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Inhibitors bound to Ca(2+)-free sarcoplasmic reticulum Ca(2+)-ATPase lock its transmembrane region but not necessarily its cytosolic region, revealing the flexibility of the loops connecting transmembrane and cytosolic domains.

Ca2+-free crystals of sarcoplasmic reticulum Ca2+-ATPase have, up until now, been obtained in 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 conformation or dynamics of the cytosolic domains (as revealed by the protection afforded against proteolysis), but it becomes unable to induce any change in the transmembrane domain (as revealed by the intrinsic fluorescence of the membranous tryptophan residues). TG also obliterates the Trp fluorescence changes normally 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 isothiocyanate fluorescence after specific labeling of this residue) is significantly different in the ATPase complex with aluminum fluoride and in the ATPase complex with beryllium fluoride, and in the latter case it is modified by TG. All these facts document the flexibility of the loops connecting the transmembrane and cytosolic domains in the ATPase. In the absence of active site ligands, TG protects the ATPase from cleavage by proteinase 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 the transmembrane region, also produce similar overall effects on Ca2+-free ATPase.