ATP binding residues of sarcoplasmic reticulum Ca2+-ATPase.
ATP-binding residues in the N and P domains of sarcoplasmic reticulum Ca-ATPase have been investigated using mutagenesis in combination with a binding assay based on the photolabeling of Lys(492) with [g-(32)P] 2',3'-O-(2,4,6 trinitrophenyl)-8-azido-ATP and competition with nucleotide. In the N domain, mutations to several residues in conserved motifs, (438)GEATE, (487)FSRDRK, (515)KGAPE, and (560)RCLALA produce nucleotide-binding defects. Key residues include Thr(441), Glu(442), Phe(487), Arg(489), Lys(492), Lys(515), Arg(560), and Leu(562). In the absence of Mg(2+), Arg(489), Lys(492), and Arg(560) are most important, whereas in its presence Thr(441) and Glu(442) also play a crucial role. In the P domain, Asp(351) is striking for its strong electrostatic repulsion of the gamma-phosphate, especially in the presence of Mg(2+). Lys(352) is a key residue, and Asp(627) and Lys(684) must come close to the nucleotide. Thr(353), Asn(359), Asp(601), and Asp(703) interact only in the presence of Mg(2+). Asn(706) and Asp(707) are unimportant for nucleotide binding. The results identify several ATP binding residues in the N and P domains and suggest that Mg(2+) changes the nucleotide/protein interaction in both. Models of bound ATP and MgATP are presented.
McIntosh
D B
DB
Division of Chemical Pathology, Department of Clinical Laboratory Sciences, Faculty of Health Sciences, University of Cape Town, South Africa. davidmci@chempath.uct.ac.za
Clausen
J D
JD
Woolley
D G
DG
MacLennan
D H
DH
Vilsen
B
B
Andersen
J P
JP
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D017124:Conserved Sequence; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D055672:Static Electricity
D010446:Peptide Fragments; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases