Mechanism of the E2 to E1 transition in Ca pump revealed by crystal structures of gating residue mutants.

Ca-ATPase of sarcoplasmic reticulum (SERCA1a) pumps two Ca per ATP hydrolyzed from the cytoplasm and two or three protons in the opposite direction. In the E2 state, after transferring Ca into the lumen of sarcoplasmic reticulum, all of the acidic residues that coordinate Ca are thought to be protonated, including the gating residue Glu309. Therefore a Glu309Gln substitution is not expected to significantly perturb the structure.

Crystal structures of the calcium pump and sarcolipin in the Mg2+-bound E1 state.

P-type ATPases are ATP-powered ion pumps that establish ion concentration gradients across biological membranes, and are distinct from other ATPases in that the reaction cycle includes an autophosphorylation step. The best studied is Ca(2+)-ATPase from muscle sarcoplasmic reticulum (SERCA1a), a Ca(2+) pump that relaxes muscle cells after contraction, and crystal structures have been determined for most of the reaction intermediates. An important outstanding structure is that of the E1 intermediate, which has empty high-affinity Ca(2+)-binding sites ready to accept new cytosolic Ca(2+).

Trinitrophenyl derivatives bind differently from parent adenine nucleotides to Ca2+-ATPase in the absence of Ca2+.

Trinitrophenyl derivatives of adenine nucleotides are widely used for probing ATP-binding sites. Here we describe crystal structures of Ca(2+)-ATPase, a representative P-type ATPase, in the absence of Ca(2+) with bound ATP, trinitrophenyl-ATP, -ADP, and -AMP at better than 2.4-Å resolution, stabilized with thapsigargin, a potent inhibitor.