Polarity of the ATP binding site of the Na+,K+-ATPase, gastric H+,K+-ATPase and sarcoplasmic reticulum Ca2+-ATPase

K.R Hossain,X Li,T Zhang,S Paula,F Cornelius,R.J Clarke

doi:10.1016/j.bbamem.2019.183138

K.R Hossain, X Li + Show 4 more

Open Access

https://doi.org/10.1016/j.bbamem.2019.183138

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Abstract

A fluorescence ratiometric method utilizing the probe eosin Y is presented for estimating the ATP binding site polarity of P-type ATPases in different conformational states. The method has been calibrated by measurements in a series of alcohols and tested using complexation of eosin Y with methyl-β-cyclodextrin. The results obtained with the Na+,K+-, H+,K+- and sarcoplasmic reticulum Ca2+-ATPases indicate that the ATP binding site, to which eosin is known to bind, is significantly more polar in the case of the Na+,K+- and H+,K+-ATPases compared to the Ca2+-ATPase. This result was found to be consistent with docking calculations of eosin with the E2 conformational state of the Na+,K+-ATPase and the Ca2+-ATPase. Fluorescence experiments showed that eosin binds significantly more strongly to the E1 conformation of the Na+,K+-ATPase than the E2 conformation, but in the case of the Ca2+-ATPase both fluorescence experiments and docking calculations showed no significant difference in binding affinity between the two conformations. This result could be due to the fact that, in contrast to the Na+,K+- and H+,K+-ATPases, the E2-E1 transition of the Ca2+-ATPase does not involve the movement of a lysine-rich N-terminal tail which may affect the overall enzyme conformation. Consistent with this hypothesis, the eosin affinity of the E1 conformation of the Na+,K+-ATPase was significantly reduced after N-terminal truncation. It is suggested that changes in conformational entropy of the N-terminal tail of the Na+, K+- and the H+,K+-ATPases during the E2-E1 transition could affect the thermodynamic stability of the E1 conformation and hence its ATP binding affinity.

Highlights

P-type ATPases play crucial roles in cell physiology
The Na+ electrochemical potential gradient created across the plasma membrane of animal cells by the Na+,K+-ATPase is used to drive nutrient reabsorption in kidney, and both the Na+ and K+ gradients it generates are essential for nerve and muscle function [1]
In this paper we report a calibration of eosin’s fluorescence excitation spectrum and provide quantitative information on the polarity of ATP binding sites of the Na+,K+-ATPase, H+,K+-ATPase and the sarcoplasmic reticulum Ca2+-ATPase under different experimental conditions

Summary

Introduction

P-type ATPases play crucial roles in cell physiology. The Na+ electrochemical potential gradient created across the plasma membrane of animal cells by the Na+,K+-ATPase (or sodium pump) is used to drive nutrient reabsorption in kidney, and both the Na+ and K+ gradients it generates are essential for nerve and muscle function [1]. The closely related enzyme, the H+,K+-ATPase (or proton pump) of gastric parietal cells creates the low pH of the stomach necessary for digestion [2]. Another P-type ATPase, the sarco(endo)plasmic reticulum Ca2+ATPase (SR calcium pump or SERCA) transports Ca2+ ions out of the cytoplasm into the sarcoplasmic reticulum, which allows muscle relaxation [3]. A key feature of the cycle is the existence of two major conformational forms of the enzyme, which can exist in both unphosphorylated and phosphorylated states, E1 and E2 (or E1P and E2P in the case of the phosphorylated states) These two forms differ significantly in their ion binding affinities. In the SR Ca2+-ATPase, the E1 form binds Ca2+ ions, and the E2 form is thought to bind H+ ions [4]

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