Conformational changes in sarcoplasmic reticulum Ca(2+)-ATPase mutants: effect of mutations either at Ca(2+)-binding site II or at tryptophan 552 in the cytosolic domain

Biochemistry
Guillaume LenoirPierre Falson

Abstract

By analyzing, after expression in yeast and purification, the intrinsic fluorescence properties of point mutants of rabbit Ca(2+)-ATPase (SERCA1a) with alterations to amino acid residues in Ca(2+)-binding site I (E(771)), site II (E(309)), in both sites (D(800)), or in the nucleotide-binding domain (W(552)), we were able to follow the conformational changes associated with various steps in the ATPase catalytic cycle. Whereas Ca(2+) binding to purified wild-type (WT) ATPase in the absence of ATP leads to the rise in Trp fluorescence expected for the so-called E2 --> E1Ca(2) transition, the Ca(2+)-induced fluorescence rise is dramatically reduced for the E(309)Q mutant. As this purified E(309)Q mutant retains the ability to bind Ca(2+) at site I (but not at site II), we tentatively conclude that the protein reorganization induced by Ca(2+) binding at site II makes the major contribution to the overall Trp fluorescence changes observed upon Ca(2+) binding to both sites. Judging from the fluorescence response of W(552)F, similar to that of WT, these changes appear to be primarily due to membranous tryptophans, not to W(552). The same holds for the fluorescence rise observed upon phosphorylation from P(i) (the so-called E2 --> E2P t...Continue Reading

References

Nov 10, 1995·The Journal of Biological Chemistry·E MintzF Guillain
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Apr 9, 2004·Nature Reviews. Molecular Cell Biology·Werner Kühlbrandt
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Jul 21, 2004·The Journal of Biological Chemistry·Cedric MontignyMarc le Maire

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Citations

Jul 17, 2010·Current Opinion in Structural Biology·Maike BublitzPoul Nissen
Jul 24, 2012·Protein Science : a Publication of the Protein Society·Peter M Kekenes-HuskeyJ Andrew McCammon
Nov 26, 2013·The EMBO Journal·Johannes D ClausenMarc le Maire
Jul 13, 2005·Current Opinion in Structural Biology·Jesper V MøllerMarc le Maire
Feb 23, 2021·Frontiers in Molecular Biosciences·Thomas BarbotLiliane Mouawad
Dec 14, 2016·Biochemistry·Edgar D Páez-PérezJosé G Sampedro

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