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.

Trans−Cis Isomerization of Proline 22 in Bovine Prothrombin Fragment 1: A Surprising Result of Structural Characterization

“…Both potential surfaces (cis and trans) appear similar, and considering that there is no significant difference in the secondary structure for the two forms, this is not surprising. The blue cavity marked by circle A is the pore (attractive to phospholipids) described by McDonald et al (25,26). This pore is also well preserved in both cis and trans conformations.…”

“…This mechanism depends at least partially on the penetration of some hydrophobic residues into the lipid surface upon calcium binding (4,5,8,20,42). In a recent model, McDonald et al (25,26) proposed a membrane contact mechanism consisting of an isolated protein-lipid ion pair. In the present study, we attempt to elucidate previously debated factors of prothrombin membrane binding using structural comparisons.…”

“…The environment of these residues may provide an opportunity to participate in the direct lipid binding through salt bridges, but whatever the interaction, it should be the same for both cis/trans-Pro22. One interesting development is the recently proposed model (see below) in which an ion pair mechanism is invoked in binding (25,26). We use the electrostatic potential surfaces using the program GRASP (27) (Figure 4) to compare cis and trans electrostatics in the context of the McDonald model.…”

Mutagenesis of the γ-Carboxyglutamic Acid Domain of Human Factor VII to Generate Maximum Enhancement of the Membrane Contact Site

“…One possible explanation for enhanced membrane binding is contact of the Tyr 4 side chain with the hydrophobic region of the phospholipid membrane. However, hydrophobic insertion by the -loop has been questioned on many grounds (3,(31)(32)(33)(34)(35). The impact of the Tyr 4 insertion was small (2-fold; ⌬⌬G ϭ 0.4 kcal/mol) compared with the free energy for transfer of a large hydrophobic group such as the phenyl side chain from aqueous solution (to octanol; ⌬⌬G ϭ Ϫ3.6 kcal/mol for ⌬K D ϭ 400-fold) (36).…”

Mouse recombinant protein C variants with enhanced membrane affinity and hyper‐anticoagulant activity in mouse plasma

“…Equilibrium data ( R eq ) was fitted to a one‐site binding hyperbola according to the relationship R eq = R max · C /( K D + C ), where R max is the binding at saturation or maximum surface coverage, C corresponds to the injected analyte concentration and K D is the equilibrium dissociation constant. We are aware of the fact that an analysis of the overall binding curve is a relatively severe simplification and that a complex set of reactions is involved in the membrane binding process of any Gla protein [33,41,48–50]. However, this simplification appears to be justified because the binding curves showed no signs of cooperativity when analyzed by scatchard plots (M. J. Krisinger, E. L. Pryzdial and R. T. MacGillivray, unpublished results).…”