ABSTRACT
The crystal structure of native chicken fibrinogen has been determined at a resolution of 5.5 A. The full-length molecule is 460 A 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 alphaC domains nestled within the confines of the sinuous coiled coils. The amino-terminal segments of the alpha- and beta-chains, including the fibrinopeptides A and B, are also disordered.
Subject(s)
Fibrinogen/chemistry , Animals , Cattle , Chickens , Crystallography, X-Ray , Protein ConformationABSTRACT
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.
Subject(s)
Fibrin Fibrinogen Degradation Products/chemistry , Fibrin Fibrinogen Degradation Products/metabolism , Oligopeptides/metabolism , Binding Sites , Calcium/physiology , Calcium-Binding Proteins/metabolism , Computer Simulation , Crystallization , Crystallography, X-Ray , Humans , Ligands , Models, Molecular , Peptide Fragments/chemistry , Protein Binding , Protein ConformationABSTRACT
Antibodies are important tools to explore receptor-ligand interactions. The anti-integrin antibody OPG2 binds in an RGD-related manner to the alphaIIb beta3 integrin as a molecular mimic of fibrinogen. The Fab fragment from OPG2 was cocrystallized with a peptide from the beta3 subunit of the integrin representing a site that binds RGD. The crystal structure of the complex was determined at 2.2-A resolution and compared with the unbound Fab. On binding the integrin peptide there were conformational changes in CDR3 of the heavy chain. Also, a significant shift across the intermolecular interface between the CH1-CL domains was observed so that the angle of rotation relating the two domains was reduced by 15 degrees. This unusual conformational adjustment represents the first example of ligand-induced conformational changes in the carboxyl domains of a Fab fragment.
Subject(s)
Binding Sites, Antibody , Immunoglobulin Fab Fragments/chemistry , Oligopeptides/metabolism , Platelet Glycoprotein GPIIb-IIIa Complex/immunology , Protein Conformation , Amino Acid Sequence , Antigen-Antibody Complex/chemistry , Antigen-Antibody Complex/metabolism , Crystallization , Immunoglobulin Fab Fragments/metabolism , Models, Molecular , Molecular Sequence Data , Platelet Glycoprotein GPIIb-IIIa Complex/chemistry , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , SolutionsABSTRACT
The crystal structure of a recombinant alphaEC domain from human fibrinogen-420 has been determined at a resolution of 2.1 A. The protein, which corresponds to the carboxyl domain of the alphaE 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 beta- and gamma-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.
Subject(s)
Fibrinogen/chemistry , Amino Acid Sequence , Animals , Crystallography, X-Ray , Fibrinogen/genetics , Humans , Molecular Sequence Data , Pichia/genetics , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Sequence Homology, Amino AcidABSTRACT
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.
