title: Ionic properties of membrane association by vitamin K-dependent proteins: the case for univalency.
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 (zazb values) of -13 to -17. Factor V showed much higher binding affinity despite nominal zazb values of about 9. Membrane-bound prothrombin and protein Z showed very low sensitivity to salt as long as calcium was at saturating levels (zazb 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 zazb 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.
authors.affiliation: Department of Biochemistry, University of Minnesota, 1479 Gortner Avenue, St. Paul, Minnesota 55108, USA.
authors.forename: J F
authors.initials: JF
authors.lastname: McDonald
authors.forename: T C
authors.initials: TC
authors.lastname: Evans
authors.forename: D B
authors.initials: DB
authors.lastname: Emeagwali
authors.forename: M
authors.initials: M
authors.lastname: Hariharan
authors.forename: N M
authors.initials: NM
authors.lastname: Allewell
authors.forename: M L
authors.initials: ML
authors.lastname: Pusey
authors.forename: A M
authors.initials: AM
authors.lastname: Shah
authors.forename: G L
authors.initials: GL
authors.lastname: Nelsestuen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002417:Cattle; D004563:Electrochemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005165:Factor V / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010455:Peptides / Q000494:pharmacology; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011516:Prothrombin / Q000737:chemistry / Q000378:metabolism*; D012542:Scattering, Radiation; D012965:Sodium Chloride / Q000494:pharmacology; D013816:Thermodynamics; D014812:Vitamin K / Q000378:metabolism*
chemical_list: D001798:Blood Proteins; D004791:Enzyme Inhibitors; D008081:Liposomes; D010455:Peptides; D010743:Phospholipids; C026080:plasma protein Z; D014812:Vitamin K; D012965:Sodium Chloride; D005165:Factor V; D011516:Prothrombin; D011493:Protein Kinase C; D002118:Calcium