Role of the hexapeptide disulfide loop in the gamma-carboxyglutamic acid domain of protein C in Ca2+-mediated structural and functional properties.
The anticoagulant and immunomodulatory effects of protein C (PC) rely on the presence of the N-terminal gamma-carboxyglutamic acid (Gla) domain. This domain is strongly conserved among vitamin K-dependent blood proteins and, in addition to a high relative content of Gla, contains a hexapeptide disulfide loop between Cys residues 17 and 22. In the present study, the contribution of the hexapeptide loop toward Gla domain structure and function was evaluated using wild-type and Cys17/Cys22-alkylated synthetic peptide analogues of the 47-residue Gla domain/helical stack of PC. Circular dichroism and intrinsic fluorescence measurements revealed significant differences in the metal ion-dependent conformations of the two peptides. Disruption of the disulfide loop slightly altered the capacity of the peptide to interact with acidic phospholipid (PL) vesicles. The affinity of the alkylated peptide for soluble endothelial protein C receptor (EPCR), as demonstrated by surface plasmon resonance studies, was increased compared with the wild-type species, although total binding was compromised. These results suggest that the disulfide loop of PC contributes to the overall Ca(2+)-dependent conformation but is not strictly required for PL membrane binding or EPCR recognition.
Dai
Qiuyun
Q
Department of Chemistry and Biochemistry and the W. M. Keck Center for Transgene Research, University of Notre Dame, Notre Dame, Indiana 46556, USA.
Prorok
Mary
M
Castellino
Francis J
FJ
D015055:1-Carboxyglutamic Acid / Q000737:chemistry*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000502:physiology*; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D007700:Kinetics; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry; D011486:Protein C / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D020349:Surface Plasmon Resonance
D004220:Disulfides; D009842:Oligopeptides; D010446:Peptide Fragments; D011486:Protein C; D015055:1-Carboxyglutamic Acid; D002118:Calcium