High-resolution NMR studies of fibrinogen-like peptides in solution: structure of a thrombin-bound peptide corresponding to residues 7-16 of the A.alpha. chain of human fibrinogen

Abstract

The interaction of the following human fibrinogen-like peptides with bovine thrombin was studied by one- and two-dimensional NMR techniques in aqueous solution: acetyl-Phe(8)-Leu(9)-Ala(10)-Glu-(11)-Gly(12)-Gly(13)-Gly(14)-Val(15)-Ar g(16)- Gly(17)-Pro(18)-NHMe (F6), acetyl-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg(16) (tF6), acetyl-Asp(7)-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg(16)-Gly(17)-Pro- Arg(19)-Val(20)-NHMe (F8), and acetyl-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg(16) (tF8). At pH 5.3 and 25 degrees C, the Arg(16)-Gly(17) peptide bonds in both F6 and F8 were cleaved instantaneously in the presence of 0.5 mM thrombin, producing truncated peptides tF6 and tF8 and other peptide fragments. On the basis of observations of line broadening, thrombin was found to bind to the cleavage products, tF6 and tF8, of peptides F6 and F8. Peptide tF8 may have a higher affinity for thrombin than peptide tF6, as suggested by the more pronounced thrombin-induced line broadening on the proton resonances in peptide tF8. Transferred NOE (TRNOE) measurements were made of the complexes between thrombin and peptides tF6 and tF8. Medium- and long-range NOE interactions were found between the NH proton of Asp(7) and the C beta H protons of Ala(10), between the C alpha H proton of Glu(11) and the NH proton of Gly(13), and between the ring protons of Phe(8) and the C alpha H protons of Gly(14) and the C gamma H protons of Val(15). Sets of structures of the decapeptide tF8 were deduced by use of distance geometry calculations based on sequential and medium- and long-range TRNOEs from the thrombin-bound peptide. A predominant feature of these structures is the nonpolar cluster formed by the side chains of residues Phe(8), Leu(9), and Val(15) that are directly involved in binding to thrombin. This structural feature is brought about by an alpha-helical segment involving residues Phe(8)-Ala(10), followed by a multiple-turn structure involving residues Glu(11)-Val(15). These results provide an explanation for the observations that Asp(7), Phe(8), and Gly(12) are strongly conserved in mammalian fibrinogens and that the mutations of Asp(7) to Asn(7) and of Gly(12) to Val(12), result in delayed release of fibrinopeptide A, producing human bleeding disorders.

Examining Thrombin Hydrolysis of the Factor XIII Activation Peptide Segment Leads to a Proposal for Explaining the Cardioprotective Effects Observed with the Factor XIII V34L Mutation

“…Table I shows sequences for Fibrinogen A␣ (amino acids 7-20) (Fbg A␣- (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)), Factor XIII activation peptide (aa 28 -41) (FXIII AP-(28 -41)), D-Phe-Pro-Arg-chloromethylketone (PPACK), and thrombin receptor (aa 32-45) (PAR1- (32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)). In each peptide, there is an Arg residue at the P 1 position 2 that binds within the catalytic cleft of thrombin forming a salt bridge with thrombin amino acid 3 Asp 189 .…”

“…X-ray crystal studies of PAR1- (32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45) and PPACK reveal that thrombin contains a hydrophobic surface that can accommodate this proline (9,10). Fbg A␣- (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), by contrast, contains a Val at the P 2 position instead of the Pro. The Phe at the P 9 position, however, is proposed to compensate for the absence of the Pro.…”

“…The Phe at the P 9 position, however, is proposed to compensate for the absence of the Pro. This Phe has been shown by x-ray crystallography (11) and NMR (12)(13)(14) studies to participate in a multiple turn conformation that brings this aromatic residue in close proximity to the thrombin cleavage site Fbg A␣ Arg 16 -Gly…”

“…An HPLC assay was used to compare the ability of thrombin to hydrolyze a segment of the Fibrinogen A␣ chain (Fbg A␣- (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)), the native Factor XIII Activation Peptide (FXIII AP-(28 -41)), and the mutant peptide (FXIII AP-(28 -41), V34L). The kinetic values K m , k cat , and k cat /K m were determined for each peptide.…”

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

“…This then places the P313 residue in a cis configuration in the i + 2 position of a type VI turn. This turn motif then permits a favorable interaction of the Ile3" residue with the thrombin hydrophobic cluster, a secondary binding interaction that has been shown to contribute greatly to the conformational specificity of thrombin cleavage [19,20,. The overlay of our proposed gp120 consensus sequence conformation with the thrombin active site is shown in Fig.…”