Importance of stalk segment S5 for intramolecular communication in the sarcoplasmic reticulum Ca2+-ATPase

Abstract

Sixteen residues in stalk segment S5 of the Ca2+-ATPase of sarcoplasmic reticulum were studied by site-directed mutagenesis. The rate of the Ca2+ binding transition, determined at 0 °C, was enhanced relative to wild type in mutants Ile743 → Ala, Val 747 → Ala, Glu748 → Ala, Glu749 → Ala, Met757 → Gly, and Gln759 → Ala and reduced in mutants Asp737 → Ala, Asp738 → Ala, Ala752 → Leu, and Tyr754 → Ala. In mutant Arg762 → Ile, the rate of the Ca2+ binding transition was wild type like at 0 °C, whereas it was 3.5-fold reduced relative to wild type at 25 °C. The rate of dephosphorylation of the ADP-insensitive phosphoenzyme was increased conspicuously in mutants Ile743 → Ala and Tyr754 → Ala (close to 20-fold in the absence of K+) and increased to a lesser extent in Asn739 → Ala, Glu749 → Ala, Gly750 → Ala, Ala752 → Gly, Met757 → Gly, and Arg762 → Ile, whereas it was reduced in mutants Asp737 → Ala, Val744 → Gly, Val744 → Ala, Val747 → Ala, and Ala752 → Leu. In mutants Ile743 → Ala, Tyr754 → Ala, and Arg762 → Ile, the apparent affinities for vanadate were enhanced 23-, 30-, and 18-fold, respectively, relative to wild type. The rate of Ca2+ dissociation was 11-fold increased in Gly750 → Ala and 2-fold reduced in Val747 → Ala. Mutants with alterations to Arg751 either were not expressed at a significant level or were completely nonfunctional. The findings show that S5 plays a crucial role in mediating communication between the Ca2+ binding pocket and the catalytic domain and that Arg751 is important for both structural and functional integrity of the enzyme.