Crystal Structure of Fragment Double-D from Human Fibrin with Two Different Bound Ligands^,

Abstract

Factor XIII-cross-linked fragment D (double-D) from human fibrin was crystallized in the presence of two different peptide ligands and the X-ray structure determined at 2.3 A. The peptide Gly-Pro-Arg-Pro-amide, which is an analogue of the knob exposed by the thrombin-catalyzed removal of fibrinopeptide A, was found to reside in the gamma-chain holes, and the peptide Gly-His-Arg-Pro-amide, which corresponds to the beta-chain knob, was found in the homologous beta-chain holes. The structure shows for the first time that the beta-chain knob does indeed bind to a homologous hole on the beta-chain. The gamma- and beta-chain holes are structurally very similar, and it is remarkable they are able to distinguish between these two peptides that differ by a single amino acid. Additionally, we have found that the beta-chain domain, like its gamma-chain counterpart, binds calcium.

Kinetics of Fibrinopeptide Release by Thrombin as a Function of CaCl2 Concentration: Different Susceptibility of FPA and FPB and Evidence for a Fibrinogen Isoform-Specific Effect at Physiological Ca2+ Concentration

“…It is tempting to speculate that this may be one of the recently discovered (37-39) additional Ca 2+ -binding sites in the D domain, as already mentioned in the previous section. In fact, one of these sites (37) has been observed in a D dimer fragment after the engagement of a synthetic B knob-like peptide in the b hole, leading to a rearrangement of the -subdomain in a way that favors the lateral aggregation of the fibrin fibers (19,37). Perhaps, at our relatively high (30 mM) Ca 2+ concentration the site becomes occupied, inducing a conformation change even without the B knob engagement.…”

“…Surprisingly, in another crystal structure consisting of a cross-linked human D dimer with two synthetic short analogues of the A and B knobs bound, a second Ca 2+ -binding site was found in a comparable position near the b polymerization hole in the C-terminal portion of the B -chain (37). However, the lack of a fourth coordinating side chain, with respect to the γ-chain site, suggests that this may be one of the controversial lower-affinity Ca 2+ -binding sites (37). Two more weak Ca 2+ -binding sites were subsequently identified by the same authors (38) in human D dimers, although only one was evident in a recombinant human fragment D crystal structure (39).…”

“…Two of the three high-affinity sites were readily localized in the outer D domains (32)(33)(34)(35), and subsequently identified by crystallography near the a polymerization hole in the C-terminal portion of the γ-chain (5,6). Surprisingly, in another crystal structure consisting of a cross-linked human D dimer with two synthetic short analogues of the A and B knobs bound, a second Ca 2+ -binding site was found in a comparable position near the b polymerization hole in the C-terminal portion of the B -chain (37). However, the lack of a fourth coordinating side chain, with respect to the γ-chain site, suggests that this may be one of the controversial lower-affinity Ca 2+ -binding sites (37).…”

Fibrin(ogen)olytic and antiplatelet activities of a subtilisin-like protease from Solanum tuberosum (StSBTc-3)

“…The approximate molecular weights of a, b and g chains are 63, 56 and 47 kDa respectively. The three chains are joined by disulfide bonds within the N-terminal of E-domain [29,30]. As illustrated in Fig.…”

Ultrathin self-assembled fibrin sheets

“…The parent protein, fibrinogen, is an elongated, sausage-shaped dimeric molecule with symmetry around a central globular domain (2)(3)(4)(5)(6). Cleavage and removal of the small peptides-fibrinopeptide A and later B-from fibrinogen by the enzyme thrombin expose specific binding sites (''knobs'') on fibrin that allow binding to corresponding regions (''holes'') on neighboring fibrin molecules (7)(8)(9)(10)(11). Fibrin can then self-associate to form elongated, sometimes twisted, often highly branched fibers and fiber networks that, together with platelets and blood cells, form clots in response to vascular injury (1,(12)(13)(14)(15).…”