Author(s): Picard M, Jensen AM, Srensen TL, Champeil P, Mller JV,
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Abstract The recently determined crystal structure of the sarcoplasmic reticulum Ca2+-ATPase (SERCA1a) with a bound ATP analogue (AMPPCP) reveals a compact state, similar to that found in the presence of ADP and aluminium fluoride. However, although the two Ca2+-binding sites in the membrane are known to be occluded in the latter state, in the AMPPCP-bound state the Ca2+-binding sites are not occluded under conditions with physiological levels of Mg2+ and Ca2+. It has been shown that the high concentration (10 mM) of Ca2+ used for crystallization (in the presence of Mg2+) may be responsible for the discrepancy. To determine whether Ca2+ competes with Mg2+ and affects the nucleotide-binding site, we have subjected the AMPPCP and ADP:AlF4- bound forms to crystallographic analysis by anomalous difference Fourier maps, and we have compared AMPPCP-bound forms crystallized in the absence or in the presence of Mg2+. We found that Ca2+ rather than Mg2+ binds together with AMPPCP at the phosphorylation site, whereas the ADP:AlF4- complex is associated with two magnesium ions. These results address the structure of the phosphorylation site before and during phosphoryl transfer. The bound CaAMPPCP nucleotide may correspond to the activated pre-complex, formed immediately before phosphorylation, whereas the Mg(2)ADP:AlF4- transition state complex reflects the preference for Mg2+ in catalysis. In addition, we have identified a phosphatidylcholine lipid molecule bound at the cytosol-membrane interface.
This article was published in J Mol Biol
and referenced in Journal of Glycomics & Lipidomics