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Publication Details
AFRICAN RESEARCH NEXUS
SHINING A SPOTLIGHT ON AFRICAN RESEARCH
biochemistry, genetics and molecular biology
Structural studies of a stabilized phosphoenzyme intermediate of Ca
2+
-ATPase
Journal of Biological Chemistry, Volume 280, No. 18, Year 2005
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Description
Ca2+-ATPase belongs to the family of P-type ATPases and maintains low concentrations of intracellular Ca2+. 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 Ca2+ gradient, and finally by removing Ca2+ 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-Å 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 crosslink 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. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
Authors & Co-Authors
Stokes, David L.
United States, New York
Nyu Grossman School of Medicine
United States, New York
New York Structural Biology Center
Delavoie, Franck
France, Paris
Inserm
Rice, William J.
United States, New York
Nyu Grossman School of Medicine
Champeil, Philippe
France, Gif-sur-yvette
Cea Saclay
McIntosh, David B.
South Africa, Cape Town
University of Cape Town
Lacapère, Jean Jacques
France, Paris
Inserm
Statistics
Citations: 22
Authors: 6
Affiliations: 5
Identifiers
Doi:
10.1074/jbc.M500031200
ISSN:
00219258