title: 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.
authors.affiliation: Institut für Biophysik, Johann Wolfgang Goethe-Universität Frankfurt, 60438 Frankfurt am Main, Germany. hauser@biophysik.uni-frankfurt.de
authors.forename: K
authors.initials: K
authors.lastname: Hauser
authors.forename: A
authors.initials: A
authors.lastname: Barth
mesh_terms: D000818:Animals; D001224:Aspartic Acid / Q000502:physiology*; D001692:Biological Transport / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D018698:Glutamic Acid / Q000502:physiology*; D007700:Kinetics; D011522:Protons; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*
chemical_list: D011522:Protons; D001224:Aspartic Acid; D018698:Glutamic Acid; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium