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 cation-bound 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 Ca2+ 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 Mg2+-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 Mg2+. 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 Ca2+ binding, and by partially opening the cytosolic headpiece prior to Ca2+ activation of nucleotide binding.

Sarcolipin and phospholamban inhibit the calcium pump by populating a similar metal ion-free intermediate state

“…1B). The dynamic nature of the cytosolic headpiece, and in particular of the A domain, is in line with previous studies showing that the headpiece is inherently flexible in the E1 state in the absence and presence of PLB [16,17]. We also evaluated the structure and mobility of SLN in the complex.…”

“…2A,B). The distances D800-E771 and D800-E908 are ∼0.3 nm longer than those expected for competent transport site geometries [16] These results indicate that SLN disfavors the formation of a competent transport site I of SERCA.…”

“…It was proposed that SLN inhibits SERCA by stabilizing the E1•Mg 2 state [14]. molecular dynamics (MD) simulation studies showed that E1•Mg 2+ is not a functional intermediate of SERCA without bound subunit [16], and that Mg 2+ ions do not bind to E1 under physiological conditions [16,17]. Based on this information, we hypothesize that SLN inhibits SERCA activity by populating a metal ion-free, protonated E1 state.…”

The N Terminus of Sarcolipin Plays an Important Role in Uncoupling Sarco-endoplasmic Reticulum Ca2+-ATPase (SERCA) ATP Hydrolysis from Ca2+ Transport

“…For the truncated version we deleted residues 2-6 of SLN after equilibration. We used the same protonation scheme as Espinoza-Fonseca et al (34), namely that, Ca 2ϩ -binding residues Glu 771 , Asp 800 , and Glu 309 were kept unprotonated, whereas Glu 908 was protonated. The system consisted of SERCA, SLN, 500 POPC molecules, and ϳ55,000 TIPS3P water molecules.…”

The Ca2+-ATPase pump facilitates bidirectional proton transport across the sarco/endoplasmic reticulum

“…The asterisk indicates the Ca 2+ -free E1 intermediate proposed by x-ray crystallography studies (E1·Mg 2+ ) 36, 37 and molecular dynamics simulations (E1·2K + ). 38 …”