SERCA mutant E309Q binds two Ca2+ ions but adopts a catalytically incompetent conformation

The sarco(endo) plasmic reticulum Ca2+-ATPase (SERCA) couples ATP hydrolysis to transport of Ca2+. This directed energy transfer requires cross-talk between the two Ca2+ sites and the phosphorylation site over 50 angstrom distance. We have addressed the mechano-structural basis for this intramolecular signal by analysing the structure and the functional properties of SERCA mutant E309Q. Glu(309) contributes to Ca2+ coordination at site II, and a consensus has been that E309Q only binds Ca2+ at site I. The crystal structure of E309Q in the presence of Ca2+ and an ATP analogue, however, reveals two occupied Ca2+ sites of a non-catalytic Ca(2)E1 state. Ca2+ is bound with micromolar affinity by both Ca2+ sites in E309Q, but without cooperativity. The Ca2+ -bound mutant does phosphorylate from ATP, but at a very low maximal rate. Phosphorylation depends on the correct positioning of the A-domain, requiring a shift of transmembrane segment M1 into an 'up and kinked position'. This transition is impaired in the E309Q mutant, most likely due to a lack of charge neutralization and altered hydrogen binding capacities at Ca2+ site II.