Roles of conserved P domain residues and Mg2+ in ATP binding in the ground and Ca2+-activated states of sarcoplasmic reticulum Ca2+-ATPase.
Residues in conserved motifs (625)TGD, (676)FARXXPXXK, and (701)TGDGVND in domain P of sarcoplasmic reticulum Ca(2+)-ATPase, as well as in motifs (601)DPPR and (359)NQR(/K)MSV in the hinge segments connecting domains N and P, were examined by mutagenesis to assess their roles in nucleotide and Mg(2+) binding and stabilization of the Ca(2+)-activated transition state for phosphoryl transfer. In the absence of Mg(2+), mutations removing the charges of domain P residues Asp(627), Lys(684), Asp(703), and Asp(707) increased the affinity for ATP and 2',3'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine 5'-triphosphate. These mutations, as well as Gly(626)--> Ala, were inhibitory for ATP binding in the presence of Mg(2+) and for tight binding of the beta,gamma-bidentate chromium(III) complex of ATP. The hinge mutations had pronounced, but variable, effects on ATP binding only in the presence of Mg(2+). The data demonstrate an unfavorable electrostatic environment for binding of negatively charged nucleotide in domain P and show that Mg(2+) is required to anchor the phosphoryl group of ATP at the phosphorylation site. Mutants Gly(626) --> Ala, Lys(684) --> Met, Asp(703) --> Ala/Ser/Cys, and mutants with alteration to Asp(707) exhibited very slow or negligible phosphorylation, making it possible to measure ATP binding in the pseudo-transition state attained in the presence of both Mg(2+) and Ca(2+). Under these conditions, ATP binding was almost completely blocked in Gly(626) --> Ala and occurred with 12- and 7-fold reduced affinities in Asp(703) --> Ala and Asp(707) --> Cys, respectively, relative to the situation in the presence of Mg(2+) without Ca(2+), whereas in Lys(684) --> Met and Asp(707) --> Ser/Asn the affinity was enhanced 14- and 3-5-fold, respectively. Hence, Gly(626) and Asp(703) seem particularly critical for mediating entry into the transition state for phosphoryl transfer upon Ca(2+) binding at the transport sites.
McIntosh
David B
DB
Chemical Pathology, Department of Clinical Laboratory Sciences, Faculty of Health Sciences, University of Cape Town, and National Health Laboratory Service, Groote Schuur Hospital, Cape Town 7925, South Africa. davidmci@chempath.uct.ac.za
Clausen
Johannes D
JD
Woolley
David G
DG
MacLennan
David H
DH
Vilsen
Bente
B
Andersen
Jens Peter
JP
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001692:Biological Transport; D019556:COS Cells; D002118:Calcium / Q000737:chemistry; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002384:Catalysis; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005998:Glycine / Q000737:chemistry; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry*; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D013997:Time Factors
D018076:DNA, Complementary; D009711:Nucleotides; D001224:Aspartic Acid; D000255:Adenosine Triphosphate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium; D005998:Glycine