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

Abstract

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 a-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 SI specificity pocket; formation of a hydrogen-bonded two-strand antiparallel 3-sheet with Ser214-Gly216) and the a-keto amide group of CtA are primarily responsible for binding to thrombin, with the a-keto amide serving as a transition-state analogue. A special interaction with the "insertion loop" of thrombin (Tyr6OA-Thr60I) is manifested through engagement of the hydroxyphenyl group of CtA with Trp6OD as part of an "aromatic stacking chain." Biochemical inhibition data (K, values at 37C) were obtained for CtA with thrombin and a diverse collection of serine proteases. Thus, CtA is just a moderate inhibitor of human ae-thrombin (K, = 0.18 ,IM) but a potent inhibitor of trypsin (Kj = 0.023 IAM) and streptokinase (K, = 0.035 ,AM). 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] fragmentcondensation approach, to serve the preparation of cyclotheonamide analogues for structure-function studies.

Amidino-Containing Schiff Base Copper(II) and Iron(III) Chelates as a Thrombin Inhibitor

“…), [11][12][13][14] aeruginosin 298-A, aeruginosins 98-A and 98-B (from the blue-green alga Microcystis aeruginosa). [15][16][17] On the other hand, many compounds having benzamidine or arginine moiety as a partial structure have been synthesized and their inhibitory effects on thrombin examined.…”

From natural to synthetic multisite thrombin inhibitors

“…76 The molecular basis of CtA-thrombin interaction has been ascertained by the crystal structure analysis of the human ␣-thrombin-CtA-hirugen complex. 9,10 CtA shows a canonical serine proteinase-inhibitor interaction mode, in which the Pro-Arg segment occupies both the apolar S2 site and S1 specificity pocket, as found in the thrombin-PPACK (D-PhePro-Arg-chloromethylketone) complex. 25,26 In particular, the peptidyl segment Dpr-Pro-Arg (Dpr: diaminopropionic acid) forms three hydrogen bonds with the backbone atoms of Ser 214 -Gly 216 segment, in a typical antiparallel alignment.…”

Structure of a Bovine Thrombin–Hirudin51−65Complex Determined by a Combination of Molecular Replacement and Graphics. Incorporation of Known Structural Information in Molecular Replacement

“…Both peptides bind to their corresponding thrombins in the fibrinogen exosite, between two surface loops formed by residues Lys70-Phe80 on one side and Phe34-Leu41 On the other, as the corresponding segments do in the hirudin-thrombin complexes (Rydel et al, 1990(Rydel et al, , 1991Griitter et al, 1990;Vitali et al, 1992) and in other thrombin-inhibitor complexes containing hirudin C-terminal peptide fragments (Skrzypczak-Jankun et al, 1991;Qiu et al , 1992;Maryanoff et al , 1993;Wu et al , 1993;Zdanov et al, 1993;Priestle et al, 1993;Stubbs et al, 1992;Banner & Hadvary, 1991). The total solvent-accessible surface area buried in the interaction between residues 51h-65h of the peptide and thrombin is 1667,~ 2 in complex 1 and 1689,~ 2 in complex 2, which is slightly less than one half of the surface area buried in the interaction between intact hirudin and bovine thrombin (Vitali et al, 1992).…”

“…Several structures of complexes of human ct-thrombin with inhibitors containing a C-terminal hirudin peptide in the exosite have been determined (Banner & Hadvary, 1991;Skrzypczak-Jankun et al, 1991;Qiu et al, 1992;Stubbs et al, 1992;Maryanoff et al, 1993;Priestle, Rahuel, Rink, Tones & Grfitter, 1993;Zdanov et al, 1993). The thrombin-hirudinsl_6S structure reported here differs from previous studies by (1) incorporating bovine thrombin, which has a lower binding affinity for hirudin instead of human thrombin, (2) using a high ionic strength precipitant instead polyethylene glycol, and (3) having the hirudin peptide complexed with both ct-thrombin and ethrombin in the asymmetric unit.…”