin, trypsin, and certain other serine proteases. Enzyme inhibition data for CtA indicate that it is a moderate inhibitor of alpha-thrombin (K(i) = 1.0 nM), but substantially more potent toward trypsin (K(i) = 0.2 nM). The comparative study of the crystal structures of the CtA complexes of alpha-thrombin and beta-trypsin reported here focuses on structure-function relationships in general and the enhanced specificity of trypsin, in particular. The crystal structures of the CtA complexes of thrombin and trypsin were solved and refined at 1.7 and 2.0 A resolution, respectively. The structures show that CtA occupies the active site with the Pro-Arg motif positioned in the S2 and S1 binding sites. The alpha-keto group of CtA is involved in a tetrahedral intermediate hemiketal structure with Ser 195 OG of the catalytic triad and is positioned within bonding distance from, and orthogonal to, the re-face of the carbonyl of the arginine of CtA. As in other productive binding modes of serine proteases, the Ser 214-Gly 216 segment runs in a twisted antiparallel beta-strand manner with respect to the diaminopropionic acid (Dpr)-Arg segment of CtA. The Tyr 60A-Thr 60I insertion loop of thrombin makes a weak aromatic stacking interaction with the v-Tyr of CtA through Trp 60D. The Glu 39 Tyr and Leu 41 Phe substitutions in trypsin produce an enhanced aromatic interaction with D-Phe of CtA, which also leads to different orientations of the side chains of D-Phe and the v-Tyr. The comparison of the CtA complexes of thrombin and trypsin shows that the gross structural features of both in the active site region are the same, whereas the differences observed are mainly due to minor insertions and substitutions. In trypsin, the substitution of Ile 174-Arg 175 by Gly 174-Gln 175 makes the S3 aryl site more polar because the Arg 175 side chain is directed away from thrombin and into the solvent, whereas Gln 175 is not. Because the site is occupied by the Dpr group of CtA, the occupancy of the S3 site is better in trypsin than in thrombin. In trypsin, the D-Phe side chain of CtA fits between Tyr 39 and Phe 41 in a favorable manner, whereas in thrombin, these residues are Glu 39 and Leu 41. The higher degree of specificity for trypsin is most likely the result of these substitutions and the absence of the fairly rigid Tyr 60A-Thr 60I insertion loop of thrombin, which narrows access to the active site and forces less favorable orientations for the D-Phe and v-Tyr residues.

Animals↗

PubMed1996

Crystal structures of thrombin with thiazole-containing inhibitors: probes of the S1' binding site.

Structures of the blood clotting enzyme thrombin complexed with hirugen and two active site inhibitors, RWJ-50353 10080(N-methyl-D-phenylalanyl-N-[5-[(aminoiminomethyl)amino]-1- [[(2-benzothiazolyl)carbonyl]butyl]-L-prolinamide trifluoroacetate hydrate) and RWJ-50215 (N-[4-(aminoiminomethyl)amino-1-[2- (thiazol-2-ylcarbonylethyl)piperidin- 1-ylcarbonyl]butyl]-5-(dimethylamino)naphthalenesulfonamide trifluoroacetate hydrate), were determined by x-ray crystallography. The refinements converged at R values of 0.158 in the 7.0-2.3-A range for RWJ-50353 and 0.155 in the 7.0-1.8-A range for RWJ-50215. Interactions between the protein and the thiazole rings of the two inhibitors provide new valuable information about the S1' binding site of thrombin. The RWJ-50353 inhibitor consists of an S1'-binding benzothiazole group linked to the D-Phe-Pro-Arg chloromethyl ketone motif. Interactions with the S1-S3 sites are similar to the D-phenylalanyl-prolyl-arginyl chloromethylketone structure. In RWJ-50215, a S1'-binding 2-ketothiazole group was added to the thrombin inhibitor-like framework of dansylarginine N-(3-ethyl-1,5-pentanediyl)amide. The geometry at the S1-S3 sites here is also similar to that of the parent compound. The benzothiazole and 2-ketothiazole groups bind in a cavity surrounded by His57, Tyr60A, Trp60D, and Lys60F. This location of the S1' binding site is consistent with previous structures of thrombin complexes with hirulog-3, CVS-995, and hirutonin-2 and -6. The ring nitrogen of the RWJ-50353 benzothiazole forms a hydrogen bond with His57, and Lys60F reorients because of close contacts. The oxygen and nitrogen of the ketothiazole of RWJ-50215 hydrogen bond with the NZ atom of Lys60F.

Binding Sites↗

PubMed1996

Molecular Interactions of Thrombin.

Thrombin possesses at least three independent binding sites for substrate, inhibitor, and co-factor molecules, four counting the Na+ ion binding site. The S1 subsite of the active site is specific for an arginine side group, while S2 is a more extended apolar site. The highly electropositive S' subsites of the fibrinogen exosite circumnavigate about a third of the thrombin surface, although evidence suggests molecular recognition of a tetra- or pentapeptide sequence is sufficient for binding. Another highly electropositive region of thrombin that binds the second kringle of prothrombin is the heparin binding site. All three of these sites display distinct binding modes with different molecules. These can arise from tolerance of imprecision of binding and/or from reversal of ligand main chain direction in active site and fibrinogen exosite binding. Preliminary indications suggest similar principles may apply to the heparin site. Such varied behavior easily accounts for the diversity of thrombin functions at the molecular level. The complexity of the behavior is compounded even more by a Na+ ion binding site that produces a procoagulant fast form of thrombin. The slow form (in the absence of Na+ ion) is anticoagulant.

Binding Sites↗

PubMed1995

The Na+ binding site of thrombin.

Thrombin is an allosteric serine protease existing in two forms, slow and fast, targeted toward anticoagulant and procoagulant activities. The slow --> fast transition is induced by Na+ binding to a site contained within a cylindrical cavity formed by three antiparallel beta-strands of the B-chain (Met180-Tyr184a, Lys224-Tyr228, and Val213-Gly219) diagonally crossed by the Glu188-Glu192 strand. The site is shaped further by the loop connecting the last two beta-strands and is located more than 15 A away from the catalytic triad. The cavity traverses through thrombin from the active site to the opposite surface and contains Asp189 of the primary specificity site near its midpoint. The bound Na+ is coordinated octahedrally by the carbonyl oxygen atoms of Tyr184a, Arg221a, and Lys224, and by three highly conserved water molecules in the D-Phe-Pro-Arg chloromethylketone thrombin. The sequence in the Na+ binding loop is highly conserved in thrombin from 11 different species and is homologous to that found in other serine proteases involved in blood coagulation. Mutation of two Asp residues flanking Arg221a (D221A/D222K) almost abolishes the allosteric properties of thrombin and shows that the Na+ binding loop is also involved in direct recognition of protein C and antithrombin.

Allosteric Regulation↗

PubMed1995

Crystallographic structure of a peptidyl keto acid inhibitor and human alpha-thrombin.

The low molecular weight alpha-keto amide inhibitor CVS-1347, benzyl-SO2-Met(O2)-Pro-Arg(CO)((CONH)CH2)-phenyl, is a slow, tight binding inhibitor of alpha-thrombin amidolytic activity having a Ki = 1.28 x 10(-10) M. A complex between human alpha-thrombin and a hydrolysis product of CVS-1347 has been determined and refined using crystallography. The crystals belong to monoclinic space group C2 with cell dimensions of a = 71.08, b = 72.05 and c = 72.98 A and beta = 100.8 degrees. The structure was solved using isomorphous replacement methods and refined with resolution limits of (8.00-1.76) A to an R-value of 0.162. The Pro-Arg core of the inhibitor binds in the S2 and S1 subsites respectively, as is usually observed for Pro-Arg thrombin inhibitors. The Met(O2) side chain does not make any close contacts with the enzyme but influences the conformation of Glu192; the N-terminal benzylsulfonyl group makes an aromatic-aromatic contact with Trp215 in the hydrophobic part of the active site. The alpha-keto carboxylic acid of the proteolyzed inhibitor binds with the carboxylate group in the oxyanion hole, demonstrating that this region can accommodate an anion in a protease-peptide complex. The alpha-keto carbonyl group interacts closely with the two most important residues in the active site: the carbon atom is within a covalent bond distance of the active site Ser195 O gamma and the carbonyl oxygen is hydrogen bonded to His57.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Functions of individual gamma-carboxyglutamic acid (Gla) residues of human protein c. Determination of functionally nonessential Gla residues and correlations with their mode of binding to calcium.

Previous studies from this laboratory have been directed toward elucidation of the roles of individual gamma-carboxyglutamic acid (Gla) residues in Gla domain-related Ca(2+)-directed properties of human protein C (PC) and activated protein C (APC). On the basis of results using recombinant variants of PC containing highly conservative (Asp) mutations of individual Gla residues, it was previously proposed that Gla6, Gla14, and Gla19 may not be essential for properties associated with the Ca(2+)-dependent conformation of the Gla domain of these proteins. In this study, we have demonstrated that radical mutations to Val of Gla residues 14 and 19 resulted in 94% and 82%, respectively, of the Gla domain-related, Ca(2+)- and phospholipid- (PL-) dependent anticoagulant (APTT) activity of wild-type recombinant (wtr) APC, while [Gla6-->Val]r-APC showed a complete loss of this same activity. The more conservative mutant [Gla6-->Gln]r-APC possessed 4% of the APTT activity of wtr-APC, whereas [Gla6-->Asp]r-APC was nearly fully active. As with wtr-PC, both [Gla6-->Val]r-PC and [Gla6-->Gln]r-PC displayed Ca(2+)-dependent intrinsic fluorescence quenching, suggesting that they adopted a Ca(2+)-induced conformation. However, Ca2+ titration data suggested that these conformations were not identical to that undergone by wtr-PC. In addition, the Ca(2+)-mediated binding parameters of [Gla6-->Val]r-PC and [Gla6-->Gln]r-PC to acidic PL vesicles were found to be defective. These data were interpreted at the molecular level using a model for the Gla domain of PC based on the X-ray crystal structure of the Ca2+/bovine prothrombin fragment 1 complex.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Carboxyglutamic Acid↗

PubMed1994

Crystallographic structure of human gamma-thrombin.

In an effort to prepare crystals and determine the structure of alpha-thrombin complexed to a synthetic peptide inhibitor (MDL-28050) of the hirudin 54-65 COOH-terminal region, it was discovered that the crystals were not those of the complex but of gamma-thrombin. Gel electrophoresis studies revealed that autolytic degradation had occurred prior to crystallization. NH2-terminal sequence analysis of these autolytic fragments confirmed the gamma-thrombin product (cleavages at Arg75-Tyr76 and/or Arg77A-Asn78, and Lys149E-Gly150; chymotrypsinogen numbering) with a minor amount of another autolysis product, beta-thrombin (first two cleavages only). The final structure has an R-factor of 0.156 for 7.0-2.5-A data, and includes 186 water molecules. A comparison of gamma-thrombin with the thrombin structure in the alpha-thrombin-hirugen complex revealed that the two structures agreed well (r.m.s. delta = 0.39 A for main chain atoms). These structures possess uninhibited active sites where the disposition of the catalytic triad residues is nearly identical. The electron density in the vicinity of the gamma-thrombin cleavage regions is poor, and only becomes well-defined several residues prior to and after the actual cleavage sites. The extensive disorder evoked by beta-cleavage(s) in the Lys70-Glu80 loop region indicates that this part of the molecule is severely disrupted by autolysis and is the reason exosite functions are dramatically impaired in beta-and gamma-thrombin. Since autolysis did not lead to a major reorganization of the folded structure of alpha-thrombin, the likely structural features of the interaction of thrombin substrate with thrombin enzyme during beta-cleavage have been modeled by docking the exosite region of one molecule at the active site of another.

Amino Acid Sequence↗

PubMed1994

Crystallographic structures of thrombin complexed with thrombin receptor peptides: existence of expected and novel binding modes.

Many of the vital actions of thrombin on platelets and other cells appear to be mediated by the recently cloned seven-transmembrane-domain thrombin receptor. Thrombin activates this receptor by a novel proteolytic mechanism. The amino-terminal exodomain of the receptor contains the sequence LDPRSFLLRNPNDKYEPF. Structure-activity studies with mutant receptors and receptor peptides suggest that this sequence binds to thrombin at two sites: LDPR with the active center of thrombin and KYEPF with the fibrinogen recognition exosite of thrombin. Thrombin then cleaves the Arg41-Ser42 bond to unmask a new amino terminus, which functions as a tethered peptide ligand binding to as yet undefined sites within the body of the receptor to effect receptor activation. We have determined eight crystal structures of thrombin complexed with receptor-based peptides. Each of the two components of the bidentate docking model was captured in individual cocrystals. In one crystal type, the LDPR sequence docked in the active center of thrombin in a manner analogous to d-PheProArg chloromethyl ketone. In other crystals, the KYEPF sequence bound in the fibrinogen anion binding exosite of thrombin in a manner analogous to the DFEEI sequence of the carboxylate-terminal peptide of hirudin. Strikingly, however, generation of a single crystal that includes both components of the anticipated bidentate binding mode was not achieved, apparently because the peptides have a dominant solution S-like conformation that does not bind in a productive way at the active center. This peptide structure apparently favored a novel alternative mode of receptor peptide-thrombin interaction in which the receptor peptides formed an intermolecular bridge between neighboring thrombin molecules, resulting in an infinite peptide thrombin chain in crystals. In this structure, the KYEPF sequence docked in the expected manner at the exosite of one thrombin molecule, but the LDPR sequence docked in an unusual nonproductive mode with the active center of a neighboring molecule. Mutations that removed important determinants of the S-like receptor peptide structure underlying the bridging mode in the receptor itself did not significantly alter thrombin signaling. Additionally, a comparison of receptor density to the responsiveness of a cell did not support a role for receptor oligomerization in signaling. The physiological role for this unexpected intermolecular binding mode, if any, remains to be identified.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1994

Differences in the metal ion structure between Sr- and Ca-prothrombin fragment 1.

The structure of Sr-prothrombin fragment 1 has been solved and refined by restrained least-squares methods at 2.5-A resolution to a crystallographic R value of 0.167. The protein structure is very similar to that of Ca-fragment 1. A polymeric array of five Sr2+ ions separated by about 4.0 A is buried among six gamma-carboxyglutamic acid (Gla) residues; three other Sr2+ ions interact with other Gla residues and are located further apart. One of these was not found in the Ca-fragment 1 structure. The coordination of the Sr2+ ions resembles that of Ca2+, but there are some significant differences between them. The most notable is the lack of water coordination with Sr2+ ions and two conformations for Gla 8, which change the coordination of Sr-2 and Sr-3. A hexose moiety of an oligosaccharide was located in the vicinity of Asn101 that was flexibly disordered in Ca-fragment 1. The new Sr2+ ion found may be involved in metal ion phospholipid binding interactions along with Sr-1, and Sr-7, Sr-8.

Amino Acid Sequence↗

PubMed1994

Conformational rearrangements required of the V3 loop of HIV-1 gp120 for proteolytic cleavage and infection.

HIV gp120 is specifically cleaved at a single site in the V3 loop between Arg315 and Ala316 by thrombin. Previous observations by others have indicated that binding to CD4 enhances the rate of V3 loop cleavage, and that this cleavage is a prerequisite for HIV infection. Other observations also suggest that the cleavage site is in a type II beta-turn centered at Pro313-Gly314. However, our docking experiments indicate that this conformation cannot dock to thrombin and other trypsin-like serine proteases. Thus, based on the thrombin-bound conformation of peptide substrates, we propose that CD4 binding, at a site remote from the V3 loop, induces and stabilizes a trans to cis isomerization of the highly conserved residue Pro313, and that this conformational shift is a prerequisite for cleavage by a 'thrombin-like' cellular protease and subsequent infection.

Binding Sites↗

PubMed1994

Kringle-kringle interactions in multimer kringle structures.

The crystal structure of a monoclinic form of human plasminogen kringle 4 (PGK4) has been solved by molecular replacement using the orthorthombic structure as a model and it has been refined by restrained least-squares methods to an R factor of 16.4% at 2.25 A resolution. The X-PLOR structure of kringle 2 of tissue plasminogen activator (t-PAK2) has been refined further using PROFFT (R = 14.5% at 2.38 A resolution). The PGK4 structure has 2 and t-PAK2 has 3 independent molecules in the asymmetric unit. There are 5 different noncrystallographic symmetry "dimers" in PGK4. Three make extensive kringle-kringle interactions related by noncrystallographic 2(1) screw axes without blocking the lysine binding site. Such associations may occur in multikringle structures such as prothrombin, hepatocyte growth factor, plasminogen (PG), and apolipoprotein [a]. The t-PAK2 structure also has noncrystallographic screw symmetry (3(1)) and mimics fibrin binding mode by having lysine of one molecule interacting electrostatically with the lysine binding site of another kringle. This ligand-like binding interaction may be important in kringle-kringle interactions involving non-lysine binding kringles with lysine or pseudo-lysine binding sites. Electrostatic intermolecular interactions involving the lysine binding site are also found in the crystal structures of PGK1 and orthorhombic PGK4. Anions associate with the cationic centers of these and t-PAK2 that appear to be more than occasional components of lysine binding site regions.

Amino Acid Sequence↗

PubMed1994

The isomorphous structures of prethrombin2, hirugen-, and PPACK-thrombin: changes accompanying activation and exosite binding to thrombin.

The X-ray crystal structure of prethrombin2 (pre2), the immediate inactive precursor of alpha-thrombin, has been determined at 2.0 A resolution complexed with hirugen. The structure has been refined to a final R-value of 0.169 using 14,211 observed reflections in the resolution range 8.0-2.0 A. A total of 202 water molecules have also been located in the structure. Comparison with the hirugen-thrombin complex showed that, apart from the flexible beginning and terminal regions of the molecule, there are 4 polypeptide segments in pre2 differing in conformation from the active enzyme (Pro 186-Asp 194, Gly 216-Gly 223, Gly 142-Pro 152, and the Arg 15-Ile 16 cleavage region). The formation of the Ile 16-Asp 194 ion pair and the specificity pocket are characteristic of serine protease activation with the conformation of the catalytic triad being conserved. With the determination of isomorphous structures of hirugen-thrombin and D-Phe-Pro-Arg chloromethyl ketone (PPACK)-thrombin, the changes that occur in the active site that affect the kinetics of chromogenic substrate hydrolysis on binding to the fibrinogen recognition exosite have been determined. The backbone of the Ala 190-Gly 197 segment in the active site has an average RMS difference of 0.55 A between the 2 structures (about 3.7 sigma compared to the bulk structure). This segment has 2 type II beta-bends, the first bend showing the largest shift due to hirugen binding. Another important feature was the 2 different conformations of the side chain of Glu 192. The side chain extends to solvent in hirugen-thrombin, which is compatible with the binding of substrates having an acidic residue in the P3 position (protein-C, thrombin platelet receptor). In PPACK-thrombin, the side chain of Asp 189 and the segment Arg 221A-Gly 223 move to provide space for the inhibitor, whereas in hirugen-thrombin, the Ala 190-Gly 197 movement expands the active site region. Although 8 water molecules are expelled from the active site with PPACK binding, the inhibitor complex is resolvated with 5 other water molecules.

Amino Acid Chloromethyl Ketones↗

PubMed1994

Structure of the non-covalent complex of prothrombin kringle 2 with PPACK-thrombin.

Prothrombin fragment 2 (the second kringle) has been co-crystallized with PPACK (D-Phe-Pro-Arg)-thrombin and the structure of the non-covalent complex has been determined and refined (R = 0.16) at 3.2 A resolution using X-ray crystallographic methods. The kringles interact with thrombin at a site that has previously been proposed to be the heparin binding region. The latter is a highly electropositive surface near the C-terminal helix of thrombin abundant in arginine and lysine residues. These form salt bridges with acidic side chains of kringle 2. Somewhat unexpectedly, the negative groups of the kringle correspond to an enlarged anionic center of the lysine binding site of lysine binding kringles such as plasminogen K1 and K4 and TPA K2. The anionic motif is DGDEE in prothrombin kringle 2. The corresponding cationic center of the lysine binding site region has an unfavorable Arg71Phe substitution but Lys35 is conserved. However, the folding of fragment 2 is different from that of prothrombin kringle 1 and other kringles: the second outer loop possesses a distorted two-turn helix and the hairpin beta-turn of the second inner loop pivots at V64 and D70 by 60 degrees. The Lys35 is located on a turn of the helix, which causes it to project into solvent space in the fragment 2-thrombin complex, thereby devastating the cationic center of the lysine binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Chloromethyl Ketones↗

PubMed1994

The structure of recombinant plasminogen kringle 1 and the fibrin binding site.

The structure of recombinant (Hoover et al. Biochemistry, 1993; 32:10936-10944) plasminogen (PG) kringle 1 (K1) has been determined and refined at 2.48 A resolution to a crystallographic R value of 0.159. In addition, 71 water molecules and two chloride ions have been located. The folding of PGK1 is very similar to that of PGK4. The lysine/fibrin binding site, however, differs from that of both PGK4 and tissue-type PG activator (t-PA) K2 at the cationic centre. Although PGK1 can potentially have a doubly charged cationic centre utilizing Arg34 and Arg71, the side chain of Arg34 is outside of Arg71 in a solvent region and its guanidino group is flexibly disordered. Moreover, site specific mutagenesis studies show unequivocally that Arg34 can be changed to glutamine without affecting the binding ability of PGK1. Thus, PGK1 only has Arg71 at the cationic site, PGK4 has Lys35/Arg71 and t-PAK2 has only Lys33. The cationic site differences may result in subtle responses in the binding affinities of the kringles. The two chloride ions are located in the lysine binding site and effectively compensate the positive charges of the region. They also appear to be involved intermolecularly in a complex way in the crystal structure. Such intermolecular anionic interactions are also found in PGK4 and t-PAK2.

Amino Acid Sequence↗

PubMed1993

Structures of thrombin complexes with a designed and a natural exosite peptide inhibitor.

The structures of two hirudin-based fibrinogen recognition exosite peptide inhibitors with significantly different sequences complexed with alpha-thrombin at a site distinct from the active site (exosite) have been determined crystallographically at 2.2 and 2.3 A resolution. One is a designed synthetic peptide with some nonconventional amino acid residues (MDL-28050), and the other is a natural COOH-terminal peptide isolated from the leech Hirudinaria manillensis (hirullin P18). The structures have been refined by restrained least squares methods to R values of 0.161 and 0.155, respectively. The first stretch of each peptide, corresponding to hirudin 55-59, associates with thrombin similar to hirudin and hirugen (hirudin 53-64). Although the remaining residues of the inhibitors interact with and bind to thrombin, the binding is accomplished. through a rigid body conformational adjustment of the peptide with respect to the conformation displayed by hirudin and hirugen (40 degrees rotation about the Ile59, CA-C bond). This causes the side groups of cyclohexylalanine 64' of MDL-28050 and Ile60, of hirullin to point in the opposite direction of the all important Tyr63, ring of hirudin and hirugen but permits the residues to penetrate and interact with the 3(10) turn hydrophobic binding pocket of thrombin. Thus, the hydrophobic interaction is accomplished in a different way by virtue of the substrate conformational readjustment. The results show that the first stretch of peptide makes concerted and efficient binding interactions with thrombin, and the peptide positions of the inhibitors are fairly specific and homologous so that the stretch appears to be related to specific recognition associated with the exosite. The relative flexibility of structure and sequence of the second stretch is a display of tolerance of imprecision by thrombin in its COOH-terminal hydrophobic association with hirudin-based inhibitors.

Amino Acid Sequence↗

PubMed1993

Molecular basis for the inhibition of human alpha-thrombin by the macrocyclic peptide cyclotheonamide A.

The macrocyclic peptide cyclotheonamide A (CtA), isolated from the marine sponge Theonella sp., represents an unusual class of serine protease inhibitor. A complex of this inhibitor with human alpha-thrombin, a protease central to the bioregulation of thrombosis and hemostasis, was studied by x-ray crystallography. This work (2.3-A resolution) confirms the structure of CtA and reveals intimate details about its molecular recognition within the enzyme active site. Interactions due to the "Pro-Arg motif" (Arg occupancy of the S1 specificity pocket; formation of a hydrogen-bonded two-strand antiparallel beta-sheet with Ser214-Gly216) and the alpha-keto amide group of CtA are primarily responsible for binding to thrombin, with the alpha-keto amide serving as a transition-state analogue. A special interaction with the "insertion loop" of thrombin (Tyr60A-Thr60I) is manifested through engagement of the hydroxyphenyl group of CtA with Trp60D as part of an "aromatic stacking chain." Biochemical inhibition data (Ki values at 37 degrees C) were obtained for CtA with thrombin and a diverse collection of serine proteases. Thus, CtA is just a moderate inhibitor of human alpha-thrombin (Ki = 0.18 microM) but a potent inhibitor of trypsin (Ki = 0.023 microM) and streptokinase (Ki = 0.035 microM). The relative lack of potency of CtA as a thrombin inhibitor is discussed with respect to certain structural features of the enzyme complex. We also report the total synthesis of CtA, by a convergent [2 + 3] fragment-condensation approach, to serve the preparation of cyclotheonamide analogues for structure-function studies.

Amino Acid Sequence↗

PubMed1993

The structure of alpha-thrombin inhibited by a 15-mer single-stranded DNA aptamer.

The structure of a complex between human alpha-thrombin and a GGTTGGTGTGGTTGG 15-nucleotide consensus sequence has been solved by x-ray crystallography and refined at 2.9-A resolution to an R value of 0.159. As in solution, in the complex the single-stranded DNA folds into a structure with two G-quartets. The DNA is sandwiched between two different positively charged regions of two symmetry-related thrombin molecules in the crystal structure making ionic and hydrophobic interactions. One region is the fibrinogen recognition exosite and the other, the putative heparin binding site. The lack of inhibition of fibrinogen clotting and platelet activation by the DNA 15-mer with the Arg75-->Glu mutant of thrombin is consistent with the several salt bridges of the DNA in the fibrinogen exosite. The association of DNA with the heparin site of a neighboring molecule appears to simply compensate residual charge. Differences in the 15-mer loop conformations between the complex and NMR solution structures can be attributed to conformational changes upon thrombin binding. Although G-quadruplexes are favored in the presence of monovalent cations, there is no evidence of the latter in the thrombin complex.

Amino Acid Sequence↗