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

Abstract

Thapsigargin (TG) is a potent inhibitor of Ca2+-ATPase from sarcoplasmic and 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 nM and that this complex closely resembles the E2 enzymatic state. We have studied the structural effects of TG binding by cryoelectron microscopy of tubular crystals, which have previously been shown to comprise Ca2+-ATPase molecules in the E2 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 the reconstructions is very similar, demonstrating that the TG/Ca2+-ATPase complex does indeed physically resemble the E2 conformation, in contrast to massive domain movements that appear to be induced by Ca2+ binding. Difference maps reveal a consistent difference on the lumenal side of the membrane, which we conclude corresponds to the thapsigargin-binding site. Modeling the atomic structure for Ca2+-ATPase into our density maps reveals that this binding site is composed of the loops between 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 changes 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 conformational coupling that drives the reaction cycle of this family of ATP-dependent ion pumps.