Roles of Conserved P Domain Residues and Mg 2+ in ATP Binding in the Ground and Ca 2+-activated States of Sarcoplasmic Reticulum Ca 2+-ATPase

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dc.contributor.authorMcIntosh, David B
dc.contributor.authorClausen, Johannes D
dc.contributor.authorWoolley, David G
dc.contributor.authorMacLennan, David H
dc.contributor.authorVilsen, Bente
dc.contributor.authorAndersen, Jens Peter
dc.date.accessioned2021-10-08T07:20:48Z
dc.date.available2021-10-08T07:20:48Z
dc.date.issued2004
dc.description.abstractResidues 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.
dc.identifier.apacitationMcIntosh, D. B., Clausen, J. D., Woolley, D. G., MacLennan, D. H., Vilsen, B., & Andersen, J. P. (2004). Roles of Conserved P Domain Residues and Mg 2+ in ATP Binding in the Ground and Ca 2+-activated States of Sarcoplasmic Reticulum Ca 2+-ATPase. <i>The Journal of Biological Chemistry</i>, 279(31), 32515 - 32523. http://hdl.handle.net/11427/35009en_ZA
dc.identifier.chicagocitationMcIntosh, David B, Johannes D Clausen, David G Woolley, David H MacLennan, Bente Vilsen, and Jens Peter Andersen "Roles of Conserved P Domain Residues and Mg 2+ in ATP Binding in the Ground and Ca 2+-activated States of Sarcoplasmic Reticulum Ca 2+-ATPase." <i>The Journal of Biological Chemistry</i> 279, 31. (2004): 32515 - 32523. http://hdl.handle.net/11427/35009en_ZA
dc.identifier.citationMcIntosh, D.B., Clausen, J.D., Woolley, D.G., MacLennan, D.H., Vilsen, B. & Andersen, J.P. 2004. Roles of Conserved P Domain Residues and Mg 2+ in ATP Binding in the Ground and Ca 2+-activated States of Sarcoplasmic Reticulum Ca 2+-ATPase. <i>The Journal of Biological Chemistry.</i> 279(31):32515 - 32523. http://hdl.handle.net/11427/35009en_ZA
dc.identifier.issn0021-9258
dc.identifier.issn1083-351X
dc.identifier.ris TY - Journal Article AU - McIntosh, David B AU - Clausen, Johannes D AU - Woolley, David G AU - MacLennan, David H AU - Vilsen, Bente AU - Andersen, Jens Peter AB - 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. DA - 2004 DB - OpenUCT DP - University of Cape Town IS - 31 J1 - The Journal of Biological Chemistry LK - https://open.uct.ac.za PY - 2004 SM - 0021-9258 SM - 1083-351X T1 - Roles of Conserved P Domain Residues and Mg 2+ in ATP Binding in the Ground and Ca 2+-activated States of Sarcoplasmic Reticulum Ca 2+-ATPase TI - Roles of Conserved P Domain Residues and Mg 2+ in ATP Binding in the Ground and Ca 2+-activated States of Sarcoplasmic Reticulum Ca 2+-ATPase UR - http://hdl.handle.net/11427/35009 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/35009
dc.identifier.vancouvercitationMcIntosh DB, Clausen JD, Woolley DG, MacLennan DH, Vilsen B, Andersen JP. Roles of Conserved P Domain Residues and Mg 2+ in ATP Binding in the Ground and Ca 2+-activated States of Sarcoplasmic Reticulum Ca 2+-ATPase. The Journal of Biological Chemistry. 2004;279(31):32515 - 32523. http://hdl.handle.net/11427/35009.en_ZA
dc.language.isoeng
dc.publisher.departmentDepartment of Clinical Laboratory Sciences
dc.publisher.facultyFaculty of Health Sciences
dc.sourceThe Journal of Biological Chemistry
dc.source.journalissue31
dc.source.journalvolume279
dc.source.pagination32515 - 32523
dc.source.urihttps://dx.doi.org/10.1074/jbc.M403242200
dc.subject.otherAdenosine Triphosphate
dc.subject.otherAmino Acid Motifs
dc.subject.otherAnimals
dc.subject.otherAspartic Acid
dc.subject.otherBiological Transport
dc.subject.otherCOS Cells
dc.subject.otherCalcium
dc.subject.otherCalcium-Transporting ATPases
dc.subject.otherCatalysis
dc.subject.otherDNA, Complementary
dc.subject.otherDose-Response Relationship, Drug
dc.subject.otherGlycine
dc.subject.otherKinetics
dc.subject.otherMagnesium
dc.subject.otherModels, Molecular
dc.subject.otherMutagenesis
dc.subject.otherMutation
dc.titleRoles of Conserved P Domain Residues and Mg 2+ in ATP Binding in the Ground and Ca 2+-activated States of Sarcoplasmic Reticulum Ca 2+-ATPase
dc.typeJournal Article
uct.type.publicationResearch
uct.type.resourceJournal Article
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