Side-Chain Protonation and Mobility in the Sarcoplasmic Reticulum Ca2+-ATPase: Implications for Proton Countertransport and Ca2+ Release

Protonation of acidic residues in the sarcoplasmic reticulum Ca2+-ATPase (SERCA 1a) was studied by multiconformation continuum electrostatic calculations in the Ca2+-bound state Ca2E1, in the Ca2+-free state E2(TG) with bound thapsigargin, and in the E2P (ADP-insensitive phosphoenzyme) analog state with MgF42−E2(TG+MgF42−). Around physiological pH, all acidic Ca2+ ligands (Glu309, Glu771, Asp800, and Glu908) were unprotonated in Ca2E1; in E2(TG) and E2(TG+MgF42−) Glu771, Asp800, and Glu908 were protonated. Glu771 and Glu908 had calculated pKa values larger than 14 in E2(TG) and E2(TG+MgF42−), whereas Asp800 titrated with calculated pKa values near 7.5. Glu309 had very different pKa values in the Ca2+-free states: 8.4 in E2(TG+MgF42−) and 4.7 in E2(TG) because of a different local backbone conformation. This indicates that Glu309 can switch between a high and a low pKa mode, depending on the local backbone conformation. Protonated Glu309 occupied predominantly two main, very differently orientated side-chain conformations in E2(TG+MgF42−): one oriented inward toward the other Ca2+ ligands and one oriented outward toward a protein channel that seems to be in contact with the cytoplasm. Upon deprotonation, Glu309 adopted completely the outwardly orientated side-chain conformation. The contact of Glu309 with the cytoplasm in E2(TG+MgF42−) makes this residue unlikely to bind lumenal protons. Instead it might serve as a proton shuttle between Ca2+-binding site I and the cytoplasm. Glu771, Asp800, and Glu908 are proposed to take part in proton countertransport.