Molecular Dynamics Simulations Support Multiple Binding Sites for Phospholamban on SERCA

Ion‐motive ATPases use the energy of ATP hydrolysis to transport ions across membrane bilayer against a concentration gradient. Here we investigate the functional regulation of the sarco(endo)plasmic reticulum calcium ATPase (SERCA), which plays a key role in cellular Ca handling. In the heart, the activity of the SERCA is regulated by its inhibitory partner phospholamban (PLB). PLB has been shown to inhibit the transporter by binding to the SERCA M6 helix, but this site is not available when SERCA is bound to Ca. Our previous fluorescence resonance energy transfer (FRET) experiments suggested that SERCA Ca binding causes PLB to translocate from the known binding site to a putative secondary site. Based on an analogous interaction of the sodium/potassium ATPase (NKA) with a regulatory partner (FXYD10), we hypothesized that PLB could interact with the M9 helix on SERCA after leaving the canonical binding site. In the present study we performed molecular dynamics simulations to compare the energetics of binding of PLB to candidate sites on SERCA. We created models of the regulatory complex using structural information from available crystal structures of the PLB‐SERCA and FXYD10‐NKA complexes. Quantification of the interaction energies of these models suggested PLB can form a stable complex with both the canonical (−856 kJ/mol) and the proposed secondary site (−510 kJ/mol). In contrast, docking of PLB to the M3 helix on the opposite side of SERCA shows a much less favorable interaction energy (−108 kJ/mol). We also used umbrella sampling to quantify the free energy of PLB bound to several candidate binding sites on SERCA transmembrane domain. The results provide insight into the relative affinity of PLB for plausible sites of interaction on SERCA. Overall, the molecular dynamics simulations support a 2‐site model in which PLB interacts first with the high affinity conventional site and then relocates to a nearby secondary site when SERCA binds calcium.