Conformation of Ca(2+)-ATPase in two crystal forms. Effects of Ca2+, thapsigargin, adenosine 5'-(beta, gamma-methylene)triphosphate), and chromium(III)-ATP on crystallization.
The structure of Ca(2+)-ATPase has been studied by electron microscopy of two different crystal forms: one tubular form induced by vanadate in native sarcoplasmic reticulum (SR) membranes and another multilamellar form grown from detergent-solubilized SR. To determine the conformation of Ca(2+)-ATPase within each crystal form, the respective effects of Ca2+, thapsigargin, adenosine 5'-(beta, gamma-methylene)triphosphate) (AMP-PCP), and chromium(III) (Cr-ATP) on crystallization have been studied. Vanadate-induced tubes were prevented from forming by micromolar Ca2+, but if preformed in the absence of Ca2_, millimolar Ca2+ was required to disrupt these crystals. Thapsigargin promoted tube formation even in the presence of 10 mM Ca2+. Neither AMP-PCP nor Cr-ATP prevented tube formation, and the Ca2+ sensitivity of tube formation from Cr-ATP-inhibited SR was identical to controls. Multilamellar crystals required at least 0.2 mM Ca2+ and were prevented from forming by thapsigargin, AMP-PCP, or Cr-ATP. It is concluded that helical tubes are composed of the Ca(2+)-free, dephosphorylated conformation (E), and the nucleotide-bound conformation (E-ATP) is also tolerated. In contrast, multilamellar crystals are composed of the Ca(2+)-bound conformation (E.Ca2) and do not tolerate nucleotide binding. Thus, comparison of structures obtained from the two crystal forms should reveal physiologically relevant conformational differences.
Stokes
D L
DL
Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville 22908.
Lacapère
J J
JJ
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D003460:Crystallization; D007700:Kinetics; D008962:Models, Theoretical; D009132:Muscles / Q000201:enzymology; D011487:Protein Conformation / Q000187:drug effects; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin
D013729:Terpenes; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; D002118:Calcium