Conformational Fluctuations of the Ca2+-ATPase in the Native Membrane Environment

Abstract

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 ...

Specificity of ligand binding to transport sites: Ca2+ binding to the Ca2+ transport ATPase and its dependence on H+ and Mg2+

“…The EF hand conformation of site II, on the other hand, appears more specific for Ca 2+ and is likely to exclude Mg 2+ . This explains how the enzyme (in the absence of Ca 2+ ) acquires an E 1 conformation at high pH and in the presence of Mg 2+ , without reaching catalytic activation [18].…”

“…The presence of protons provides important stabilization of the E 2 state by the establishment of hydrogen bonding. In the absence of TG and BHQ, gradual H + dissociation and destabilization of the E 2 state are expected as the pH is raised from 6 to 8 [18,27].…”

“…It has also been reported that Mg 2+ , which is an absolute requirement at the catalytic site for utilization of ATP, can also bind competitively to the Ca 2+ sites under certain conditions [9]. We recently observed that Mg 2+ , at high pH and in the absence of Ca 2+ , confers to the ATPase conformational features (E 1 state) similar to those produced by Ca 2+ binding, but without catalytic activation [18]. We report here new measurements of Ca 2+ binding to the ATPase activation/transport sites of the SERCA protein, and its dependence on H + and Mg 2+ .…”

“…The EF hand conformation of site II, on the other hand, appears more specific for Ca 2+ and is likely to exclude Mg 2+ . This explains how the enzyme (in the absence of Ca 2+ ) acquires an E 1 conformation at high pH and in the presence of Mg 2+ , without reaching catalytic activation [18].It is noteworthy that when enzyme turnover occurs in the presence of ATP, Ca 2+ /H + exchange occurs as the phosphorylated enzyme dissociates bound Ca 2+ on the luminal side of the membrane in exchange for H + . However, exchange of all four H + for the 2 Ca 2+ transported per each ATPase cycle is not expected since the Glu309 side chain orientation makes unlikely Zafar et al…”

“…The presence of protons provides important stabilization of the E 2 state by the establishment of hydrogen bonding. In the absence of TG and BHQ, gradual H + dissociation and destabilization of the E 2 state are expected as the pH is raised from 6 to 8 [18,27].On the other hand, similar calculations predict that the four acid residues participating in Ca 2+ binding are all unprotonated in the E 1 ·2Ca 2+ state around physiological pH [26]. However, Glu908 may dissociate its H + only as the pH is raised to 8 [24], while Glu58, which does not participate directly in binding, retains a H + by interacting with the Glu309 carbonyl oxygen.…”