Microsecond Molecular Dynamics Simulations of Mg(2+)- and K(+)- Bound E1 Intermediate States of the Calcium Pump

We have performed microsecond molecular dynamics (MD) simulations to characterize the structural dynamics of cation-bound E1 intermediate states of the calcium pump (sarcoendoplasmic reticulum Ca(2+)-ATPase, SERCA) in atomic detail, including a lipid bilayer with aqueous solution on both sides. X-ray crystallography with 40 mM Mg(2+) in the absence of Ca(2+) has shown that SERCA adopts an E1 structure with transmembrane Ca(2+)-binding sites I and II exposed to the cytosol, stabilized by a single Mg(2+) bound to a hybrid binding site I′. This Mg(2+)-bound E1 intermediate state, designated E1•Mg(2+), is proposed to constitute a functional SERCA intermediate that catalyzes the transition from E2 to E1•2Ca(2+) by facilitating H(+)/Ca(2+) exchange. To test this hypothesis, we performed two independent MD simulations based on the E1•Mg(2+) crystal structure, starting in the presence or absence of initially-bound Mg(2+). Both simulations were performed for 1 µs in a solution containing 100 mM K(+) and 5 mM Mg(2+) in the absence of Ca(2+), mimicking muscle cytosol during relaxation. In the presence of initially-bound Mg(2+), SERCA site I′ maintained Mg(2+) binding during the entire MD trajectory, and the cytosolic headpiece maintained a semi-open structure. In the absence of initially-bound Mg(2+), two K(+) ions rapidly bound to sites I and I′ and stayed loosely bound during most of the simulation, while the cytosolic headpiece shifted gradually to a more open structure. Thus MD simulations predict that both E1•Mg(2+) and E•2K(+) intermediate states of SERCA are populated in solution in the absence of Ca(2+), with the more open 2K(+)-bound state being more abundant at physiological ion concentrations. We propose that the E1•2K(+) state acts as a functional intermediate that facilitates the E2 to E1•2Ca(2+) transition through two mechanisms: by pre-organizing transport sites for Ca(2+) binding, and by partially opening the cytosolic headpiece prior to Ca(2+) activation of nucleotide binding.