The contributions of individual gamma-carboxyglutamic acid residues in the calcium-dependent binding of recombinant human protein C to acidic phospholipid vesicles

Abstract

The dependence on the integrity of the human protein C (PC) gamma-carboxyglutamic acid (Gla) domain on its Ca(2+)-dependent binding properties to acidic phospholipid (PL) vesicles has been examined by analysis of these interactions with recombinant (r)-PC Gla domain muteins. The concentration of Ca2+ that results in 50% saturation (C50-Ca) of PL with wild-type (wt) r-PC was not altered by more than 2-fold for the following r-variants of PC, viz. [Gla6-->Asp]r-PC, [Gla14-->Asp]r-PC, [Gla19-->Asp]r-PC, [Gla25-->Asp]r-PC, [Gla29-->Asp]r-PC, and [Gln32-->Gla]r-PC. The C50-Ca was substantially higher than that of wtr-PC for [Gla7-->Asp]r-PC (8.2 mM), [Arg15-->Leu]r-PC (4.5 mM), [Gla16-->Asp]r-PC (> 10 mM), [Gla20-->Asp]r-PC (> 10 mM), and [Gla26-->Asp]r-PC (> 10 mM), indicating that the Ca(2+)-induced conformations of these latter variants interacted poorly with these acidic PL vesicles. Titration of the PL vesicles with wtr-PC at a constant concentration of 20 mM Ca2+ leads to calculation of a concentration of PC that results in 50% saturation of the PL (C50-PC) of 0.38 microM. Essentially this same value was determined for the r-mutants, [Gla7-->Asp]r-PC and [Gln32-->Gla]r-PC. An approximate 2-fold lower C50-PC was obtained for [Gla14-->Asp]r-PC (0.14 microM), [Gla25-->Asp]r-PC (0.16 microM), and [Gla29-->Asp]r-PC (0.15 microM). Somewhat higher C50-PC values were found for [Gla6-->Asp]r-PC (1.2 microM), [Arg15-->Leu]r-PC (1.2 microM), [Gla16-->Asp]r-PC (1.2 microM), [Gla19-->Asp]r-PC (1.8 microM), [Gla20-->Asp]r-PC (1.1 microM), and [Gla26-->Asp]r-PC (1.6 microM). The results of this investigation, in conjunction with other structural and functional studies with these mutants, and the x-ray crystallographic structure of the prothrombin Gla domain-Ca2+ complex, show that Gla16 and Gla26 are the most indispensable Gla residues for maintenance of the functional Ca(2+)-dependent structure of the Gla domain of PC, whereas Gla14 is the least critical Gla residue in this regard. Of the non-Gla residues investigated, Arg15 is of great importance for maintenance of a functional Ca(2+)-dependent structure of PC, and insertion of a Gla residue at position 32, a situation that exists in the cases of some other proteins of this type, does not significantly alter these characteristics of r-PC.