Structural Studies of a Stabilized Phosphoenzyme Intermediate of Ca 2+ -ATPase

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dc.contributor.authorStokes, David L
dc.contributor.authorDelavoie, Franck
dc.contributor.authorRice, William J
dc.contributor.authorChampeil, Philippe
dc.contributor.authorMcIntosh, David B
dc.contributor.authorLacapère, Jean-Jacques
dc.date.accessioned2021-10-08T07:22:51Z
dc.date.available2021-10-08T07:22:51Z
dc.date.issued2005
dc.description.abstractCa(2+)-ATPase belongs to the family of P-type ATPases and maintains low concentrations of intracellular Ca(2+). Its reaction cycle consists of four main intermediates that alternate ion binding in the transmembrane domain with phosphorylation of an aspartate residue in a cytoplasmic domain. Previous work characterized an ultrastable phosphoenzyme produced first by labeling with fluorescein isothiocyanate, then by allowing this labeled enzyme to establish a maximal Ca(2+) gradient, and finally by removing Ca(2+) from the solution. This phosphoenzyme is characterized by very low fluorescence and has specific enzymatic properties suggesting the existence of a high energy phosphoryl bond. To study the structural properties of this phosphoenzyme, we used cryoelectron microscopy of two-dimensional crystals formed in the presence of decavanadate and determined the structure at 8-A resolution. To our surprise we found that at this resolution the low fluorescence phosphoenzyme had a structure similar to that of the native enzyme crystallized under equivalent conditions. We went on to use glutaraldehyde cross-linking and proteolysis for independent structural assessment and concluded that, like the unphosphorylated native enzyme, Ca(2+) and vanadate exert a strong influence over the global structure of this low fluorescence phosphoenzyme. Based on a structural model with fluorescein isothiocyanate bound at the ATP site, we suggest that the stability as well as the low fluorescence of this phosphoenzyme is due to a fluorescein-mediated cross-link between two cytoplasmic domains that prevents hydrolysis of the aspartyl phosphate. Finally, we consider the alternative possibility that phosphate transfer to fluorescein itself could explain the properties of this low fluorescence species.
dc.identifier.apacitationStokes, D. L., Delavoie, F., Rice, W. J., Champeil, P., McIntosh, D. B., & Lacapère, J. (2005). Structural Studies of a Stabilized Phosphoenzyme Intermediate of Ca 2+ -ATPase. <i>The Journal of Biological Chemistry</i>, 280(18), 18063 - 18072. http://hdl.handle.net/11427/35010en_ZA
dc.identifier.chicagocitationStokes, David L, Franck Delavoie, William J Rice, Philippe Champeil, David B McIntosh, and Jean-Jacques Lacapère "Structural Studies of a Stabilized Phosphoenzyme Intermediate of Ca 2+ -ATPase." <i>The Journal of Biological Chemistry</i> 280, 18. (2005): 18063 - 18072. http://hdl.handle.net/11427/35010en_ZA
dc.identifier.citationStokes, D.L., Delavoie, F., Rice, W.J., Champeil, P., McIntosh, D.B. & Lacapère, J. 2005. Structural Studies of a Stabilized Phosphoenzyme Intermediate of Ca 2+ -ATPase. <i>The Journal of Biological Chemistry.</i> 280(18):18063 - 18072. http://hdl.handle.net/11427/35010en_ZA
dc.identifier.issn0021-9258
dc.identifier.issn1083-351X
dc.identifier.ris TY - Journal Article AU - Stokes, David L AU - Delavoie, Franck AU - Rice, William J AU - Champeil, Philippe AU - McIntosh, David B AU - Lacapère, Jean-Jacques AB - Ca(2+)-ATPase belongs to the family of P-type ATPases and maintains low concentrations of intracellular Ca(2+). Its reaction cycle consists of four main intermediates that alternate ion binding in the transmembrane domain with phosphorylation of an aspartate residue in a cytoplasmic domain. Previous work characterized an ultrastable phosphoenzyme produced first by labeling with fluorescein isothiocyanate, then by allowing this labeled enzyme to establish a maximal Ca(2+) gradient, and finally by removing Ca(2+) from the solution. This phosphoenzyme is characterized by very low fluorescence and has specific enzymatic properties suggesting the existence of a high energy phosphoryl bond. To study the structural properties of this phosphoenzyme, we used cryoelectron microscopy of two-dimensional crystals formed in the presence of decavanadate and determined the structure at 8-A resolution. To our surprise we found that at this resolution the low fluorescence phosphoenzyme had a structure similar to that of the native enzyme crystallized under equivalent conditions. We went on to use glutaraldehyde cross-linking and proteolysis for independent structural assessment and concluded that, like the unphosphorylated native enzyme, Ca(2+) and vanadate exert a strong influence over the global structure of this low fluorescence phosphoenzyme. Based on a structural model with fluorescein isothiocyanate bound at the ATP site, we suggest that the stability as well as the low fluorescence of this phosphoenzyme is due to a fluorescein-mediated cross-link between two cytoplasmic domains that prevents hydrolysis of the aspartyl phosphate. Finally, we consider the alternative possibility that phosphate transfer to fluorescein itself could explain the properties of this low fluorescence species. DA - 2005 DB - OpenUCT DP - University of Cape Town IS - 18 J1 - The Journal of Biological Chemistry LK - https://open.uct.ac.za PY - 2005 SM - 0021-9258 SM - 1083-351X T1 - Structural Studies of a Stabilized Phosphoenzyme Intermediate of Ca 2+ -ATPase TI - Structural Studies of a Stabilized Phosphoenzyme Intermediate of Ca 2+ -ATPase UR - http://hdl.handle.net/11427/35010 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/35010
dc.identifier.vancouvercitationStokes DL, Delavoie F, Rice WJ, Champeil P, McIntosh DB, Lacapère J. Structural Studies of a Stabilized Phosphoenzyme Intermediate of Ca 2+ -ATPase. The Journal of Biological Chemistry. 2005;280(18):18063 - 18072. http://hdl.handle.net/11427/35010.en_ZA
dc.language.isoeng
dc.publisher.departmentInstitute of Infectious Disease and Molecular Medicine
dc.publisher.facultyFaculty of Health Sciences
dc.sourceThe Journal of Biological Chemistry
dc.source.journalissue18
dc.source.journalvolume280
dc.source.pagination18063 - 18072
dc.source.urihttps://dx.doi.org/10.1074/jbc.M500031200
dc.subject.otherAnimals
dc.subject.otherCalcium-Transporting ATPases
dc.subject.otherEnzyme Stability
dc.subject.otherPhosphates
dc.subject.otherProtein Conformation
dc.subject.otherRabbits
dc.subject.otherSarcoplasmic Reticulum
dc.subject.otherPhosphates
dc.subject.otherCalcium-Transporting ATPases
dc.titleStructural Studies of a Stabilized Phosphoenzyme Intermediate of Ca 2+ -ATPase
dc.typeJournal Article
uct.type.publicationResearch
uct.type.resourceJournal Article
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