Biophysical Society Conference | Tahoe 2023

Proton Reactions: From Basic Science to Biomedical Applications

Tuesday Speaker Abstracts

CRYO-EM, MOLECULAR DYNAMICS SIMULATIONS AND FUNCTIONAL ASSAYS REVEAL UNANTICIPATED INSIGHTS INTO CHLORIDE/PROTON ANTIPORT MECHANISM OF CLC-EC1. Amy R Nava 1 ; Jeurgen Kreiter 1 ; Chih-Ta Chien 2 ; Deniz Aydin 3 ; Wah Chiu 2 ; Ron O Dror 3 ; Merritt C Maduke 1 ; 1 Stanford University, Molecular and Cellular Physiology, Stanford, CA, USA 2 Stanford University, Department of Bioengineering, Stanford, CA, USA 3 Stanford University, Department of Computer Science, Stanford, CA, USA The CLC membrane transporters exchange two chloride (Cl - ) ions against one proton (H + ). They are the only transporters known to exchange anions for cations. CLC transporters are present in all cells in the human body, where they play critical roles in endocytosis, vesicle recycling, and lysosome acidification. CLC-ec1 is a bacterial homolog, essential for extreme acid tolerance, that has served as a paradigm for the family. Understanding the CLC-ec1 mechanisms will provide fundamental biological knowledge for how the human CLC proteins function. Structures and functional studies of CLC-ec1 have led to proposed mechanisms for this unusual protein family. However, questions remain. Previously, we determined the crystallographic structure of a triple mutant (“QQQ”), which we proposed to represent the outward-facing open conformational state of the transporter. Based on this structure, together with molecular dynamics (MD) simulations and functional studies, we proposed a novel H + permeation mechanism involving water wires connecting the intracellular bulk water directly to “Glu gate ”, a conserved residue that gates the anion pathway and is essential for H + transport. However, since this QQQ protein has a non-titratable Gln at the Glu gate site, the question remains whether its structure is relevant to the WT transport mechanism. Here, we present cryo-EM structures of wild-type CLC-ec1 at pH 3 and 7, to favor protonation and deprotonation of Glu gate respectively. The pH 7 structure closely resembles the WT CLC-ec1 conformation solved by X-ray crystallography; the pH 3 structure, the QQQ conformation. Using MD simulations, we confirmed the water wires observed in the QQQ simulations. Strikingly, we found a new water wire in our WT CLC-ec1 simulations, which occurs more frequently than the previously observed water wires. Guided by the simulations, we mutated residues lining this new water wire and evaluated the effect of these mutations on function. Our results provide new insights into the CLC transporter mechanism.

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