The E2P-like state induced by magnesium fluoride complexes in the 2 Na,K-ATPase. Kinetics of formation and interaction with Rb+

MONTES, MÓNICA R.; FERREIRA GOMES, MARIELA; CENTENO, MERCEDES; ROSSI ROLANDO

BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES

ELSEVIER SCIENCE BV

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

0005-2736

The first X-ray crystal structures of the Na,K-ATPase were obtained in the presence of magnesium and fluoride as E2(K2)Mg?MgF4, an E2∙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 magnesium fluoride complexes on the E1?E2 conformational transition and the kinetics of Rb+ exchange between the medium and the E2(Rb2)Mg?MgF4 state. Our results show that both in the absence and in the presence of Rb+, simultaneous addition of magnesium and fluoride stabilizes the Na,K-ATPase in an E2 conformation, presumably the E2Mg?MgF4 complex, that is unable to shift to E1 upon addition of Na+. The time course of conformational change suggests the action of fluoride and magnesium at different steps of the E2Mg?MgF4 formation. Increasing concentrations of fluoride revert along a sigmoid curve the drop in the level of occluded Rb+ caused by Mg2 +. Na+-induced release of Rb+ from E2(Rb2)Mg?MgF4 occurs at the same rate as from E2(Rb2) but is insensitive to ADP. The rate of Rb+ occlusion into the E2Mg?MgF4 state is 5?8 times lower than that described for the E2Mg?vanadate complex. Since the E2Mg?MgF4 and E2Mg?vanadate complexes represent different intermediates in the E2-P → E2 dephosphorylation sequence, the variation in occlusion rate could provide a tool to discriminate between these intermediates.