The PT1-Ca2+ Gla domain binds to a membrane through two dipalmitoylphosphatidylserines. A computational study.

Binding of vitamin K-dependent proteins to cell membranes containing phosphatidylserine (PS) via gamma-carboxyglutamic acid (Gla) domains is one of the essential steps in the blood coagulation pathway. During activation of the coagulation cascade, prothrombin is converted to thrombin by prothrombinase, a complex consisting of serine protease FXa and cofactor FVa, anchored to anionic phospholipids on the surface of activated platelets in the presence of calcium ions. To investigate the binding of the Gla domain of prothrombin fragment 1 (PT1) to anionic lipids in the presence of Ca2+, we have conducted MD simulations of the protein with one and two dipalmitoylphosphatidylserines (DPPS) in a dipalmitoylphosphatidylcholine (DPPC) bilayer membrane. The results show a well-defined phosphatidylserine binding site, which agrees generally with crystallographic studies [Huang, M., et al. (2003) Nat. Struct. Biol. 10, 751-756]. However, in the presence of the lipid membrane, some of the interactions observed in the crystal structure adjust during the simulations possibly because in our system the PT1-Ca2+ complex is embedded in a DPPC lipid membrane. Our simulations confirm the existence of a second phospholipid headgroup binding site on the opposite face of the PT1-Ca2+ complex as suggested by MacDonald et al. [(1997) Biochemistry 36, 5120-5127]. The serine headgroup in the second site binds through a Gla domain-bound calcium ion Ca1, Gla30, and Lys11. On the basis of free energy simulations, we estimate the energy of binding of the PT1-Ca2+ complex to a single DPPS to be around -11.5 kcal/mol. The estimated free energy of binding of a DPPS lipid to the second binding site is around -8.8 kcal/mol and is in part caused by the nature of the second site and in part by entropic effects.