Polymerization-defective fibrinogen variant gammaD364A binds knob "A" peptide mimic.
Fibrin polymerization is supported in part by interactions called "A:a". Crystallographic studies revealed gamma364Asp is part of hole "a" that interacts with knob "A" peptide mimic, GPRP. Biochemical studies have shown gamma364Asp is critical to polymerization, as polymerization of variants gammaD364A, gammaD364H, and gammaD364V is exceptionally impaired. To understand the molecular basis for the aberrant function, we solved the crystal structure of fragment D from gammaD364A. Surprisingly, the structure (rfD-gammaD364A+GP) showed near normal "A:a" interactions with GPRP bound to hole "a" and no change in the overall structure of gammaD364A. Of note, inspection of the structure showed negative electrostatic potential inside hole "a" was diminished by this substitution. We examined GPRP binding to the gamma364Asp variants in solution by plasmin protection assay. We found no protection of either gammaD364H or gammaD364V but partial protection of gammaD364A, indicating the peptide does not bind to either gammaD364H or gammaD364V and binds more weakly than normal to gammaD364A. 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 gamma364Asp 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.
Bowley
Sheryl R
SR
Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Merenbloom
Betsy K
BK
Okumura
Nobuo
N
Betts
Laurie
L
Heroux
Annie
A
Gorkun
Oleg V
OV
Lord
Susan T
ST
D015241:Fibrinogens, Abnormal / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D011108:Polymers; D011485:Protein Binding; D011487:Protein Conformation; D055672:Static Electricity
D015241:Fibrinogens, Abnormal; D009842:Oligopeptides; D011108:Polymers; C048770:glycyl-histidyl-arginyl-proline; C034585:glycyl-prolyl-arginyl-proline