The binding energy of human coagulation protein C to acidic phospholipid vesicles contains a major contribution from leucine 5 in the gamma-carboxyglutamic acid domain.
A mutant cDNA of recombinant (r)-human protein C (PC) encoding a Leu5-->Gln substitution ([Leu5-->Gln]r-PC) has been constructed and expressed in human 293 cells. A subpopulation of molecules was then purified that contained fully gamma-carboxylated [Leu5-->Gln]r-PC. Intrinsic fluorescence quenching studies with this mutant protein revealed a C50 for Ca2+ binding (0.24 mM) that was essentially the same as that of wild-type (wt) r-PC (0.35 mM). Additionally, the C50 for the Ca2+ dependence of binding of a Ca(2+)-specific monoclonal antibody to [Leu5-->Gln]r-PC, of 5.4 mM-6.5 mM, was similar to that of wtr-PC (4.5 mM). On the other hand, the anticoagulant activity of the activated form of this mutant ([Leu5-->Gln]r-APC) was only 2% of that of activated wtr-PC (wtr-APC), and the inactivation rate of human factor Va catalyzed by this same mutant enzyme was approximately 10% of that of wtr-APC, at maximal levels of Ca2+. This substantial loss of activity was reconciled with the apparent retention of the integrity of the Ca(2+)-dependent conformation of this mutant by the finding that this Ca(2+)-dependent conformation of [Leu5-->Gln]r-PC interacted poorly with mixed (60:40, w/w) phosphatidylcholine/phosphatidylserine (PL) vesicles. These results suggest that Leu5, a residue strictly conserved in vitamin K-dependent proteins, is required for functional binding of PC to PL vesicles. These findings lend support to recent observations that have shown the importance of hydrophobic interactions in the binding of coagulation factors to PL vesicles. This current work further implicates Leu5 as a possible key participant in this hydrophobic binding energy.
Zhang
L
L
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
Castellino
F J
FJ
D015055:1-Carboxyglutamic Acid / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D006801:Humans; D007700:Kinetics; D007930:Leucine; D008433:Mathematics; D008958:Models, Molecular; D008962:Models, Theoretical; D010743:Phospholipids / Q000378:metabolism*; D017354:Point Mutation; D011486:Protein C / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011516:Prothrombin / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014961:X-Ray Diffraction
D010743:Phospholipids; D011486:Protein C; D011994:Recombinant Proteins; D015055:1-Carboxyglutamic Acid; D011516:Prothrombin; D007930:Leucine; D002118:Calcium