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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.
Highlights
Dynamic interactions of proteins with lipid membranes are essential regulatory events in biology, but remain rudimentarily understood and overlooked in membrane proteins
Despite many investigations underscoring the relevance of various lipids for Na+/H+-exchanger 1 (NHE1) function, and the previous identification of the NHE1-lipid-interacting domain (LID) as a key region for NHE1 regulation, no structural data exist for this part of NHE1 (Fig. 1)
Unraveling the dynamics in membrane proteins is essential for understanding their functions[54]
Summary
Dynamic interactions of proteins with lipid membranes are essential regulatory events in biology, but remain rudimentarily understood and overlooked in membrane proteins. 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. 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. In addition to the highly structured regions amenable to such analyses, intrinsically disordered N- and C-terminals appear frequently in the human transmembrane proteome[1] Such intrinsically disordered regions (IDR) play key roles in membrane protein function[2], adding an additional layer of complexity to the mechanistic understanding of these proteins. The functional importance of NHE1:PI(4,5)P2 interaction was underscored by the finding that in kidney glomerular injury, accumulating amphipathic long-chain acyl-CoA (LC-CoA)
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