The bacterial potassium channel KcsA is gated by pH, opening for conduction under acidic conditions. Molecular determinants responsible for this effect have been identified at the extracellular selectivity filter, at the membrane–cytoplasm interface (TM2 gate), and in the cytoplasmic C‐terminal domain (CTD), an amphiphilic four‐helix bundle mediated by hydrophobic and electrostatic interactions. Here we have employed NMR and EPR to provide a structural view of the pH‐induced open‐to‐closed CTD transition. KcsA was embedded in lipoprotein nanodiscs (LPNs), selectively methyl‐protonated at Leu/Val residues to allow observation of both states by NMR, and spin‐labeled for the purposes of EPR studies. We observed a pHinduced structural change between an associated structured CTD at neutral pH and a dissociated flexible CTD at acidic pH, with a transition in the 5.0–5.5 range, consistent with a stabilization of the CTD by channel architecture. A double mutant constitutively open at the TM2 gate exhibited reduced stability of associated CTD, as indicated by weaker spin–spin interactions, a shift to higher transition pH values, and a tenfold reduction in the population of the associated “closed” channels. We extended these findings for isolated CTD‐derived peptides to full‐length KcsA and have established a contribution of the CTD to KcsA pH‐controlled gating, which exhibits a strong correlation with the state of the proximal TM2 gate.
Membrane-embedded proteins (MPs) are central to a wide range of cellular processes. Despite their importance, structural studies of MPs are hindered by expression difficulties and the need for stabilization in a membranemimicking environment. High-resolution NMR methods can investigate structure and function of MPs due to methodological advances and new membrane-like assemblies for stabilization of MPs. In this perspective of the field, we introduce the challenges and opportunities of NMR studies of membrane proteins, briefly surveying membrane-mimicking systems and their application in structure determination. A case study then focuses on the C-terminal domain of the bacterial potassium channel KcsA, describing how improvements in membrane-mimicking conditions eventually enabled us to present a structural view of the pH-dependent behavior of this cytoplasmic channel domain. The results highlight prerequisites for a successful study of MPs and the potential for future investigations.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.