Locating the thapsigargin-binding site on Ca2+-ATPase by cryoelectron microscopy

Thapsigargin (TG) is a potent inhibitor of Ca2+-ATPase from sarcoplasmic an d endoplasmic reticula. Previous enzymatic studies have concluded that Ca2-ATPase is locked in a dead-end complex upon binding TG with an affinity of <1 nhl and that this complex closely resembles the E-2 enzymatic state. We have studied the structural effects of TG binding by cryoelectron microsco py of tubular crystals, which have previously been shown to comprise Ca2+-A TPase molecules in the E., conformation. In particular, we have compared 3D reconstructions of Ca2+-ATPase in the absence and presence of either TG or its dansylated derivative. The overall molecular shape of Ca2+-ATPase in t he reconstructions is very similar, demonstrating that the TG/Ca2+-ATPase c omplex does indeed physically resemble the E-2 conformation, in contrast to massive domain movements that appear to be induced by Ca2+ binding. Differ ence maps reveal a consistent difference on the lumenal side of the membran e, which we conclude corresponds to the thapsigargin-binding site. Modeling the atomic structure for Ca2+-ATPase into our density maps reveals that th is binding site is composed of the loops bet been transmembrane segments M3 /M4 and M7/M8. indirect effects are proposed to explain the effects of the S3 stalk segment on thapsigargin affinity as well as thapsigargin-induced c hanges in ATP affinity. Indeed, a second difference density was observed at the decavanadate-binding site within the three cytoplasmic domains, which we believe reflects an altered affinity as a result of the long-range confo rmational coupling that drives the reaction cycle of this family of ATP-dep endent ion pumps. <(c)> 2001 Academic Press.