Premium
SERCA mutant E309Q binds two Ca 2+ ions but adopts a catalytically incompetent conformation
Author(s) -
Clausen Johannes D,
Bublitz Maike,
Arnou Bertrand,
Montigny Cédric,
Jaxel Christine,
Møller Jesper Vuust,
Nissen Poul,
Andersen Jens Peter,
le Maire Marc
Publication year - 2013
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1038/emboj.2013.250
Subject(s) - biology , mutant , serca , ion , crystallography , biophysics , biochemistry , enzyme , physics , gene , atpase , chemistry , quantum mechanics
The sarco(endo)plasmic reticulum Ca 2+ ‐ATPase (SERCA) couples ATP hydrolysis to transport of Ca 2+ . This directed energy transfer requires cross‐talk between the two Ca 2+ sites and the phosphorylation site over 50 Å 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 Ca 2+ coordination at site II, and a consensus has been that E309Q only binds Ca 2+ at site I. The crystal structure of E309Q in the presence of Ca 2+ and an ATP analogue, however, reveals two occupied Ca 2+ sites of a non‐catalytic Ca 2 E 1 state. Ca 2+ is bound with micromolar affinity by both Ca 2+ sites in E309Q, but without cooperativity. The Ca 2+ ‐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 Ca 2+ site II.