Abstract

Factor Va binds to membranes exposing phosphatidylserine (PS) with nanomolar affinity. This high affinity interaction plays a vital role in the assembly of membrane-bound prothrombinase thereby supporting robust thrombin formation at the site of vascular damage. Although the importance of the C1 and C2 domains of FVa in membrane binding has long been recognized, mechanistic details are incompletely understood. Unlike human prothrombinase, Pseudonaja textilis (common brown snake, P. tex) has evolved a membrane-independent form of prothrombinase composed of a FVa-like protein (VPtex) tightly bound to a FXa-like protein in solution. Our structural advances with FVPtex provide new tools to address major unresolved questions related to membrane binding by FVa. The high resolution (1.95Å) structure of VPtex resembles previously published structures of inactivated bovine FVa and human FVIII of lower resolution with all the structural features considered critical for membrane binding by FVa. However, VPtex bound to PS-containing membranes with very poor affinity (Kd > 1 µM). Substitution of 9 residues in the C1 and C2 domains of VPtex with residues present in the hemostatic form of FV present in the plasma of the snake yielded a derivative (VPtexC1C2) that bound to PS-containing membranes with nanomolar affinity equivalent to hFV. Interestingly, the newly acquired function of membrane binding in VPtexC1C2 does not affect its ability to function in solution, suggesting that membrane binding and solution-phase function are controlled independently. Variants containing substitutions in the individual C domains (VPtexC1 and VPtexC2) exhibited intermediate affinities (Kd=100 nM and Kd=50 nM) for binding to PS-containing membranes. However, the binding energy contributions from the individual C-domains did not additively explain the affinity of VPtexC1C2 for membranes. The large connection energy (-8.7 kcal/mole) implies substantial energetic expenditure, possibly through a conformational rearrangement, upon membrane binding. This correlates well with higher thermal factors observed in the C1 and C2 domains of structures of VPtexC1C2 and VPtexC2 as compared to VPtex. It is also supported by rapid kinetic studies illustrating equivalence in the bimolecular association rate constants for human Va and VPtex variants regardless of their membrane affinity. Thus, high affinity membrane binding results from large decreases in the dissociation rate constant expected from a conformational change that allows the protein to adopt a new stable membrane-bound configuration. A second explanation for the lack of an obvious correlation between x-ray structures of VPtexC1C2, VPtexC2 and VPtex and their affinity for membranes lies in the possibility that their solution-phase conformations differ. We explored this using small angle x-ray scattering (SAXS) of VPtex, VPtexC2 and VPtexC1C2 in solution. The low resolution SAXS envelope for VPtex could be accounted for by minor shifts in the individual domains, particularly in C1 and C2, as seen in the crystal structure. However, the SAXS envelopes for membrane binding variants (VPtexC2 and VPtexC1C2) showed major shape changes in the C-domains. Rigid body modeling revealed an increasingly extended end-on arrangement, rather than a side-by-side configuration, of the C1 and C2 domains seen in the x-ray structure and in the SAXS envelope for FVPtex. The C2-domain was found to extend away from the base of the C1 domain along the long axis of the molecule correlating major structural differences in these VPtex variants with increasing affinity for membranes. Accordingly, the spatial disposition of the C1 and C2 domains in VPtexC2 appears intermediate to their arrangement in VPtex and VPtexC1C2.These findings contrast to the arrangement seen in the crystal structures of all factor V forms, where the C-domains are arranged side-by-side, probably due to limitations imposed by crystal packing. Our SAXS studies provide clear evidence of an unforeseen framework of C-domains associated with the ability of factor V forms to bind to membranes with high affinity. The findings reveal new mechanistic insights into the structural correlates of the membrane binding function of factor V.

Disclosures

Camire:Pfizer: Consultancy, Patents & Royalties, Research Funding.

Author notes

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Asterisk with author names denotes non-ASH members.