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 hydropho bic amino acids, viz., F-4, L(5), and L(8), present at the amino-termi nus of the Ca2+-bound form of gamma-carboxyglutamic acid domain (GD) o f human protein C (PC), contributes a substantial portion of the total functional binding energy of PC and its activated form, APC, to acidi c 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 th e properties of a series of mutant proteins containing A(5), V-5, I-5, and W-5 as replacements for L(5) in recombinant (r)-PC and APC. The C a2+- and PL-dependent plasma-based anticoagulant activities of [L(5)A] r-APC, [L(5)V]r-APC, [L(5)I]r-APC, and [L(5)W]r-APC were determined to be approximately 28%, 51%, 98%, and 105%, respectively, of that of wi ld-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 a ssays. Apparently normal Ca2+-dependent conformations were adopted by each of the mutant proteins, but the Ca2+-bound form of [L(5)A]r-PC wa s relatively the most defective of the mutants in its binding to FL. T hese results confirm the importance of the hydrophobic character at se quence position 5 as critical to the functional binding of PC to FL.