Biophysical Society Conference | Tahoe 2023

Proton Reactions: From Basic Science to Biomedical Applications

Tuesday Speaker Abstracts

CONFORMATIONAL CHANGES AND THE ROLE OF INTERNAL GLUTAMATE IN CL – /H + ANTIPORTERS Zhi Yue 1 ; Gregory A Voth 1 ; 1 The University of Chicago, Department of Chemistry, Chicago, IL, USA Cl – /H + antiporters have long been viewed to not operate through alternating between inward- and outward-facing states, but rather to move ions stepwise within the protein, with the only required conformational change being an external glutamate (Glu ex ) rotation. This notion is supported by crystallography, which captures little backbone arrangement, but has been challenged by low-pH spectroscopic studies indicating substantial conformational change and a high-pH triple glutamine (Q) mutant (QQQ) structure of a Cl – /H + antiporter from Escherichia coli (ClC-ec1) displaying considerable backbone adjustment. In addition, mutagenesis of ClC-ec1 and sequence inspection of other ClC antiporters have raised questions about the role of an internal glutamate (Glu in , E203 in ClC-ec1) in H + transport. To investigate if ClC-ec1 operates through a Glu in independent alternating-access mechanism, we employed continuous constant-pH molecular dynamics to study the H + -coupled dynamics of ClC-ec1. We found that while acid-induced large-scale conformational rearrangements do occur due to the accumulation of excess charge within the protein, an occluded ClC-ec1 within the active pH range of 3.5–7.5 requires a protonated E148 (Glu ex ) to facilitate extracellular Cl – release. This indicates that the conformational rearrangements are insufficient to classify ClC-ec1 as a canonical transporter, reinforcing the long-standing belief that ClC-ec1 does not function through an alternating-access mechanism. Furthermore, we revealed for the first time that the internal H + transport between E148 and the intracellular solution is likely achieved through coupled H + titration of the E113/E203 dyad. This highlights the necessity of having a protonatable residue at the E203 position in ClC-ec1 and underscores the need for case-by-case analyses in other ClC antiporters. In addition, we found that the conformational dynamics are not QQQ-like, indicating that the QQQ mutant does not accurately represent the behavior of the wild-type ClC-ec1.

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