The Hydrophobic Nature of Residue-5 of Human Protein C Is a Major Determinant of Its Functional Interactions with Acidic Phospholipid Vesicles

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Abstract
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 Ca2+-bound form of γ-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 Ca2+- 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 Ca2+-dependent conformations were adopted by each of the mutant proteins, but the Ca2+-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.