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.

The structure of fragment double-D from human fibrin has been solved in the presence and absence of the peptide ligands that simulate the two knobs exposed by the removal of fibrinopeptides A and B, respectively. All told, six crystal structures have been determined, three of which are reported here for the first time: namely, fragments D and double-D with the peptide GHRPam alone and double-D in the absence of any peptide ligand. Comparison of the structures has revealed a series of conformational changes that are brought about by the various knob-hole interactions. Of greatest interest is a moveable "flap" of two negatively charged amino acids (Glubeta397 and Aspbeta398) whose side chains are pinned back to the coiled coil with a calcium atom bridge until GHRPam occupies the beta-chain pocket. Additionally, in the absence of the peptide ligand GPRPam, GHRPam binds to the gamma-chain pocket, a new calcium-binding site being formed concomitantly.

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The crystal structure of native chicken fibrinogen has been determined at a resolution of 5.5 Å. The full-length molecule is 460 Å in length and sigmoidally shaped. The structure includes the full sweep of the coiled coils that connect the central and terminal domains; the chain paths of the central domain confirm a predicted scheme of planar disulfide rings in apposition with each other. Electron density maps have revealed the outlines of disordered ␣C domains nestled within the confines of the sinuous coiled coils. The amino-terminal segments of the ␣-and ␤-chains, including the fibrinopeptides A and B, are also disordered.central domain ͉ coiled coils ͉ x-ray structure F ibrinogen is a large (molecular mass ϭ 340 kDa) hexameric (␣ 2 ␤ 2 ␥ 2 ) glycoprotein found in the blood plasma of all vertebrate animals; it is converted into fibrin clots by the thrombin-catalyzed removal of peptides from the aminoterminal regions of the ␣-and ␤-chains. Tens of thousands of studies have been conducted on this system in the past, and its general properties are well known. Shadow-cast electron microscope images long ago revealed a triglobular structure approximately 470 Å in length (1), the subdomainal structure of which has been partially visualized by negative staining electron microscopy and low resolution x-ray crystallography (2, 3). The native protein has proved very difficult to crystallize, however, and no diffraction-grade crystals have been reported. There have been some recent successes in determining x-ray structures of certain core fragments. The first of these to be reported was a 30-kDa recombinant protein corresponding to the carboxylterminal domain of the ␥-chain of human fibrinogen (4); it was followed shortly thereafter by a structure for the 86-kDa fragment D, also from human fibrinogen, and its crosslinked equivalent from fibrin (5-7). The D fragments account for about half the mass of the fibrinogen molecule. Most recently, the partial structure of a 285-kDa moiety prepared from bovine fibrinogen was reported (8).One of the problems in obtaining good crystals of native fibrinogen has been attributed to the presumed mobility of the carboxyl domains of the ␣-chains. These domains (␣C) are the most variable parts of the molecule on a species-to-species basis (9). They are easily trimmed away by proteolysis and are often referred to as ''free-swimming appendages.'' Moreover, most contain a series of repeated sequences (10, 11) that are thought to add to the flexible nature of the region. As it happens, chicken fibrinogen ␣-chains lack these repeats (12), an attribute that led us to attempt crystallization of the native protein. Indeed, we were able to obtain large, easily managed crystals in short order. Like crystals of modified bovine fibrinogen (3, 8), these crystals have a high solvent content and diffract anisotropically.We now report a structure for native chicken fibrinogen at 5.5 Å, determined by the method of molecular replacement by using high-resolution structures of fragment D (5-7) as search mod...

The crystal structure of a recombinant ␣ E C domain from human fibrinogen-420 has been determined at a resolution of 2.1 Å. The protein, which corresponds to the carboxyl domain of the ␣ E chain, was expressed in and purified from Pichia pastoris cells. Felicitously, during crystallization an amino-terminal segment was removed, apparently by a contaminating protease, allowing the 201-residue remaining parent body to crystallize. An x-ray structure was determined by molecular replacement. The electron density was clearly defined, partly as a result of averaging made possible by there being eight molecules in the asymmetric unit related by noncrystallographic symmetry (P1 space group). Virtually all of an asparagine-linked sugar cluster is present. Comparison with structures of the ␤-and ␥-chain carboxyl domains of human fibrinogen revealed that the binding cleft is essentially neutral and should not bind Gly-Pro-Arg or Gly-His-Arg peptides of the sort bound by those other domains. Nonetheless, the cleft is clearly evident, and the possibility of binding a carbohydrate ligand like sialic acid has been considered.