Crystal structure of the central region of bovine fibrinogen (E ₅ fragment) at 1.4-Å resolution

Abstract

The high-resolution crystal structure of the N-terminal central region of bovine fibrinogen (a 35-kDa E 5 fragment) reveals a remarkable dimeric design. The two halves of the molecule bond together at the center in an extensive molecular ''handshake'' by using both disulfide linkages and noncovalent contacts. On one face of the fragment, the A␣ and B␤ chains from the two monomers form a funnel-shaped domain with an unusual hydrophobic cavity; here, on each of the two outer sides there appears to be a binding site for thrombin. On the opposite face, the N-terminal ␥ chains fold into a separate domain. Despite the chemical identity of the two halves of fibrinogen, an unusual pair of adjacent disulfide bonds locally constrain the two ␥ chains to adopt different conformations. The striking asymmetry of this domain may promote the known supercoiling of the protofibrils in fibrin. This information on the detailed topology of the E 5 fragment permits the construction of a more detailed model than previously possible for the critical trimolecular junction of the protofibril in fibrin. F ibrinogen, the key structural protein in blood clotting, has a unique and complex dimeric structure: the central so-called ''E'' region, critical for fibrin formation, contains a nexus of chains that bond the two identical halves of the molecule together in a small globular region (Fig. 1). Each monomer of this large (340-kDa) elongated (450-Å-long) molecule consists of three nonidentical chains, A␣, B␤, and ␥, and the N-terminal portions of the six chains are linked together by 11 disulfide bonds at the center. The C termini of each of the three chains also end in globular domains: those of the B␤ and ␥ chains are located at the ends, or D regions, and those of the A␣ chains, the ␣C domains, appear to interact with each other close to the central E region. Except for an extended flexible portion of the ␣C domain, the regions between the globular domains in each half-molecule form ␣-helical coiled-coil structures, so that the E region consists of a globular region with two coiled-coil extensions (for review, see ref. 1).When clotting occurs, thrombin cleaves two pairs of small negatively charged fibrinopeptides from the central E region, and soluble fibrinogen is converted into a relatively insoluble fibrin molecule, which self assembles to form the clot. In this process, the exposed N-terminal ''knobs'' of the ␣ chains in one molecule of fibrin bind to receptor pockets in the terminal ␥C domains of adjacent molecules, leading to the formation of two-stranded half-staggered protofibrils (2, 3). The N-terminal knobs of the ␤ chains are also exposed, and interactions between these knobs and receptor pockets in the ␤C domains may promote assembly of the protofibrils into fibers (4-6). Moreover, release of the fibrinopeptides by thrombin also appears to result in the dissociation of the ␣C domains from the central E region, and these domains can then promote assembly of protofibrils into fibers (7,8). Lateral association of the protofibrils ...

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

“…Furthermore, they subsequently showed (42) that the third pH-dependent site was present in human FG (K d ∼ 9 × 10 -6 M) only when the AR-chains were intact, suggesting that it could be, for symmetry reasons, a composite site involving the two halves of the molecule. In addition, no Ca 2+ ions were found in the recently published FG central E domain structure (80), although it lacked residues AR1-28 and B 1-60. In the end, a consensus exists only for the two high-affinity Ca 2+ -binding sites, which were clearly localized in the FG outer D domains (5-8), confirming previous biochemical work (see ref 35, and references therein).…”

Crystal structure of the complex between thrombin and the central “E” region of fibrin

“…22) and the bovine fibrinogen E 5 fragment (PDB ID 1JY2; ref. 33) were taken as probe models. The rotation search was conducted in the resolution range of 15-3.65 Å, independently for each model.…”

“…The disulfide-linked NH 2 -terminal portions of all six chains form the central E region, whereas COOH-terminal portions form two terminal D regions and two ␣C domains (28)(29)(30). Recent x-ray studies of fibrinogen and its fragments established the high-resolution structure of most of the molecule (31)(32)(33)(34). Specifically, it was found that, in the central region, the NH 2 -terminal portions of the A␣͞B␤ chains and the ␥ chain form the funnel-shaped domain on one side of the molecule and the ␥N-domain on the other side, respectively, while the remaining portions of all three chains in both subunits form triple helical coiled coils (33,34).…”

“…Recent x-ray studies of fibrinogen and its fragments established the high-resolution structure of most of the molecule (31)(32)(33)(34). Specifically, it was found that, in the central region, the NH 2 -terminal portions of the A␣͞B␤ chains and the ␥ chain form the funnel-shaped domain on one side of the molecule and the ␥N-domain on the other side, respectively, while the remaining portions of all three chains in both subunits form triple helical coiled coils (33,34). Fibrin contains two types of thrombin binding sites, termed ''low'' and ''high'' affinity (35).…”

“…The B␤ chain Ala-68 was also suggested to be involved in this interaction because its mutation to Thr in fibrinogen Naples I results in reduced thrombin binding (16). Crystal structures of the bovine fibrinogen E 5 fragment (33) and chicken fibrinogen (34) revealed the spatial positioning of B␤Ala-68 and most of the residues in the thrombin binding portion of the A␣ chain, enabling the prediction of the location of the thrombin binding sites on the surface of the E region (33,34). In the present study, we crystallized thrombin in complex with human fibrinogen fragment E ht , which is devoid of FPA and FPB and established the structure of the complex at 3.65-Å resolution.…”