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 [Ca 2+ ] AMPPCP versus ADP.AlFx, and Ca ion occlusion in SERCA1a - Article - Mai 2005

Martin Picard, Chikashi Toyoshima, Philippe Champeil

Martin Picard, Chikashi Toyoshima, Philippe Champeil, « 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 [Ca 2+ ]  », Journal of Biological Chemistry, mai 2005, pp. 18745-18754. ISSN 0021-9258

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, ADP.fluoroaluminate, 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 E1.AMPPCP and the E1.ADP.AlFx 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 crystallisation media) than when they are examined in the presence of the few µM 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 towards SH-directed Ellman’s reagent, ATPase intrinsic fluorescence properties (here described for the E1.ADP.AlFx 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 Ca 2+-ATPase.AMPPCP crystals should be re-investigated. A contrario, they emphasize the fact that the average conformation of the E1.AMPPCP complex under usual conditions in the test tube differs from that found in the crystalline form. The extended conformation of nucleotide revealed by the E1.AMPPCP crystalline form might be only indicative of the requirements for further processing of the complex, towards the transition state leading to phosphorylation and Ca 2+ occlusion.

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