ATP‐binding to P‐type ATPases as revealed by biochemical, spectroscopic, and crystallographic experiments

Abstract

P-type ATPases form a large family of cation translocating ATPases. Recent progress in crystallography yielded several high-resolution structures of Ca(2+)-ATPase from sarco(endo)plasmic reticulum (SERCA) in various conformations. They could elucidate the conformational changes of the enzyme, which are necessary for the translocation of cations, or the mechanism that explains how the nucleotide binding is coupled to the cation transport. However, crystals of proteins are usually obtained only under conditions that significantly differ from the physiological ones and with ligands that are incompatible with the enzyme function, and both of these factors can inevitably influence the enzyme structure. Biochemical (such as mutagenesis, cleavage, and labeling) or spectroscopic experiments can yield only limited structural information, but this information could be considered relevant, because measurement can be performed under physiological conditions and with true ligands. However, interpretation of some biochemical or spectroscopic data could be difficult without precise knowledge of the structure. Thus, only a combination of both these approaches can extract the relevant information and identify artifacts. Briefly, there is good agreement between crystallographic and other experimental data concerning the overall shape of the molecule and the movement of cytoplasmic domains. On the contrary, the E1-AMPPCP crystallographic structure is, in details, in severe conflict with numerous spectroscopic experiments and probably does not represent the physiological state. Notably, the E1-ADP-AlF(4) structure is almost identical to the E1-AMPPCP, again suggesting that the structure is primarily determined by the crystal-growth conditions. The physiological relevance of the E2 and E2-P structures is also questionable, because the crystals were prepared in the presence of thapsigargin, which is known to be a very efficient inhibitor of SERCA. Thus, probably only crystals of E1-2Ca conformation could reflect some physiological state. Combination of biochemical, spectroscopic, and crystallographic data revealed amino acids that are responsible for the interaction with the nucleotide. High sequence homology of the P-type ATPases in the cytoplasmic domains enables prediction of the ATP-interacting amino acids also for other P-type ATPases.

Highly Cooperative Dependence of Sarco/Endoplasmic Reticulum Calcium ATPase (SERCA) 2a Pump Activity on Cytosolic Calcium in Living Cells

“…Channels in the plasma membrane and sarco/endoplasmic reticulum (SR/ER) 8 membrane are responsible for the Ca 2ϩ influx, whereas pumps and exchangers carry out the Ca 2ϩ efflux. SR/ER Ca 2ϩ -ATPase (SERCA) is a Ca 2ϩ pump that transfers Ca 2ϩ from the cytosol to the lumen of the SR/ER at the expense of ATP hydrolysis (3). SERCA functions to determine the resting level of intracellular Ca 2ϩ (4) and to control the spatiotemporal profile of Ca 2ϩ transients and the frequency of Ca 2ϩ oscillations (5).…”

Characterization of the Saccharomyces cerevisiae ATP-Interactome using the iTRAQ-SPROX Technique

“…There are currently over 20 X-ray crystallographic structures of Saccharomyces cerevisiae proteins that have been solved with ATP [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25]. A significantly larger number of Saccharomyces cerevisiae proteins have been predicted to bind ATP using bioinformatics approaches.…”

Elucidation of the ATP7B N-Domain Mg2+-ATP Coordination Site and Its Allosteric Regulation

“…Compared to other P-ATPases, structural information is lacking for the P Ib -ATPase subtype concerning the direct interaction between the N-domain and ATP molecule [20]. This emphasizes the question about the definition of the ATP coordination site of ATP7B N-domain as well as the role of magnesium and the two glycine amino acids (G1099, G1101) previously mentioned.…”

“…The scientific literature is abundant on P-ATPases. Many studies are focused on their catalytic cycle with an emphasis on the phosphorylation step [25] and the general role of ATP [20]. Less attention has been paid to the nucleotide-binding step.…”