Modulatory and catalytic modes of ATP binding by the calcium pump.
We present crystal structures of the calcium-free E2 state of the sarcoplasmic reticulum Ca2+ -ATPase, stabilized by the inhibitor thapsigargin and the ATP analog AMPPCP. The structures allow us to describe the ATP binding site in a modulatory mode uncoupled from the Asp351 phosphorylation site. The Glu439 side chain interacts with AMPPCP via an Mg2+ ion in accordance with previous Fe2+ -cleavage studies implicating this residue in the ATPase cycle and in magnesium binding. Functional data on Ca2+ mediated activation indicate that the crystallized state represents an initial stage of ATP modulated deprotonation of E2, preceding the binding of Ca2+ ions in the membrane from the cytoplasmic side. We propose a mechanism of Ca2+ activation of phosphorylation leading directly from the compact E2-ATP form to the Ca2E1-ATP state. In addition, a role of Glu439 in ATP modulation of other steps of the functional cycle is suggested.
Jensen
Anne-Marie Lund
AM
Department of Molecular Biology, Aarhus University, Denmark.
Sørensen
Thomas Lykke-Møller
TL
Olesen
Claus
C
Møller
Jesper Vuust
JV
Nissen
Poul
P
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011522:Protons; D011817:Rabbits; D019284:Thapsigargin / Q000378:metabolism
D011522:Protons; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium