IONIC PROPERTIES OF MEMBRANE ASSOCIATION BY VITAMIN-K-DEPENDENT PROTEINS - THE CASE FOR UNIVALENCY

Ionic properties of membrane interaction by prothrombin, protein Z, an d other vitamin K-dependent proteins were studied to determine the rel evance of a monovalent membrane contact mechanism between one phosphol ipid headgroup and a calcium-lined pore in the protein [McDonald, J. F ., Shah, A. M., Schwalbe, R. A., Kisiel, W., Dahlback, B., and Nelsest uen, G. L. (1997) Biochemistry 36, 5120-5127]. For comparison, multiva lent ionic interaction was illustrated by peptides of +3 to +5 net cha rge and by blood clotting factor V. As expected, the peptides were eas ily dissociated by salt and gave nominal charge-charge interactions (z (a)z(b) values) of -13 to -17. Factor V showed much higher binding aff inity despite nominal z(a)z(b) values of about 9. Membrane-bound proth rombin and protein Z showed very low sensitivity to salt as long as ca lcium was at saturating levels (z(a)z(b) values of approximately -1.3 to -1.4), appropriate for univalent ionic attraction. Prothrombin cont ains +3 charge groups (Lys-2, Lys-ll, Arg-10) that are absent from the GLA domain (residues 1-35) of protein Z, while protein Z contains -4 charge groups (Gla-ll, Asp-34, Asp-35) that are absent in prothrombin. Thus, similar z(a)z(b) relationships indicated little role for these surface charges in direct membrane contact. Calcium-saturated protein Z bound to phosphatidylcholine (PC) in a manner which indicated the ad dition of one calcium ion, bringing the total calcium stoichiometry in the protein-membrane complex to at least 8. Protein Z bound to phosph atidic acid (PA) in a manner suggesting the need for a fully ionized p hosphate headgroup, a property expected by ion pairing in an isolated environment. Electrostatic calculations showed that the proposed prote in site for phosphate interaction was electropositive. The cluster of hydrophobic amino acids (Phe-5, Leu-6, and Val-9) on the surface of pr othrombin was electronegative, suggesting a role in the electrostatic architecture of the GLA domain. Overall, membrane binding by vitamin K -dependent proteins appeared consistent with the formation of an ion p air in an isolated environment.