Conformational changes induced by magnesium fluoride complexes in the Na,K-ATPase and H,K-ATPase

ROSSI, RC; CENTENO, MERCEDES; FERREIRA GOMES, MARIELA; MONTI, JOSE LUIS; SAINT MARTIN, MALÉN; MONTES, MÓNICA R.

Salto

Congreso; Latin American Crosstalk In Biophysics And Physiology; 2015

Na,K-ATPase and H,K-ATPase are representative members of the P-typeion-transporting ATPases. By using the energy derived from ATPhydrolysis, they generate electrochemical gradients for ions across theplasma membranes of animal cells. Fluorinated complexes such asmagnesium-, aluminium- and beryllium fluoride have been employed forstructural analysis of the E2P states of the sarcoplasmic reticulum Ca-ATPase, SERCA [1, 2], since they seem to mimic the phosphoryl group inthe ground (BeFx), transition (AlFx and vanadate) and product (MgFx)states during the dephosphorylation sequence:E2-P (ground state) → E2∙P (transition state) → E2∙Pi (product state) → E2.The first X-ray crystal structures of the Na,K-ATPase were obtained in the, magnesium and fluoride as E2[K2]Mg-MgF4, an E2Pi-like state capable to occlude K+presence of K+several fluoride analogues on the Na,K-ATPase and H,K-ATPase appear tobe similar to those observed in SERCA [4] but the interactions between theinhibitors and these enzymes are less characterized.This work presents a functional characterization of the crystallized form ofthe enzyme and proposes a model to explain the interaction betweenmagnesium, fluoride and Rb+conformational change show that both in the absence and in the presence of, simultaneous addition of magnesium and fluoride stabilizes the Na,K-ATPase in an E2∙Pi-like conformation, presumably the E2Mg-MgF4Rb+complex, that is unable to shift to E1 upon addition of Na+suggest that MgF4phosphate binding site of the enzyme. Additionally, we performedexperiments with the H,K-ATPase in order to compare the behavior of bothenzymes.1] Danko S, et al. J. Biol. Chem 279:14991?98, 2004.[2] Clausen J.D., et al, J. Biol. Chem. 286:11792?802, 2011[3] Shinoda T, et al, Nature 459:446?450, 2009[4] Murphy A.J., et al, J. Biol. Chem. 267:16995-16700, 1992.With grants from ANPCYT, CONICET and UBACYTor its congener Rb+(or Rb+with the Na,K-ATPase. The time courses of2- is not formed in aqueous solution but only in the