Calcium-binding site beta 2, adjacent to the "b" polymerization site, modulates lateral aggregation of protofibrils during fibrin polymerization.
Structural analysis of recombinant fibrinogen fragment D revealed that the calcium-binding site (beta2-site) composed of residues BbetaAsp261, BbetaAsp398, BbetaGly263, and gammaGlu132 is modulated by the "B:b" interaction. To determine the beta2-site's role in polymerization, we engineered variant fibrinogen gammaE132A in which calcium binding to the beta2-site was disrupted by replacing glutamic acid at gamma132 with alanine. We compared polymerization of gammaE132A to normal fibrinogen as a function of calcium concentration. Polymerization of gammaE132A at concentrations of calcium <or=1 mM exhibited an uncharacteristic 2-3-fold increase in lateral aggregation and fiber thickness compared to normal fibrinogen, while polymerization of variant and normal were indistinguishable at 10 mM calcium. These results suggest that the beta2-site controls the extent of lateral aggregation. That is, when the calcium anchor (beta2-site) is eliminated before "B:b" interactions occur then lateral aggregation is enhanced. We solved structures of fragment D of gammaE132A fibrinogen (rfD-gammaE132A) with and without Gly-His-Arg-Pro-amide (GHRPam) and found no change to the global structure. X-ray diffraction data showed GHRPam binding in the "a" and "b" polymerization sites and that calcium could still bind to the beta2-site of gammaE132A fibrinogen at 70 mM calcium. We found that the gamma2 calcium-binding site (in loop gamma294-301) did not have calcium bound in the structure of fragment D of gammaE132A fibrinogen with GHRPam bound (rfD-gammaE132A+GH). Analysis of structures rfD-gammaE132A+GH and rfD-BbetaD398A+GH indicated that differences in calcium occupation of the gamma2-site resulted from minor conformational changes provoked by crystal packing and GHRPam binding to the "a" site did not directly modulate calcium binding to this site.
Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-7525, USA.
Michael S
MS
Kostelansky
Karim C
KC
Lounes
Li Fang
LF
Ping
Sarah K
SK
Dickerson
Oleg V
OV
Gorkun
Susan T
ST
Lord
D000409:Alanine / Q000235:genetics; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D004151:Dipeptides / Q000378:metabolism; D005337:Fibrin / Q000737:chemistry* / Q000378:metabolism*; D005338:Fibrin Fibrinogen Degradation Products / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005344:Fibrinopeptide A / Q000378:metabolism; D005345:Fibrinopeptide B / Q000378:metabolism; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism
D002135:Calcium-Binding Proteins; D004151:Dipeptides; D005338:Fibrin Fibrinogen Degradation Products; D021122:Protein Subunits; D011994:Recombinant Proteins; C015214:fibrinogen D fragment; C027787:glycylhistidine; D005344:Fibrinopeptide A; D005345:Fibrinopeptide B; D018698:Glutamic Acid; D005337:Fibrin; D013917:Thrombin; D000409:Alanine; D002118:Calcium