The intracellular lipid-binding domain of human Na+/H+ exchanger 1 forms a lipid-protein co-structure essential for activity

Abstract

Dynamic interactions of proteins with lipid membranes are essential regulatory events in biology, but remain rudimentarily understood and particularly overlooked in membrane proteins. The ubiquitously expressed membrane protein Na+/H+-exchanger 1 (NHE1) regulates intracellular pH (pHi) with dysregulation linked to e.g. cancer and cardiovascular diseases. NHE1 has a long, regulatory cytosolic domain carrying a membrane-proximal region described as a lipid-interacting domain (LID), yet, the LID structure and underlying molecular mechanisms are unknown. Here we decompose these, combining structural and biophysical methods, molecular dynamics simulations, cellular biotinylation- and immunofluorescence analysis and exchanger activity assays. We find that the NHE1-LID is intrinsically disordered and, in presence of membrane mimetics, forms a helical αα-hairpin co-structure with the membrane, anchoring the regulatory domain vis-a-vis the transport domain. This co-structure is fundamental for NHE1 activity, as its disintegration reduced steady-state pHi and the rate of pHi recovery after acid loading. We propose that regulatory lipid-protein co-structures may play equally important roles in other membrane proteins.

Dynamic Na+/H+ exchanger 1 (NHE1) – calmodulin complexes of varying stoichiometry and structure regulate Ca2+-dependent NHE1 activation

“…Finally, given the key role of anionic lipids including phosphatidyl-inositol(4,5)-diphosphate (PI[4,5]P 2 ) in regulating NHE1 by interacting with the C-tail ( Aharonovitz et al, 2000 ; Shimada-Shimizu et al, 2014 ), Ca 2+ -CaM could also regulate NHE1 through electrostatic tuning of the NHE1:PI(4,5)P 2 interaction. Such mechanisms are reported for several other membrane proteins ( Cao et al, 2013 ; Monteiro et al, 2014 ) and would be consistent with the folding of the NHE1-LID domain upon membrane interaction, bringing the CaM-binding region in close proximity to the NHE1-LID and the membrane ( Hendus-Altenburger et al, 2020 ).…”

“…Our results agree with earlier work showing that cellular NHE1:CaM interaction is partially retained in an NHE1 variant functionally similar to our H1-CR variant ( Bertrand et al, 1994 ). Additionally, cellular NHE1:CaM interactions may occur through C-tail interactions with other proteins (e.g., calcineurin) or membrane lipids ( Hendus-Altenburger et al, 2019 ; Hendus-Altenburger et al, 2020 ), independent of the residues studied here but also giving rise to PLA signals. Finally, while NHE1:CaM proximity in the absence of interaction cannot be excluded, the observed binding of NHE1 to apo-CaM in vitro and the lack of effect of ionomycin on the number of proximity events observed in cells indicate that a fraction of NHE1:CaM complexes are present at all times.…”

“…This would require each CaM to span two NHE1 C-tails, binding to two different sites on two tails, forming a crossover structure (not shown). Whether this is feasible in a given cellular context will depend on the structural organization of the C-tail complexed with its binding partners and on the interaction of the NHE1 lipid interaction domain (LID) with the plasma membrane ( Hendus-Altenburger et al, 2020 ). At the conditions studied here, we did not detect the 2:2 complex.…”

Principal Components of Genetic Sequences: Correlations and Significance

“…N-терминальный трансмембранный домен много раз пересекает мембрану в обоих направлениях, поэтому характерным элементом его ВС является большое количество α-спиралей. Кроме того, несколько α-спиралей входят в состав Cтерминального цитозольного домена, находящегося вне мембраны [7].…”

Strong relationship between cholesterol, low-density lipoprotein receptor, Na+/H+ exchanger, and SARS-COV-2: this association may be the cause of death in the patient with COVID-19

“…Recently, it has been suggested that NHE may increase susceptibility to SARS-CoV-2 and the severity of COVID-19 [ 5 , 6 ]. Interestingly, there is also an interaction between lipids and NHE [ 7 ]. Here, we examined whether there is a triple interaction between SARS-CoV-2, lipids, and NHE.…”