Biomedical subjects

P D Martin

Publications and source records attributed to P D Martin.

12 recordsLinked to original sources

PubMed1992

Solution structure of a platelet receptor peptide bound to bovine alpha-thrombin.

NMR experiments were carried out to study the interaction of thrombin with a synthetic peptide, ESKATNATLDPR, derived from the newly-identified platelet receptor for thrombin [Vu, T.-K. H., Hung, D. T., Wheaton, V. I., & Coughlin, S. R. (1991) Cell 64, 1057-1068]. On the basis of the observation of the thrombin-induced line broadening and transferred NOEs, binding of the peptide was found to be located exclusively within residues LDPR of the proteolytic cleavage site LDPR/S essential for receptor activation by thrombin. Measurement of transferred NOEs and molecular modeling indicate that the side chain of the Asp(P3) residue may form a hydrogen bond with thrombin and, by doing so, it is brought near a positively-charged thrombin residue Arg(221A), thereby partially neutralizing the negative charge of an Asp residue at this site of protein substrates. The hydrophobic side chains of residues Leu(P4) and Pro(P2) reside on the same side of the peptide backbone as indicated by transferred NOEs and were found by modeling to fit into a hydrophobic cage around the thrombin active site. These results suggest that the interaction of thrombin with protein substrates such as prothrombin, protein C, protein S, the platelet receptor, and the A alpha- and B beta-chains of fibrinogen all follow the same canonical binding mode in that the substrate forms an antiparallel beta-strand with thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

The structure of a complex of bovine alpha-thrombin and recombinant hirudin at 2.8-A resolution.

Crystals of the complex of bovine alpha-thrombin with recombinant hirudin variant 1 have space group C222(1) with cell constants a = 59.11, b = 102.62, and c = 143.26 A. The orientation and position of the thrombin component was determined by molecular replacement and the hirudin molecule was fit in 2 magnitude of Fo - magnitude of Fc electron density maps. The structure was refined by restrained least squares and simulated annealing to R = 0.161 at 2.8-A resolution. The binding of hirudin to thrombin is generally similar to that observed in the crystals of human thrombin-hirudin. Several differences in the interactions of the COOH-terminal polypeptide of hirudin, specifically of residues Asp-55h, Phe-56h, Glu-57h, and Glu-58h, and a few differences in the interactions of the hirudin core, specifically of residues Asp-5h, Ser-19h, and Asn-20h, with thrombin from human thrombin-hirudin suggest that there is some flexibility in the binding of these 2 molecules. Most of the residues in the 9 subsites that bind fibrinopeptide A7-16 to thrombin also interact with the NH2-terminal domain of hirudin. The S1 subsite is a notable exception in that only 1 of its 6 residues, namely Ser-214, interacts with hirudin. The only difference between human and bovine thrombins that appears to influence the binding of hirudin is the replacement of Lys-149E by an acidic glutamate in the bovine enzyme.

Amino Acid Sequence↗

PubMed1992

Refined 2.3 A X-ray crystal structure of bovine thrombin complexes formed with the benzamidine and arginine-based thrombin inhibitors NAPAP, 4-TAPAP and MQPA. A starting point for improving antithrombotics.

Well-diffracting crystals of bovine epsilon-thrombin in complex with several "non-peptidic" benzamidine and arginine-based thrombin inhibitors have been obtained by co-crystallization. The 2.3 A crystal structures of three complexes formed either with NAPAP, 4-TAPAP, or MQPA, were solved by Patterson search methods and refined to crystallographic R-values of 0.167 to 0.178. The active-site environment of thrombin is only slightly affected by binding of the different inhibitors; in particular, the exposed "60-insertion loop" essentially maintains its typical projecting structure. The D-stereoisomer of NAPAP and the L-stereoisomer of MQPA bind to thrombin with very similar conformations, as previously inferred from their binding to bovine trypsin; the arginine side-chain of the latter inserts into the specificity pocket in a "non-canonical" manner. The L-stereoisomer of 4-TAPAP, whose binding geometry towards trypsin was only poorly defined, is bound to the thrombin active-site in a compact conformation. In contrast to NAPAP, the distal p-amidino/guanidino groups of 4-TAPAP and MQPA do not interact with the carboxylate group of Asp189 in the thrombin specificity pocket in a "symmetrical" twin N-twin O manner, but through "lateral" single N-twin O contacts; in contrast to the p-amidino group of 4-TAPAP, however, the guanidyl group of MQPA packs favourably in the pocket due to an elaborate hydrogen bond network, which includes two entrapped water molecules. These thrombin structures confirm previous conclusions of the important role of the intermolecular hydrogen bonds formed with Gly216, and of the good sterical fit of the terminal bulky hydrophobic inhibitor groups with the hydrophobic aryl binding site and the S2-cavity, respectively, for tight thrombin active site binding of these non-peptidic inhibitors. These accurate crystal structures are presumed to be excellent starting points for the design and the elaboration of improved antithrombotics.

Amidines↗

PubMed1979