Refined 2·3ÅX-ray crystal structure of bovine thrombin complexes formed with the benzamidine and arginine-based thrombin inhibitors NAPAP, 4-TAPAP and MQPA

Brandstetter, Hans; Turk, Dušan; Hoeffken, H.W.; Grosse, D.; Stürzebecher, Jörg; Martin, Philip D.; Edwards, Brian F.P.; Bode, Wolfram

doi:10.1016/0022-2836(92)91054-s

Cited by 193 publications

(179 citation statements)

References 49 publications

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“…Our results show that, in contrast to the compact folded conformations observed for thrombin specific inhibitors [4,[15][16][17][18][19], DX9065a stretches along the active site cleft in an extended manner. This conformation is stabilised by the expected salt bridge formation of the napthamidine group to Asp-189, by hydrophobic interactions between the benzyl spacer and the side of the pyrrolidinyl group with the enzyme surface and by a weak electrostatic interaction of the basic pyrrolidinyl/acetimidoyl group with a previously unidentified electronegative cavity of factor Xa.…”

Section: Discussionmentioning

confidence: 68%

“…This has been shown to be the case for thrombin specific inhibitors [16][17][18][19], even though thrombin and trypsin exhibit even weaker similarity.…”

Section: Discussionmentioning

confidence: 97%

“…However, the active site of factor Xa is blocked in the crystal form examined and no crystals of inhibited factor Xa have been reported as yet. The success in determining the mode of action of thrombin specific inhibitors by introducing them into crystals of trypsin [15][16][17], subsequently verified by their crystal structures in complex with thrombin [18,19] prompted us to investigate the binding of factor Xa inhibitors to the active site of trypsin. In this communication, we present three structures of compounds of the Daiichi class in complex with bovine fl-trypsin, indicating structural requirements for the inhibition of factor Xa for the first time.…”

Section: Introductionmentioning

confidence: 99%

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Crystal structures of factor Xa specific inhibitors in complex with trypsin: structural grounds for inhibition of factor Xa and selectivity against thrombin

1995

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Crystal structures of DX9065a and a related bisamidino-aryl inhibitor specific for the blood-clotting factor Xa have been solved in complex with bovine ~-trypsin to a resolution of 1.9 A. Each inhibitor exhibits an extended conformation along the active site, in contrast to the compact folded structures observed for thrombin specific inhibiturs. Few direct contacts (predominantly in the S1 pocke0 are made between trypsin and the inhibitors. Transfer of the inhibitors to the active site of factor Xa suggests a three-site interaction: salt bridge formation at the base of the primary specificity pocket, extensive hydrophobie surface burial and a weak electrostatic interaction between the distal basic component of the inhibitor and an electronegative cavity of factor Xa formed by three backbone carbonyl oxygens. Additivity of these three interactions is the basis for the observed strong inhibition of factor Xa and provides a framework for the design of novel factor Xa inhibitors. A propionic acid group of the inhibitor would clash with the thrombin specific '60-insertion loop', thus conferring selectivity against thrombin.

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Section: Discussionmentioning

confidence: 68%

“…This has been shown to be the case for thrombin specific inhibitors [16][17][18][19], even though thrombin and trypsin exhibit even weaker similarity.…”

Section: Discussionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

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Crystal structures of factor Xa specific inhibitors in complex with trypsin: structural grounds for inhibition of factor Xa and selectivity against thrombin

1995

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“…The structure was solved by Patterson search techniques with bovine thrombin as obtained in complex with N ␣ -(2-naphthylsulfonylglycyl)-DL-p-amidinophenylalanylpiperidine (19) as a search model. For the monoclinic data with reflections up to 2.6 Å the model was refined to a R value of 0.189 using X-PLOR (20) with the parameters of Engh and * The costs of publication of this article were defrayed in part by the payment of page charges.…”

Section: Methodsmentioning

confidence: 99%

Crystallographic Evidence That the F2 Kringle Catalytic Domain Linker of Prothrombin Does Not Cover the Fibrinogen Recognition Exosite

Locht

Stubbs

Bauer

et al. 1996

Journal of Biological Chemistry

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The 2.6-Å x-ray crystal structure of bovine ␣-thrombin in complex with rhodniin, a protein inhibitor isolated from the bug Rhodnius prolixus, has been solved and refined. The structure has enabled us to trace the Nterminal part of the 49-residue A-chain of bovine ␣-thrombin for the first time, which is fixed in a Ushaped loop on the molecular surface opposite the active site canyon. Model building shows that the 25 amino acid residues that link the A-chain and F2 kringle cannot run through the fibrinogen recognition exosite. This demonstrates that this fibrinogen recognition exosite is available in prothrombin and meizothrombin.Thrombin plays a central role in hemostasis and thrombosis, performing both coagulatory and regulatory functions (see Ref. 1). Prothrombin circulates in the plasma as a 579-residue (in man; 582 residues in bovine) 4-domain zymogen, consisting of an N-terminal Gla (␥-carboxyglutamic acid) domain, two tandem kringle domains, and the catalytic domain (see Fig. 1). Activation of prothrombin via two cleavages at Arg-PT271 (human; Arg-PT274 in bovine) and Arg-15 yields the two-chain ␣-thrombin (for thrombin, A-chain residues from Cys-1 to Arg-15 and the B-chain are identified via chymotrypsinogen numbers deduced from topological equivalence (2, 3); residues preceding Cys-1 are numbered according to human prothrombin and designated by the prefix PT (see Table I)). Depending on the reaction conditions, either bond may be cleaved first (4,5). Under physiological conditions (i.e. in the presence of Ca 2ϩ and negatively charged phospholipids), activation is effected largely via the prothrombinase complex (factors Xa and Va). In this case, the first cleavage occurs at Arg-15, resulting in the catalytically active membrane-bound intermediate meizothrombin. Subsequent cleavage at Arg-PT271 releases the mature two-chain procoagulant ␣-thrombin into the blood vessel. In the absence of factor Va, factor Xa performs these cleavages in reverse order at a much slower rate (4). Due to an additional autocatalytic cleavage at Arg-PT285-Thr-PT286, the human ␣-thrombin A-chain is further truncated to 36 residues.Crystallographic studies of ␣-thrombin reveal a compact molecule, with the B-chain and the central portion of the Achain forming a single globular domain (1-3). The ␣-thrombin molecule is characterized by a deep narrow canyon-like active site cleft, together with two positively charged surface patches, representing the "fibrinogen recognition exosite" and the "heparin binding site" (3). These two sites are of great functional significance in thrombin's interactions with macromolecular substrates and inhibitors (1). Recent crystal structures of ␣-thrombin in complex with F2 kringle (6) reveal that this kringle domain binds to the heparin binding site, largely via specific electrostatic interactions. The structure of prethrombin 2 has also recently been solved (7), exhibiting an overall organization identical to that of ␣-thrombin; the C-terminal part of the A-chain shows some displacement due to the intac...

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“…For example, the Phe8 aromatic ring of the ®brinogen substrate , the Tyr3 side chain of the thrombin inhibitor hirudin (Rydel et al, 1990) and the (d)Phe rings of the peptidomimetic inhibitors (d)Phe-Pro-Arg chloromethylketone (PPACK; Bode et al, 1989) and Ac-(d)Phe-Pro-boroArg-OH (DuP714, Weber et al, 1995), all form edge-to-face interactions with Trp215. To utilize this preferred interaction, aromatic ring systems have been incorporated into many synthetic thrombin inhibitors and the existence of the interaction has been con®rmed in the crystallographic structures of thrombin complexed with several inhibitors, including sodium [(2-naphthyl sulfonyl)glycyl]-dl-p-amidinophenylalanylpiperidide (NAPAP; Brandstetter et al, 1992), a retro-binding peptide inhibitor (Tabernero et al, 1995) and a designed non-peptide inhibitor (Obst et al, 1995).…”

Section: Introductionmentioning

confidence: 99%

Biochemical and Crystallographic Characterization of Homologous Non-peptidic Thrombin Inhibitors Having Alternate Binding Modes

Strickland¹,

Fevig

Galemmo

et al. 1998

Acta Crystallogr D Biol Cryst

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The X-ray crystallographic structure of [N-(3-phenylpropionyl)-N-(phenethyl)]-Gly-boroLys-OH (HPBK, K i = 0.42 nM, crystallographic R factor to 1.8 A Ê resolution, 19.6%) complexed with human -thrombin shows that the boron adopts a tetrahedral geometry and is covalently bonded to the active serine, Ser195. The HPBK phenethyl aromatic ring forms an edge-to-face interaction with the indole side chain of Trp215. Four HPBK analogs containing either electron-withdrawing or electron-donating substitutents at the 3 H position of the phenethyl ring were synthesized in an attempt to modulate ligand af®nity by inductive stabilization of the edge-to-face interaction. Re®ned crystallographic structures of the tri¯uoromethyl (K i = 0.37 nM, crystallographic R factor to 2.0 A Ê resolution = 18.7%),¯uoro (K i = 0.60; R factor to 2.3 A Ê resolution = 18.4%), methoxy (K i = 0.91 nM, R factor to 2.2 A Ê resolution = 19.8%) and methyl (K i = 0.20 nM, R factor to 2.5 A Ê resolution = 16.9%) HPBK analogs complexed with thrombin revealed two binding modes for the closely related compounds. A less than 1.5-fold variation in af®nity was observed for analogs (tri¯uoromethyl-HPBK and¯uoro-HPBK) binding with the edge-to-face interaction. The slight inductive modulation is consistent with the overall weak nature of the edge-to-face interaction. Owing to an unexpected rotation of the phenethyl aromatic ring, the 3 H substituent of two analogs, methoxy-HPBK and methyl-HPBK, made direct contact with the Trp215 indole side chain. Increased af®nity of the 3 H methyl analog is attributed to favorable interactions between the methyl group and the Trp215 indole ring. Differences in inhibitor, thrombin and solvent structure are discussed in detail. These results demonstrate the subtle interplay of weak forces that determine the equilibrium binding orientation of inhibitor, solvent and protein.

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Refined 2·3ÅX-ray crystal structure of bovine thrombin complexes formed with the benzamidine and arginine-based thrombin inhibitors NAPAP, 4-TAPAP and MQPA

Cited by 193 publications

References 49 publications

Crystal structures of factor Xa specific inhibitors in complex with trypsin: structural grounds for inhibition of factor Xa and selectivity against thrombin

Crystal structures of factor Xa specific inhibitors in complex with trypsin: structural grounds for inhibition of factor Xa and selectivity against thrombin

Crystallographic Evidence That the F2 Kringle Catalytic Domain Linker of Prothrombin Does Not Cover the Fibrinogen Recognition Exosite

Biochemical and Crystallographic Characterization of Homologous Non-peptidic Thrombin Inhibitors Having Alternate Binding Modes

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