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

K. Hauser ^* and A. Barth 

^* Institut für Biophysik, Johann Wolfgang Goethe-Universität Frankfurt, 60438 Frankfurt am Main, Germany; and Department of Biochemistry and Biophysics, Stockholm University, Arrhenius Laboratories for Natural Sciences, SE-106 91 Stockholm, Sweden

Correspondence: Address reprint requests to K. Hauser, Institut für Biophysik, Johann Wolfgang Goethe-Universität Frankfurt, Max-von-Laue-Str.1, 60438 Frankfurt am Main, Germany. Tel.: 49-69-798-46407; Fax: 49-69-798-46421; E-mail: hauser{at}biophysik.uni-frankfurt.de.

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 Around physiological pH, all acidic Ca²⁺ ligands (Glu³⁰⁹, Glu⁷⁷¹, Asp⁸⁰⁰, and Glu⁹⁰⁸) were unprotonated in Ca₂E1; in E2(TG) and Glu⁷⁷¹, Asp⁸⁰⁰, and Glu⁹⁰⁸ were protonated. Glu⁷⁷¹ and Glu⁹⁰⁸ had calculated pK_a values larger than 14 in E2(TG) and whereas Asp⁸⁰⁰ titrated with calculated pK_a values near 7.5. Glu³⁰⁹ had very different pK_a values in the Ca²⁺-free states: 8.4 in 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 pK_a mode, depending on the local backbone conformation. Protonated Glu³⁰⁹ occupied predominantly two main, very differently orientated side-chain conformations in 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 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.

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