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.
Highlights
P-type ATPases are responsible for active transport of a specific ion, such as Ca2+, Na+, or K+, against its concentration gradient [1,2]
Preliminary rounds of short molecular dynamics (MD) simulations showed that this difference in A domain orientation between native and recombinant E1NMg2+ is small, indicating that the crystal structure of recombinant E1NMg2+ is an adequate starting structure to simulate the dynamics of E1
To determine the effect of metal ion binding on the structural dynamics of E1, we removed the ATP analog trinitrophenyl adenosine monophosphate (TNP-AMP) and the Mg2+ ion bound to the phosphate group of TNP-AMP
Summary
P-type ATPases are responsible for active transport of a specific ion, such as Ca2+, Na+, or K+, against its concentration gradient [1,2]. The catalytic cycle of SERCA involves a major structural transition between two key conformations: low Ca2+ affinity E2, with binding sites exposed to the lumen, and high Ca2+ affinity E1, with binding sites exposed to the cytosol. This E2RE1 transition is driven by Ca2+/H+ exchange and may include steps facilitated by other cations [7,8,9]
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