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 Ca(2+)-ATPase (SERCA 1a) was studied by multiconformation continuum electrostatic calculations in the Ca(2+)-bound state Ca(2)E1, in the Ca(2+)-free state E2(TG) with bound thapsigargin, and in the E2P (ADP-insensitive phosphoenzyme) analog state with MgF(4)(2-) E2(TG+MgF(4)(2-)). Around physiological pH, all acidic Ca(2+) ligands (Glu(309), Glu(771), Asp(800), and Glu(908)) were unprotonated in Ca(2)E1; in E2(TG) and E2(TG+MgF(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 E2(TG+MgF(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 E2(TG+MgF(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 E2(TG+MgF(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 E2(TG+MgF(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.

FIGURE 1

Most occupied conformers of the Ca²⁺ carboxyl ligands (Glu³⁰⁹, Glu⁷⁷¹, Asp⁸⁰⁰, Glu⁹⁰⁸) in the state at pH 6. Glu³⁰⁹ has two, almost equally occupied conformers at pH 6; the inwardly oriented conformer is similar in structure to the crystal structure and represented as solid; the outwardly oriented conformer is shown as transparent. See text for further explanations. M indicates transmembrane helices. Only a part of the transmembrane region of the Ca²⁺-ATPase is shown. The view is approximately parallel to the membrane. The cytoplasmic side is on the top of the figure, the lumenal side on the bottom. At pH 9, the ionized side chain of Glu³⁰⁹ adopts the same conformation as the outwardly oriented conformer at pH 6.

FIGURE 2

The different conformations of the Glu³⁰⁹ side chain. (Blue) E2(TG) structure (1IWO.pdb) having outwardly oriented Glu³⁰⁹; (dark green) the structure (1WPG.pdb), having inwardly oriented Glu³⁰⁹; (light green) the outwardly oriented, protonated conformer of Glu³⁰⁹ from our calculations with The hydrogen atoms are omitted for clarity. Structures were overlaid by superposition of the backbone atoms of residues 305–315. (A) Side view approximately parallel to the membrane surface. Shown is the backbone of residues 307–311 of M4 and the side chain of Glu³⁰⁹. The conformational change of the backbone around Glu³⁰⁹ is clearly seen. The arrows point toward the backbone oxygen of Glu³⁰⁹. (B) Top view approximately parallel to M4 toward the lumenal side. Shown are residues Glu³⁰⁹ and Ile⁹⁷. The latter is closest to the outwardly oriented Glu³⁰⁹ conformer obtained in our calculations with the structure. The arrows point toward the C_α-C_β bond of Glu³⁰⁹, which is oriented differently in the E2(TG) and crystal structures. The orientation of this bond is the same for all conformers generated by MCCE with a given backbone structure.

FIGURE 3

The suggested Ca²⁺ release path between transmembrane helices M1, M2, and M4. Shown is the suggested Ca²⁺ exit path within the membrane domain of the structure (21) in van der Waals representation viewed approximately parallel to the membrane surface. The left-hand side shows a cut through the structure and the right-hand side a slice of the structure. The arrow indicates the Ca²⁺ path. Residue numbers are given for a selection of residues. Two glutamic acids are highlighted by cpk colors.