Side-Chain Protonation and Mobility in the Sarcoplasmic Reticulum Ca 2+-ATPase: Implications for Proton Countertransport and Ca 2+ Release

Abstract

Protonation of acidic residues in the sarcoplasmic reticulum Ca 2+-ATPase (SERCA 1a) was studied by multiconformation continuum electrostatic calculations in the Ca 2+-bound state Ca 2E1, in the Ca 2+-free state E2(TG) with bound thapsigargin, and in the E2P (ADP-insensitive phosphoenzyme) analog state with Mg F 4 2 − E 2 ( TG + Mg F 4 2 − ) . Around physiological pH, all acidic Ca 2+ ligands (Glu 309, Glu 771, Asp 800, and Glu 908) were unprotonated in Ca 2E1; in E2(TG) and E 2 ( TG + Mg F 4 2 − ) Glu 771, Asp 800, and Glu 908 were protonated. Glu 771 and Glu 908 had calculated pK a values larger than 14 in E2(TG) and E 2 ( TG + Mg F 4 2 − ) , whereas Asp 800 titrated with calculated pK a values near 7.5. Glu 309 had very different pK a values in the Ca 2+-free states: 8.4 in E 2 ( TG + Mg F 4 2 − ) and 4.7 in E2(TG) because of a different local backbone conformation. This indicates that Glu 309 can switch between a high and a low pK a mode, depending on the local backbone conformation. Protonated Glu 309 occupied predominantly two main, very differently orientated side-chain conformations in E 2 ( TG + Mg F 4 2 − ) : one oriented inward toward the other Ca 2+ ligands and one oriented outward toward a protein channel that seems to be in contact with the cytoplasm. Upon deprotonation, Glu 309 adopted completely the outwardly orientated side-chain conformation. The contact of Glu 309 with the cytoplasm in E 2 ( TG + Mg F 4 2 − ) makes this residue unlikely to bind lumenal protons. Instead it might serve as a proton shuttle between Ca 2+-binding site I and the cytoplasm. Glu 771, Asp 800, and Glu 908 are proposed to take part in proton countertransport.