Ionic properties of membrane association by vitamin K-dependent proteins: The case for univalency

John F. McDonald, Thomas C. Evans, Dale Brown Emeagwali, Meena Hariharan, Norma M. Allewell, Marc L. Pusey, Amit M. Shah, Gary L Nelsestuen

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Abstract

Ionic properties of membrane interaction by prothrombin, protein Z, and other vitamin K-dependent proteins were studied to determine the relevance of a monovalent membrane contact mechanism between one phospholipid headgroup and a calcium-lined pore in the protein [McDonald, J. F., Shah, A.M., Schwalbe, R. A., Kisiel, W., Dahlback, B., and Nelsestuen, G. L. (1997) Biochemistry 36, 5120-5127]. For comparison, multivalent ionic interaction was illustrated by peptides of +3 to +5 net charge and by blood clotting factor V. As expected, the peptides were easily dissociated by salt and gave nominal charge-charge interactions (Z(a)Z(b) values) of -13 to -17. Factor V showed much higher binding affinity despite nominal Z(a)Z(b) values of about 9. Membrane-bound prothrombin and protein Z showed very low sensitivity to salt as long as calcium was at saturating levels (Z(a)Z(b) values of approximately -1.3 to -1.4), appropriate for univalent ionic attraction. Prothrombin contains +3 charge groups (Lys-2, Lys-11, Arg-10) that are absent from the GLA domain (residues 1-35) of protein Z, while protein Z contains - 4 charge groups (Gla-11, 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 addition of one calcium ion, bringing the total calcium stoichiometry in the protein-membrane complex to at least 8. Protein Z bound to phosphatidic acid (PA) in a manner suggesting the need for a fully ionized phosphate headgroup, a property expected by ion pairing in an isolated environment. Electrostatic calculations showed that the proposed protein site for phosphate interaction was electropositive. The cluster of hydrophobic amino acids (Phe-5, Leu-6, and Val-9) on the surface of prothrombin 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 pair in an isolated environment.

Original languageEnglish (US)
Pages (from-to)15589-15598
Number of pages10
JournalBiochemistry
Volume36
Issue number50
DOIs
StatePublished - Dec 16 1997

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