The conformation of the activation peptide of protein C is influenced by Ca2+ and Na+ binding

Abstract

Previous studies have suggested that the conformation of the activation peptide of protein C is influenced by the binding of Ca2+. To provide direct evidence for the linkage between Ca2+ binding and the conformation of the activation peptide, we have constructed a protein C mutant in the γ-carboxyglutamic acid-domainless form in which the P1 Arg 169 of the activation peptide is replaced with the fluorescence reporter Trp. Upon binding off Ca2+, the intrinsic fluorescence of the mutant decreases ∼30%, as opposed to only 5% for the wild-type, indicating that Trp169 is directly influenced by the divalent cation. The Kd of Ca2+ binding for the mutant protein C was impaired ∼4-fold compared with wild-type. Interestingly, the conformation of the activation peptide was also found to be sensitive to the binding of Na +, and the affinity for Na+ binding increased ∼5-fold in the presence of Ca2+. These findings suggest that Ca2+ changes the conformation of the activation peptide of protein C and that protein C is also capable of binding Na+, although with a weaker affinity compared with the mature protease. The mutant protein C can no longer be activated by thrombin but remarkably it can be activated efficiently by chymotrypsin and by the thrombin mutant D189S. Activation of the mutant protein C by chymotrypsin proceeds at a rate comparable to the activation of wild-type protein C by the thrombin-thrombomodulin complex.