Inhibitors Bound to Ca2+-Free Sarcoplasmic Reticulum Ca2+-ATPase Lock Its Transmembrane Region but Not Necessarily Its Cytosolic Region, Revealing the Flexibility of the Loops Co

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• 作者：Cé ; dric Montigny ; Martin Picard ; Guillaume Lenoir ; Carole Gauron ; Chikashi Toyoshima ; Philippe Champeil
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：December 25, 2007
• 年：2007
• 卷：46
• 期：51
• 页码：15162 - 15174
• 全文大小：794K
• 年卷期：v.46,no.51(December 25, 2007)
• ISSN：1520-4995

文摘

Ca²⁺-free crystals of sarcoplasmic reticulum Ca²⁺-ATPase have, up until now, been obtainedin the presence of inhibitors such as thapsigargin (TG), bound to the transmembrane region of this protein.Here, we examined the consequences of such binding for the protein. We found that, after TG binding,an active site ligand such as beryllium fluoride can still bind to the ATPase and change the conformationor dynamics of the cytosolic domains (as revealed by the protection afforded against proteolysis), but itbecomes unable to induce any change in the transmembrane domain (as revealed by the intrinsicfluorescence of the membranous tryptophan residues). TG also obliterates the Trp fluorescence changesnormally induced by binding of MgATP or metal-free ATP, as well as those induced by binding of Mg²⁺alone. In the nucleotide binding domain, the environment of Lys⁵¹⁵ (as revealed by fluorescein isothiocyanatefluorescence after specific labeling of this residue) is significantly different in the ATPase complex withaluminum fluoride and in the ATPase complex with beryllium fluoride, and in the latter case it is modifiedby TG. All these facts document the flexibility of the loops connecting the transmembrane and cytosolicdomains in the ATPase. In the absence of active site ligands, TG protects the ATPase from cleavage byproteinase K at Thr²⁴²-Glu²⁴³, suggesting TG-induced reduction in the mobility of these loops. 2,5-Di-tert-butyl-1,4-dihydroxybenzene or cyclopiazonic acid, inhibitors which also bind in or near thetransmembrane region, also produce similar overall effects on Ca²⁺-free ATPase.