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

ROLANDO ROSSI ; MERCEDES CENTENO ; MARIELA FERREIRA GOMES; JOSE LUIS MONTI; MALÉN SAINT MARTIN; MÓNICA RAQUEL MONTES

Salto

Congreso; Latin American Crosstalk in Biophysics and Physiology 2015; 2015

Sociedad Uruguaya de Biociencias y Sociedad Argentina de Biofísica

Na,K-ATPase and H,K-ATPase are representative members of the P-type ion-transporting ATPases. By using the energy derived from ATP hydrolysis, they generate electrochemical gradients for ions across the plasma membranes of animal cells. Fluorinated complexes such as magnesium-, aluminium- and beryllium fluoride have been employed for structural analysis of the E2P states of the sarcoplasmic reticulum Ca-ATPase, SERCA [1, 2], since they seem to mimic the phosphoryl group in the 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 presence of K+ or its congener Rb+, magnesium and fluoride as E2[K2]Mg-MgF4, an E2P like state capable to occlude K+ (or Rb+) [3]. The effects of several fluoride analogues on the Na,K-ATPase and H,K-ATPase appear to be similar to those observed in SERCA [4] but the interactions between the inhibitors and these enzymes are less characterized.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. The time courses of conformational change 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∙Pi-like conformation, presumably the E2Mg-MgF4 complex, that is unable to shift to E1 upon addition of Na+. Our results suggest that MgF42- is not formed in aqueous solution but only in the phosphate binding site of the enzyme. Additionally, we performed experiments with the H,K-ATPase in order to compare the behavior of both enzymes.