Locating the thapsigargin-binding site on Ca(2+)-ATPase by cryoelectron microscopy.
Thapsigargin (TG) is a potent inhibitor of Ca(2+)-ATPase from sarcoplasmic and endoplasmic reticula. Previous enzymatic studies have concluded that Ca(2+)-ATPase is locked in a dead-end complex upon binding TG with an affinity of <1 nM and that this complex closely resembles the E(2) enzymatic state. We have studied the structural effects of TG binding by cryoelectron microscopy of tubular crystals, which have previously been shown to comprise Ca(2+)-ATPase molecules in the E(2) conformation. In particular, we have compared 3D reconstructions of Ca(2+)-ATPase in the absence and presence of either TG or its dansylated derivative. The overall molecular shape of Ca(2+)-ATPase in the reconstructions is very similar, demonstrating that the TG/Ca(2+)-ATPase complex does indeed physically resemble the E(2) conformation, in contrast to massive domain movements that appear to be induced by Ca(2+) 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 Ca(2+)-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.
Young
H S
HS
Skirball Institute of Biomolecular Medicine, Department of Cell Biology, NYU School of Medicine, New York, NY 10016, USA.
Xu
C
C
Zhang
P
P
Stokes
D L
DL
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy; D003460:Crystallization; D003619:Dansyl Compounds / Q000378:metabolism; D005583:Fourier Analysis; D007091:Image Processing, Computer-Assisted; D008958:Models, Molecular; D011487:Protein Conformation; D051381:Rats; D012519:Sarcoplasmic Reticulum; D019284:Thapsigargin / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology
D003619:Dansyl Compounds; C093012:dansylthapsigargin; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases