Polymerization-Defective Fibrinogen Variant γD364A Binds Knob “A” Peptide Mimic

Fibrin polymerization is supported in part by interactions called "A:a". Crystallographic studies revealed γ364Asp is part of hole "a" that interacts with knob "A" peptide mimic, GPRP. Biochemical studies have shown γ364Asp is critical to polymerization, as polymerization of variants γD364A, γD364H, and γD364V is exceptionally impaired. To understand the molecular basis for the aberrant function, we solved the crystal structure of fragment D from γD364A. Surprisingly, the structure (rfD-γD364A+GP) showed near normal "A:a" interactions with GPRP bound to hole "a" and no change in the overall structure of γD364A. Of note, inspection of the structure showed negative electrostatic potential inside hole "a" was diminished by this substitution. We examined GPRP binding to the γ364Asp variants in solution by plasmin protection assay. We found no protection of either γD364H or γD364V but partial protection of γD364A, indicating the peptide does not bind to either γD364H or γD364V and binds more weakly than normal to γD364A. We also examined protection by calcium and found all variants were indistinguishable from normal, suggesting the global structures of the variants are not markedly different from normal. Our data imply that γ364Asp per se is not required for knob "A" binding to hole "a"; rather, this residue's negative charge has a critical role in the electrostatic interactions that facilitate the important first step in fibrin polymerization.