Structural and energetic analysis of metastable intermediate states in the E1P-E2P transition of Ca²⁺-ATPase

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.