The E2P-like state induced by magnesium fluoride complexes in the Na,K-ATPase. Kinetics of formation and interaction with Rb+

MÓNICA R. MONTES; MARIELA S. FERREIRA-GOMES; MERCEDES CENTENO; ROLANDO C. ROSSI

BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2015 vol. 1848 p. 1514 - 1523

0005-2736

Thefirst X-ray crystal structures of the Na,K-ATPase were obtained in the presence of magnesium andfluoride as E2(K2)Mg?MgF4,anE2∙Pi-like state capable to occlude K+(or Rb+). This work presents a functional characterization of the crystallized form of the enzyme and proposes a model to explain the interaction between magnesium,fluoride and Rb+ with the Na,K-ATPase. We studied the effect of magnesium and magnesiumfluoride complexes on theE1?E2 conformational transition and the kinetics of Rb+ exchange between the medium and theE2(Rb2)Mg?MgF4state. Our results show that both in the absence and in the presence of Rb+,simultaneous addition of magnesium andfluoride stabilizes the Na,K-ATPase in anE2 conformation, presumably theE2Mg?MgF4complex, that is unable to shift toE1 upon addition of Na+. The time course of conformational change suggests the action offluoride and magnesium at different steps of theE2Mg?MgF4formation. Increasing concentrations offluoride revert along a sigmoid curve the drop in the level of occluded Rb+ caused by Mg2+.Na+-inducedrelease of Rb+ fromE2(Rb2)Mg?MgF4occurs at the same rate as fromE2(Rb2) but is insensitive to ADP. The rate of Rb+ occlusion into theE2Mg?MgF4state is 5?8 times lower than that described for theE2Mg?vanadate complex. Since theE2Mg?MgF4andE2Mg?vanadate complexes represent different intermediates in theE2-P→E2 dephosphorylation sequence, the variation in occlusion rate could provide a tool to discriminate between these intermediates.