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+). In the absence of Ca(2+) and at pH 7 or higher, the ATPase is predominantly in E1, not in E2 (low affinity for Ca(2+)), and if millimolar Mg(2+) is present, one Mg(2+) is expected to occupy one of the Ca(2+)-binding sites with a millimolar dissociation constant. This Mg(2+) accelerates the reaction cycle, not permitting phosphorylation without Ca(2+) binding. Here we describe the crystal structure of native SERCA1a (from rabbit) in this E1·Mg(2+) state at 3.0 Å resolution in addition to crystal structures of SERCA1a in E2 free from exogenous inhibitors, and address the structural basis of the activation signal for phosphoryl transfer. Unexpectedly, sarcolipin, a small regulatory membrane protein of Ca(2+)-ATPase, is bound, stabilizing the E1·Mg(2+) state. Sarcolipin is a close homologue of phospholamban, which is a critical mediator of β-adrenergic signal in Ca(2+) regulation in heart (for reviews, see, for example, refs 8-10), and seems to play an important role in muscle-based thermogenesis. We also determined the crystal structure of recombinant SERCA1a devoid of sarcolipin, and describe the structural basis of inhibition by sarcolipin/phospholamban. Thus, the crystal structures reported here fill a gap in the structural elucidation of the reaction cycle and provide a solid basis for understanding the physiological regulation of the calcium pump.

2؉ to the E2-P pattern, whereby alkaline pH will limit this conformational transition. Complementary experiments on digestion with trypsin exhibit high temperature dependence, indicating that, in the E1 and E2 ground states, the ATPase conformation undergoes strong fluctuations related to internal protein dynamics. The fluctuations are tightly constrained by ATP binding and phosphoenzyme formation, and this constraint must be overcome by thermal activation and substrate-free energy to allow enzyme turnover. In fact, a substantial portion of ATP free energy is utilized for conformational work related to the E1ϳP⅐2Ca 2؉ to E2-P transition, thereby disrupting high affinity binding and allowing luminal diffusion of Ca 2؉ . The E2 state and luminal path closure follow removal of conformational constraint by phosphate.The Ca 2ϩ -ATPase of sarcoendoplasmic reticulum membranes (SERCA) 2 includes multiple isoforms and splice variants with variable tissue distribution. In this study, we used the SERCA1a isoform of skeletal muscle, a well characterized enzyme (1, 2) that utilizes the free energy of ATP for Ca 2ϩ transport against a concentration gradient. The functional unit is a protein monomer consisting of 994 amino acid residues. The sequence is folded into a cluster of 10 segments forming a transmembrane region, and three relatively large domains ("N", "P," and "A") protruding from the cytosolic surface of the membrane (3, 4). The ATPase cycle begins with high affinity binding of two Ca 2ϩ derived from the cytosolic medium ("outside"), followed by ATP utilization to form a phosphorylated enzyme intermediate. Isomerization of the phosphoenzyme intermediate is then coupled to active transport of the bound Ca 2ϩ across the membrane ("inside"). Hydrolytic cleavage of the phosphoenzyme is the final step that allows enzyme turnover.The cooperative character of Ca 2ϩ binding as well as the relatively large distance between the catalytic site in the headpiece and the Ca 2ϩ -binding sites of the ATPase within the transmembrane region imply that conformational rearrangements of the ATPase protein are involved in the mechanism of catalytic activation and energy transduction. Within the general context of cation transport, these rearrangements were envisioned as interconversions of the E1 and E2 conformations in the ground state of the enzyme and the E1-P to E2-P conformations of the phosphorylated intermediate. In fact, conformational changes were initially detected by spectroscopic experimentation (5). High resolution structures were then obtained by crystallographic studies and attributed to different catalytic intermediates (6). On the other hand, the occurrence of conformational transitions in the native membrane environment is revealed by changes in the patterns of proteolysis (7). We report here a series of experiments on limited proteolysis with proteinase K or trypsin, yielding complementary information on the conformational effects of pH, temperature, catalytic ligands, and the specific inhibitor thapsigargin (TG). The ...

Recombinant and purified Thermotoga maritima CopA sustains ATPase velocity of 1.78 -2.73 mol/mg/min in the presence of Cu ؉ (pH 6, 60°C) and 0.03-0.08 mol/mg/min in the absence of Cu ؉ . High levels of enzyme phosphorylation are obtained by utilization of [␥-32 P]ATP in the absence of Cu ؉ . This phosphoenzyme decays at a much slower rate than observed with Cu⅐E1 ϳ P. In fact, the phosphoenzyme is reduced to much lower steady state levels upon addition of Cu ؉ , due to rapid hydrolytic cleavage. Negligible ATPase turnover is sustained by CopA following deletion of its N-metal binding domain (⌬NMBD) or mutation of NMBD cysteines (CXXC). Nevertheless, high levels of phosphoenzyme are obtained by utilization of [␥-32 P]ATP by the ⌬NMBD and CXXC mutants, with no effect of Cu ؉ either on its formation or hydrolytic cleavage. Phosphoenzyme formation (E2P) can also be obtained by utilization of P i , and this reaction is inhibited by Cu ؉ (E2 to E1 transition) even in the ⌬NMBD mutant, evidently due to Cu ؉ binding at a (transport) site other than the NMBD. E2P undergoes hydrolytic cleavage faster in ⌬NMBD and slower in CXXC mutant. We propose that Cu ؉ binding to the NMBD is required to produce an "active" conformation of CopA, whereby additional Cu ؉ bound to an alternate (transmembrane transport) site initiates faster cycles including formation of Cu⅐E1 ϳ P, followed by the E1 ϳ P to E2-P conformational transition and hydrolytic cleavage of phosphate. An H479Q mutation (analogous to one found in Wilson disease) renders CopA unable to utilize ATP, whereas phosphorylation by P i is retained.Cation transport ATPases utilize ATP-free energy for transport of specific ions across biological membranes, against electrochemical gradients. They are referred to as P-type ATPases when enzyme phosphorylation, obtained by phosphoryl transfer from ATP to the enzyme protein, is an obligatory intermediate step in the mechanism of ATP utilization and coupled cation transport (1-3). The P-type ATPase family is divided into five branches referred to as I-V (4), including the important PII-type ATPases, which are specific for H , and Co 2ϩ (5). The PIB ATPases sustain important roles in accumulation and tolerance of heavy metal in biological systems (6 -8), as well as for delivery of copper to metalloenzymes (9). Two ATPases of this subgroup serve as copper transporters in humans (10). Mutations of these proteins are involved in the etiology of Menkes and Wilson diseases (8,(11)(12)(13). The catalytic mechanism of the PIB ATPases has been the subject of preliminary studies (14 -19).For a comparative evaluation, it is useful to consider that the structure of PII-type ATPases, originally established for the Ca 2ϩ -ATPase by sequence analysis (20) and crystallography (21), includes three cytosolic domains referred to as N (nucleotide binding), P (phosphorylation), and A (actuator) domains, and 10 transmembrane helices containing the specific cation binding site for catalytic activation and transport. On the other hand, the structure of the...

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Junctional adhesion molecule-A (JAM-A