The Average Conformation at Micromolar [Ca2+] of Ca2+-ATPase with Bound Nucleotide Differs from That Adopted with the Transition State Analog ADP·AlFx or with AMPPCP under Crystallization Conditions at Millimolar [Ca2+]

Abstract

Crystalline forms of detergent-solubilized sarcoplasmic reticulum Ca 2؉ -ATPase, obtained in the presence of either a substrate analog, AMPPCP, or a transition state complex, ADP⅐fluoroaluminate, were recently described to share the same general architecture despite the fact that, when studied in a test tube, these forms show different functional properties. Here, we show that the differences in the properties of the E1⅐AMPPCP and the E1⅐ADP⅐AlFx membraneous (or solubilized) forms are much less pronounced when these properties are examined in the presence of 10 mM Ca 2؉ (the concentration prevailing in the crystallization media) than when they are examined in the presence of the few micromolar of Ca 2؉ known to be sufficient to saturate the transport sites. This concerns various properties, including ATPase susceptibility to proteolytic cleavage by proteinase K, ATPase reactivity toward SH-directed Ellman's reagent, ATPase intrinsic fluorescence properties (here described for the E1⅐ADP⅐AlFx complex for the first time), and also the rates of 45 Ca 2؉ -40 Ca 2؉ exchange at site "II." These results solve the above paradox at least partially and suggest that the presence of a previously unrecognized Ca 2؉ ion in the E1⅐AMPPCP crystals should be re-investigated. A contrario, they emphasize the fact that the average conformation of the E1⅐AMPPCP complex under usual conditions in the test tube differs from that found in the crystalline form. The extended conformation of nucleotide revealed by the E1⅐AMPPCP crystalline form might be only indicative of the requirements for further processing of the complex, toward the transition state leading to phosphorylation and Ca 2؉ occlusion.

Comprehensive Analysis of Expression and Function of 51 Sarco(endo)plasmic Reticulum Ca2+-ATPase Mutants Associated with Darier Disease

“…Deletions ⌬L41 and ⌬P42 on this linker also inhibited the dephosphorylation process. These results are entirely consistent with our previous mutation study with SERCA1a and the prediction (61) that the proper structure of the A domain at the junctional residue Asn 39 and the appropriate length of the A domain/M1 linker (Glu 40 -Ser 48 ) are critical in the dephosphorylation process probably for inducing the A domain motion. Asn 39 also forms a hydrogen-bonding network with the top part of the A domain/M3 linker, the strain of which has also been predicted to be critical as a moving force for the A domain rotation in the dephosphorylation process (53).…”

“…It is possible that the coordination of Ca 2ϩ became more favored as compared with Mg 2ϩ (especially at the lower temperature) in the mutated catalytic site and inhibited the phosphorylation. In phosphoserine phosphatase, a member of the haloacid dehalogenase superfamily of SERCAs, the coordination geometry of Ca 2ϩ at the catalytic site was previously revealed to be different from that of the native cofactor Mg 2ϩ and therefore not suitable for catalysis (46 (38,39) was recently interpreted as a transient conformation for phosphoryl transfer (48). Consistently, the rate-limiting step for EP formation from E1Ca 2 and MgATP was previously shown to be the conformational change in the E1Ca 2 ⅐MgATP complex (to bring ␥-phosphate to Asp 351 (50)) preceding the phosphoryl transfer (50 -52).…”

Domain Organization and Movements in Heavy Metal Ion Pumps

“…In contrast, if the Ca 2ϩ concentration is low (e.g. 0.1 mM), AMPPCP rather enhances exchange of transmembranebound Ca 2ϩ with those in the cytoplasm (41), presumably because approaches of ATP (analogs) to the P-domain agitate large structural changes in both cytoplasmic and transmembrane domains. Thus, with CopA, where the Cu ϩ concentration is low, the enhanced susceptibility of the A-domain-M3 loop in E1⅐AMPPCP is not unexpected.…”

“…The protection of SERCA1 in E1⅐AMPPCP is due to a stabilization of the same closed configuration of the cytoplasmic domains as in E1⅐AlF x ⅐ADP by the binding of Ca 2ϩ to the Mg 2ϩ site in the P-domain (8,10,24). Thus, the protection of this site by AMPPCP requires millimolar Ca 2ϩ (24,41) and causes occlusion of Ca 2ϩ in the transmembrane binding sites. In contrast, if the Ca 2ϩ concentration is low (e.g.…”

Concerted Conformational Effects of Ca2+and ATP Are Required for Activation of Sequential Reactions in the Ca2+ATPase (SERCA) Catalytic Cycle,

“…In fact, the enzyme with bound nucleotide (AMPPCP-E1-2Ca 2+ ) and the phosphoenzyme analog with bound ADP (ADP-E1-AlF x -2Ca 2+ ) yield quite similar crystal structures, including evidence for an occluded state of bound Ca 2+ (10,12). On the other hand, the effects of these ligands in solution are quite different (27,28), since Ca 2+ occlusion can be easily demonstrated in the presence of AlF x. and ADP, but it is very difficult to demonstrate in the presence of AMPPCP. Finally, a related issue is the lack of any data on ADP binding by the ATPase in solution.…”