Critical Interaction of Actuator Domain Residues Arginine 174, Isoleucine 188, and Lysine 205 with Modulatory Nucleotide in Sarcoplasmic Reticulum Ca 2+ -ATPase

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dc.contributor.authorClausen, Johannes D
dc.contributor.authorMcIntosh, David B
dc.contributor.authorWoolley, David G
dc.contributor.authorAndersen, Jens Peter
dc.date.accessioned2021-10-08T07:22:52Z
dc.date.available2021-10-08T07:22:52Z
dc.date.issued2008
dc.description.abstractATP plays dual roles in the reaction cycle of the sarcoplasmic reticulum Ca2+-ATPase by acting as the phosphorylating substrate as well as in nonphosphorylating (modulatory) modes accelerating conformational transitions of the enzyme cycle. Here we have examined the involvement of actuator domain residues Arg174, Ile188, Lys204, and Lys205 by mutagenesis. Alanine mutations to these residues had little effect on the interaction of the Ca2E1 state with nucleotide or on the HnE 2 to Ca2E1 transition of the dephosphoenzyme. The phosphoenzyme processing steps, Ca2E1P to E2P and E2P dephosphorylation, and their stimulation by MgATP/ATP were markedly affected by mutations to Arg174, Ile188, and Lys205. Replacement of Ile188 with alanine abolished nucleotide modulation of dephosphorylation but not the modulation of the Ca2E1P to E2P transition. Mutation to Arg174 interfered with nucleotide modulation of either of the phosphoenzyme processing steps, indicating a significant overlap between the modulatory nucleotide-binding sites involved. Mutation to Lys205 enhanced the rates of the phosphoenzyme processing steps in the absence of nucleotide and disrupted the nucleotide modulation of the Ca2E1P to E2P transition. Remarkably, the mutants with alterations to Lys205 showed an anomalous inhibition by ATP of the dephosphorylation, and in the alanine mutant the affinity for the inhibition by ATP was indistinguishable from that for stimulation by ATP of the wild type. Hence, the actuator domain is an important player in the function of ATP as modulator of phosphoenzyme processing, with Arg174, Ile188, and Lys205 all being critically involved, although in different ways. The data support a variable site model for the modulatory effects with the nucleotide binding somewhat differently in each of the conformational states occurring during the transport cycle.
dc.identifier.apacitationClausen, J. D., McIntosh, D. B., Woolley, D. G., & Andersen, J. P. (2008). Critical Interaction of Actuator Domain Residues Arginine 174, Isoleucine 188, and Lysine 205 with Modulatory Nucleotide in Sarcoplasmic Reticulum Ca 2+ -ATPase. <i>The Journal of Biological Chemistry</i>, 283(51), 35703 - 35714. http://hdl.handle.net/11427/35014en_ZA
dc.identifier.chicagocitationClausen, Johannes D, David B McIntosh, David G Woolley, and Jens Peter Andersen "Critical Interaction of Actuator Domain Residues Arginine 174, Isoleucine 188, and Lysine 205 with Modulatory Nucleotide in Sarcoplasmic Reticulum Ca 2+ -ATPase." <i>The Journal of Biological Chemistry</i> 283, 51. (2008): 35703 - 35714. http://hdl.handle.net/11427/35014en_ZA
dc.identifier.citationClausen, J.D., McIntosh, D.B., Woolley, D.G. & Andersen, J.P. 2008. Critical Interaction of Actuator Domain Residues Arginine 174, Isoleucine 188, and Lysine 205 with Modulatory Nucleotide in Sarcoplasmic Reticulum Ca 2+ -ATPase. <i>The Journal of Biological Chemistry.</i> 283(51):35703 - 35714. http://hdl.handle.net/11427/35014en_ZA
dc.identifier.issn0021-9258
dc.identifier.issn1083-351X
dc.identifier.ris TY - Journal Article AU - Clausen, Johannes D AU - McIntosh, David B AU - Woolley, David G AU - Andersen, Jens Peter AB - ATP plays dual roles in the reaction cycle of the sarcoplasmic reticulum Ca2+-ATPase by acting as the phosphorylating substrate as well as in nonphosphorylating (modulatory) modes accelerating conformational transitions of the enzyme cycle. Here we have examined the involvement of actuator domain residues Arg174, Ile188, Lys204, and Lys205 by mutagenesis. Alanine mutations to these residues had little effect on the interaction of the Ca2E1 state with nucleotide or on the HnE 2 to Ca2E1 transition of the dephosphoenzyme. The phosphoenzyme processing steps, Ca2E1P to E2P and E2P dephosphorylation, and their stimulation by MgATP/ATP were markedly affected by mutations to Arg174, Ile188, and Lys205. Replacement of Ile188 with alanine abolished nucleotide modulation of dephosphorylation but not the modulation of the Ca2E1P to E2P transition. Mutation to Arg174 interfered with nucleotide modulation of either of the phosphoenzyme processing steps, indicating a significant overlap between the modulatory nucleotide-binding sites involved. Mutation to Lys205 enhanced the rates of the phosphoenzyme processing steps in the absence of nucleotide and disrupted the nucleotide modulation of the Ca2E1P to E2P transition. Remarkably, the mutants with alterations to Lys205 showed an anomalous inhibition by ATP of the dephosphorylation, and in the alanine mutant the affinity for the inhibition by ATP was indistinguishable from that for stimulation by ATP of the wild type. Hence, the actuator domain is an important player in the function of ATP as modulator of phosphoenzyme processing, with Arg174, Ile188, and Lys205 all being critically involved, although in different ways. The data support a variable site model for the modulatory effects with the nucleotide binding somewhat differently in each of the conformational states occurring during the transport cycle. DA - 2008 DB - OpenUCT DP - University of Cape Town IS - 51 J1 - The Journal of Biological Chemistry LK - https://open.uct.ac.za PY - 2008 SM - 0021-9258 SM - 1083-351X T1 - Critical Interaction of Actuator Domain Residues Arginine 174, Isoleucine 188, and Lysine 205 with Modulatory Nucleotide in Sarcoplasmic Reticulum Ca 2+ -ATPase TI - Critical Interaction of Actuator Domain Residues Arginine 174, Isoleucine 188, and Lysine 205 with Modulatory Nucleotide in Sarcoplasmic Reticulum Ca 2+ -ATPase UR - http://hdl.handle.net/11427/35014 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/35014
dc.identifier.vancouvercitationClausen JD, McIntosh DB, Woolley DG, Andersen JP. Critical Interaction of Actuator Domain Residues Arginine 174, Isoleucine 188, and Lysine 205 with Modulatory Nucleotide in Sarcoplasmic Reticulum Ca 2+ -ATPase. The Journal of Biological Chemistry. 2008;283(51):35703 - 35714. http://hdl.handle.net/11427/35014.en_ZA
dc.language.isoeng
dc.publisher.departmentDivision of Chemical Pathology
dc.publisher.facultyFaculty of Health Sciences
dc.sourceThe Journal of Biological Chemistry
dc.source.journalissue51
dc.source.journalvolume283
dc.source.pagination35703 - 35714
dc.source.urihttps://dx.doi.org/10.1074/jbc.M806795200
dc.subject.otherAmino Acid Substitution
dc.subject.otherAnimals
dc.subject.otherBinding Sites
dc.subject.otherCOS Cells
dc.subject.otherCercopithecus aethiops
dc.subject.otherMuscle Proteins
dc.subject.otherMutagenesis, Site-Directed
dc.subject.otherPhosphorylation
dc.subject.otherProtein Structure, Tertiary
dc.subject.otherRabbits
dc.subject.otherSarcoplasmic Reticulum
dc.subject.otherSarcoplasmic Reticulum Calcium-Transporting ATPases
dc.subject.otherMuscle Proteins
dc.subject.otherSarcoplasmic Reticulum Calcium-Transporting ATPases
dc.titleCritical Interaction of Actuator Domain Residues Arginine 174, Isoleucine 188, and Lysine 205 with Modulatory Nucleotide in Sarcoplasmic Reticulum Ca 2+ -ATPase
dc.typeJournal Article
uct.type.publicationResearch
uct.type.resourceJournal Article
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