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

TY - JOUR

T1 - SERCA mutant E309Q binds two Ca ions but adopts a catalytically incompetent conformation

AU - Clausen, Johannes D.

AU - Bublitz, Maike

AU - Arnou, Bertrand

AU - Montigny, Cédric

AU - Jaxel, Christine

AU - Møller, Jesper Vuust

AU - Nissen, Poul

AU - Andersen, Jens Peter

AU - le Maire, Marc

PY - 2013/12/11

Y1 - 2013/12/11

N2 - 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 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. Glu309 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 Ca2E1 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.

AB - 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 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. Glu309 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 Ca2E1 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.

U2 - 10.1038/emboj.2013.250

DO - 10.1038/emboj.2013.250

M3 - Journal article

C2 - 24270570

SN - 0261-4189

VL - 32

SP - 3231

EP - 3243

JO - E M B O Journal

JF - E M B O Journal

IS - 24

ER -