The role of conformational change and key glutamic acid residues in the ClC-ec1 antiporter

Biophys J. 2023 Mar 21;122(6):1068-1085. doi: 10.1016/j.bpj.2023.01.025. Epub 2023 Jan 25.


The triple glutamine (Q) mutant (QQQ) structure of a Cl-/H+ antiporter from Escherichia coli (ClC-ec1) displaying a novel backbone arrangement has been used to challenge the long-held notion that Cl-/H+ antiporters do not operate through large conformational motions. The QQQ mutant substitutes the glutamine residue for an external glutamate E148, an internal glutamate E203, and a third glutamate E113 that hydrogen-bonds with E203. However, it is unknown if QQQ represents a physiologically relevant state, as well as how the protonation of the wild-type glutamates relates to the global dynamics. We herein apply continuous constant-pH molecular dynamics to investigate the H+-coupled dynamics of ClC-ec1. Although any large-scale conformational rearrangement upon acidification would be due to the accumulation of excess charge within the protein, protonation of the glutamates significantly impacts mainly the local structure and dynamics. Despite the fact that the extracellular pore enlarges at acidic pHs, an occluded ClC-ec1 within the active pH range of 3.5-7.5 requires a protonated E148 to facilitate extracellular Cl- release. E203 is also involved in the intracellular H+ transfer as an H+ acceptor. The water wire connection of E148 with the intracellular solution is regulated by the charge states of the E113/E203 dyad with coupled proton titration. However, the dynamics extracted from our simulations are not QQQ-like, indicating that the QQQ mutant does not represent the behavior of the wild-type ClC-ec1. These findings reinforce the necessity of having a protonatable residue at the E203 position in ClC-ec1 and suggest that a higher level of complexity exists for the intracellular H+ transfer in Cl-/H+ antiporters.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Antiporters* / genetics
  • Antiporters* / metabolism
  • Chloride Channels / metabolism
  • Chlorides / metabolism
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Escherichia coli Proteins* / genetics
  • Escherichia coli Proteins* / metabolism
  • Glutamic Acid / chemistry
  • Glutamine
  • Protons


  • Antiporters
  • Glutamic Acid
  • Glutamine
  • Chlorides
  • Protons
  • Chloride Channels
  • CLC-ec1 protein, E coli
  • Escherichia coli Proteins