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

Abstract

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 View the MathML sourceView the MathML source Around physiological pH, all acidic Ca2+ ligands (Glu309, Glu771, Asp800, and Glu908) were unprotonated in Ca2E1; in E2(TG) and View the MathML source Glu771, Asp800, and Glu908 were protonated. Glu771 and Glu908 had calculated pKa values larger than 14 in E2(TG) and View the MathML source whereas Asp800 titrated with calculated pKa values near 7.5. Glu309 had very different pKa values in the Ca2+-free states: 8.4 in View the MathML source 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 View the MathML source 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 View the MathML source 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.