Involvement of the alpha-subunit N-terminus in the mechanism of the Na+,K+-ATPase

Previous studies have shown that cytoplasmic K⁺ release and the associated E2 → E1 conformational change of the Na⁺,K⁺-ATPase is a major rate-determining step of the enzyme's ion pumping cycle and hence a prime site of acute regulatory intervention. From the ionic strength dependence of the enzyme's distribution between the E2 and E1 states, it has also been found that E2 is stabilized by an electrostatic attraction. Any disruption of this electrostatic attraction would, thus, have profound effects on the rate of ion pumping. The aim of this paper is to identify the location of this interaction. Using enhanced-sampling molecular dynamics simulations with a predicted N-terminal structure added to the X-ray crystal structure of the Na⁺,K⁺-ATPase, a previously postulated salt bridge between Lys32 and Glu233 (rat sequence numbering) of the enzyme's α-subunit can be excluded. The residues never approach closely enough to form a salt bridge. In contrast, strong interactions with anionic lipid head groups were seen. To investigate the possibility of a protein-lipid interaction experimentally, the surface charge density of Na⁺,K⁺-ATPase-containing membrane fragments was estimated from zeta potential measurements to be 0.019 (± 0.001) C m^?2. This is in good agreement with the charge density previously determined to be responsible for stabilization of the E2 state of 0.023 (± 0.009) C m^?2 and the membrane charge density estimated here from published electron-microscopic images of 0.018C m^?2. The results are, therefore, consistent with an interaction of the Na⁺,K⁺-ATPase α-subunit N-terminus with negatively-charged lipid head groups of the neighbouring cytoplasmic membrane surface as the origin of the electrostatic interaction stabilising the E2 state.