Ca 2+versus Mg 2+ Coordination at the Nucleotide-binding site of the Sarcoplasmic Reticulum Ca 2+-ATPase

Abstract

The recently determined crystal structure of the sarcoplasmic reticulum Ca 2+-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 Ca 2+-binding sites in the membrane are known to be occluded in the latter state, in the AMPPCP-bound state the Ca 2+-binding sites are not occluded under conditions with physiological levels of Mg 2+ and Ca 2+. It has been shown that the high concentration (10 mM) of Ca 2+ used for crystallization (in the presence of Mg 2+) may be responsible for the discrepancy. To determine whether Ca 2+ competes with Mg 2+ and affects the nucleotide-binding site, we have subjected the AMPPCP and ADP:AlF 4 − 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 Mg 2+. We found that Ca 2+ rather than Mg 2+ binds together with AMPPCP at the phosphorylation site, whereas the ADP:AlF 4 − 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 2ADP:AlF 4 − transition state complex reflects the preference for Mg 2+ in catalysis. In addition, we have identified a phosphatidylcholine lipid molecule bound at the cytosol–membrane interface.