Abstract
Sarcoplasmic reticulum (SR) Ca2+-ATPase transports two Ca2+ ions from the cytoplasm to the SR lumen against a large concentration gradient. X-ray crystallography has revealed the atomic structures of the protein before and after the dissociation of Ca2+, while biochemical studies have suggested the existence of intermediate states in the transition between E1P⋅ADP⋅2Ca2+ and E2P. Here, we explore the pathway and free energy profile of the transition using atomistic molecular dynamics simulations with the mean-force string method and umbrella sampling. The simulations suggest that a series of structural changes accompany the ordered dissociation of ADP, the A-domain rotation, and the rearrangement of the transmembrane (TM) helices. The luminal gate then opens to release Ca2+ ions toward the SR lumen. Intermediate structures on the pathway are stabilized by transient sidechain interactions between the A- and P-domains. Lipid molecules between TM helices play a key role in the stabilization. Free energy profiles of the transition assuming different protonation states suggest rapid exchanges between Ca2+ ions and protons when the Ca2+ ions are released toward the SR lumen.
Highlights
Sarcoplasmic reticulum (SR) Ca2+-ATPase transports two Ca2+ ions from the cytoplasm to the SR lumen against a large concentration gradient
We investigate the effect of protonation states in the E1P–E2P transitions from atomistic molecular dynamics (MD) simulations, which is difficult via experimental studies
Large deviations are observed only in the N-domain between the three MD simulations when representative snapshots are superimposed on the starting crystal structures
Summary
Sarcoplasmic reticulum (SR) Ca2+-ATPase transports two Ca2+ ions from the cytoplasm to the SR lumen against a large concentration gradient. We explore the pathway and free energy profile of the transition using atomistic molecular dynamics simulations with the mean-force string method and umbrella sampling. Crystal structures that represent E1P·ADP·2Ca2+ and E2P (Fig. 1 A and B) reveal important conformational changes to release Ca2+ toward the SR lumen: 1) the A-domain rotates ∼90°; 2) the threonine–glycine–glutamate–serine (TGES) loop in the A-domain reaches the phosphorylated Asp351 in the P-domain [13,14,15]; 3) M1-M6 are rearranged to open the luminal gate for the dissociation of Ca2+. We perform atomistic MD simulations with an enhanced conformational sampling method to investigate the conformational pathway and free energy profile in the transition between E1P·ADP·2Ca2+ and E2P. The same approach has been previously applied to adenylate kinase in Significance
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