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

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 degrees C, was enhanced relative to wild type in mutants Ile(743) --> Ala, Val(747) --> Ala, Glu(748) --> Ala, Glu(749) --> Ala, Met(757) --> Gly, and Gln(759) --> Ala and reduced in mutants Asp(737) --> Ala, Asp(738) --> Ala, Ala(752) --> Leu, and Tyr(754) --> Ala, In mutant Arg(762) --> Ile, the rate of the Ca2+ binding transition was wild type like at 0 degrees C, whereas it was 3.5-fold reduced relative to wild type at 25 degrees C. The rate of dephosphorylation of the ADP-insensitive phosphoenzyme was increased conspicuously in mutants Ile(743) --> Ala and Tyr(754) --> Ala (close to 10-fold in the absence of K+) and increased to a lesser extent in Asn(739) --> Ala, Glu(749) --> Ala, Gly(760) --> Ala, Ala(752) --> Gly, Met(757) --> Gly, and Arg(762) --> Ile, whereas it was reduced in mutants Asp(737) --> Ala, Val(744) --> Gly, Val(744) --> Ala, Val(747) --> Ala, and Ala(752) --> Leu. In mutants Ile(743) --> Ale, Tyr754, Ala, and Arg(762) --> Ile, the apparent affinities for vanadate were enhanced 23-, 30-, and le-fold, respectively, relative to wild type, The rate of Ca2+ dissociation was Ii-fold increased in Gly(750) --> Ala and 2-fold reduced in Val(747) --> Ala. Mutants with alterations to Arg(751) 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 Arg(751) is important for both structural and functional integrity of the enzyme.