The hydrophobic nature of residue-5 of human protein C is a major determinant of its functional interactions with acidic phospholipid vesicles.
We have previously proposed that a cluster of surface-exposed hydrophobic amino acids, viz., F4, L5, and L8, present at the amino-terminus of the Ca(2+)-bound form of gamma-carboxyglutamic acid domain (GD) of human protein C (PC), contributes a substantial portion of the total functional binding energy of PC and its activated form, APC, to acidic phospholipid (PL) vesicles. A deeper understanding of the importance of the hydrophobic nature of sequence position 5, and the particular relevance of leucine at that location, was sought by examination of the properties of a series of mutant proteins containing A5, V5, I5, and W5 as replacements for L5 in recombinant (r)-PC and APC. The Ca(2+)- and PL-dependent plasma-based anticoagulant activities of [L5A]r-APC, [L5V]r-APC, [L5I]r-APC, and [L5W]r-APC were determined to be approximately 28%, 51%, 98%, and 105%, respectively, of that of wild-type r-APC. A similar trend in activities of the mutant enzymes was observed in in vitro factor V/Va and factor VIII/VIIIa inactivation assays. Apparently normal Ca(2+)-dependent conformations were adopted by each of the mutant proteins, but the Ca(2+)-bound form of [L5A]r-PC was relatively the most defective of the mutants in its binding to PL. These results confirm the importance of the hydrophobic character at sequence position 5 as critical to the functional binding of PC to PL.
Jalbert
L R
LR
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556, USA.
Chan
J C
JC
Christiansen
W T
WT
Castellino
F J
FJ
D015055:1-Carboxyglutamic Acid / Q000378:metabolism; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000925:Anticoagulants / Q000494:pharmacology; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D018076:DNA, Complementary; D005165:Factor V / Q000037:antagonists & inhibitors; D005169:Factor VIII / Q000037:antagonists & inhibitors; D005453:Fluorescence; D006801:Humans; D008081:Liposomes / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010314:Partial Thromboplastin Time; D010713:Phosphatidylcholines / Q000378:metabolism*; D011485:Protein Binding; D011486:Protein C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins
D000911:Antibodies, Monoclonal; D000925:Anticoagulants; D018076:DNA, Complementary; D008081:Liposomes; D010713:Phosphatidylcholines; D011486:Protein C; D011994:Recombinant Proteins; D015055:1-Carboxyglutamic Acid; D005165:Factor V; D005169:Factor VIII; D002118:Calcium