Abstract
Crystalline forms of detergent-solubilized sarcoplasmic reticulum Ca2+-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 Ca2+ (the concentration prevailing in the crystallization media) than when they are examined in the presence of the few micromolar of Ca2+ 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 E1.ADP.AlFx complex for the first time), and also the rates of 45Ca2+-40Ca2+ exchange at site "II." These results solve the above paradox at least partially and suggest that the presence of a previously unrecognized Ca2+ ion in the E1.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, toward the transition state leading to phosphorylation and Ca2+ occlusion.
Highlights
After the initial description of the high resolution structures of two crystalline forms of the sarcoplasmic reticulum calcium pump [1, 2], additional forms of this Ca2ϩ-ATPase were recently crystallized, with the hope of characterizing as many as possible of the different intermediates formed in sequence during the catalytic cycle of this enzyme and to provide a structural basis for the mechanistic description of ion pumping [3,4,5,6,7]
ADP1⁄7AlF4 Blocks 45Ca2ϩ Dissociation from SR Ca2ϩ-ATPase, but under Ordinary Conditions, AMPPCP Does Not—Sørensen et al [4] and Toyoshima and Mizutani [5] found that the presence of AMPPCP or ADP1⁄7fluoroaluminate in the crystalline forms of Ca2ϩ-ATPase resulted in structural changes indicative of occlusion of the bound Ca2ϩ ions in both cases, but they already noted that this was somewhat dissonant with a previous experimental finding deduced from rapid filtration experiments with 45Ca2ϩ: the half-time for the dissociation of 45Ca2ϩ from the transport sites of non-phosphorylated ATPase only increases by 50% in the presence of 250 M AMPPCP, at pH 6 in the presence of 20 mM Mg2ϩ [12], and dissociation remains relatively fast in the presence of AMPPCP
Depending on Mg2ϩ, AMPPCP May Either Stimulate or Slightly Reduce the Rate of Overall Ca2ϩ Dissociation from Ca2ϩ-ATPase, and the Modulatory Effect of Mg2ϩ Differs for Various Nucleotides—We re-investigated the effect of AMPPCP under the previous conditions at pH 6, using a stopped-flow assay in which Ca2ϩ dissociation from Ca2ϩATPase was triggered by mixing Ca2ϩ-equilibrated SR vesicles with the fluorescent chelator quin2, whose fluorescence changes allowed us to monitor the rate of this Ca2ϩ dissociation
Summary
After the initial description of the high resolution structures of two crystalline forms of the sarcoplasmic reticulum calcium pump (the membranous Ca2ϩ-dependent P-type ATPase SERCA1a) [1, 2], additional forms of this Ca2ϩ-ATPase were recently crystallized, with the hope of characterizing as many as possible of the different intermediates formed in sequence during the catalytic cycle of this enzyme and to provide a structural basis for the mechanistic description of ion pumping [3,4,5,6,7] Among these forms, one has its two transport sites occupied by Ca2ϩ, and its nucleotide binding site occupied by a non-hydrolyzable analog of ATP, AMPPCP1; it is referred to as “E11⁄7AMPPCP.”. We discuss various possibilities to explain, a contrario, the differences between the fluoroaluminate complex and the AMPPCP complex under more usual conditions: among these, the average conformation of the E11⁄7AMPPCP complex under such conditions might well be different from the one found in the crystal, a fact that should not be overlooked in future descriptions of ATP binding to Ca2ϩ-ATPase during the normal cycle
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