Microsecond molecular dynamics simulations of Mg2+- And K +- Bound E1 intermediate states of the calcium pump

Abstract

We have performed microsecond molecular dynamics (MD) simulations to characterize the structural dynamics of cationbound E1 intermediate states of the calcium pump (sarcoendoplasmic reticulum Ca2+-ATPase, SERCA) in atomic detail, including a lipid bilayer with aqueous solution on both sides. X-ray crystallography with 40 mM Mg2+ in the absence of Ca 2+ has shown that SERCA adopts an E1 structure with transmembrane Ca2+-binding sites I and II exposed to the cytosol, stabilized by a single Mg2+ bound to a hybrid binding site I′. This Mg 2+-bound E1 intermediate state, designated E1•Mg2+, is proposed to constitute a functional SERCA intermediate that catalyzes the transition from E2 to E1•2Ca2+ by facilitating H +/Ca2+ exchange. To test this hypothesis, we performed two independent MD simulations based on the E1•Mg2+ 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 Mg2+ in the absence of Ca2+, mimicking muscle cytosol during relaxation. In the presence of initially-bound Mg2+, SERCA site I′ maintained Mg2+ binding during the entire MD trajectory, and the cytosolic headpiece maintained a semi-open structure. In the absence of initially-bound Mg2+, 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•Mg2+ and E•2K+ intermediate states of SERCA are populated in solution in the absence of Ca2+, 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•2Ca2+ transition through two mechanisms: by pre-organizing transport sites for Ca 2+ binding, and by partially opening the cytosolic headpiece prior to Ca2+ activation of nucleotide binding. © 2014 Espinoza-Fonseca et al.