Charge Transport in the ClC-type Chloride-Proton Anti-porter from Escherichia coli

Abstract

The first chloride transporter identified in the superfamily of ClC chloride channels was from Escherichia coli (EClC) (Accardi, A., and Miller, C. (2004) Nature 427, 803-807). Pathways, energetics, and mechanism of proton and chloride translocation and their coupling are up to now unclear. To bridge the hydrophobic gap of proton transport, we modeled four stable buried waters into both subunits of the WT EClC structure. Together they form a "water wire" connecting Glu-203 with the chloride at the central site, which in turn connects to Glu-148, the hypothetical proton exit site. Assuming the transient production of hydrochloride in the central chloride binding site of EClC, the water wire could establish a transmembrane proton transport pathway starting from Glu-203 all the way downstream onto Glu-148. We demonstrated by electrostatic and quantum chemical computations that protonation of the central chloride is energetically feasible. We characterized all chloride occupancies and protonation states possibly relevant for the proton-chloride transport cycle in EClC and constructed a working model. Accordingly, EClC evolves through states involving up to two excess protons and between one and three chlorides, which was required to fulfill the experimentally observed 2:1 stoichiometry. We show that the Y445F and E203H mutants of EClC can operate similarly, thus explaining why they exhibit almost WT activity levels. The proposed mechanism of coupled chloride-proton transport in EClC is consistent with available experimental data and allows predictions on the importance of specific amino acids, which may be probed by mutation experiments.

Exploring the Possible Role of Glu286 in CcO by Electrostatic Energy Computations Combined with Molecular Dynamics

“…To determine an initial protonation pattern in CcO, we evaluated all pK A values by electrostatic energy computations using karlsberg+ 37,38 for the CcO crystal structure (PDB: 2GSM 11 ) as done in previous applications 36,39 . The resulting protonation pattern was applied for all MD runs, if not stated otherwise.…”

Water access points and hydration pathways in CLC H + /Cl − transporters

“…Neutralization of either glutamate eliminates H + translocation by ClC-ec1 (9, 28). However, the discovery of these H + binding sites also raised a mechanistic puzzle (3, 23): How do protons translocate between the two sites, which are separated by a ∼15-Å-long, largely hydrophobic region within the lumen of the protein?Since the report of its first crystal structure, a large number of computational studies have aimed at investigating various molecular details related to the CLC H + transport mechanism (27,(29)(30)(31)(32)(33)(34). One model emerging from these studies proposes that water molecules may connect the two H + sites (Glu ex and Glu in ) and, thereby, facilitate H + transport (29,30,34).…”

Identification of a proton-chloride antiporter (EriC) by Himar1 transposon mutagenesis in Lactobacillus reuteri and its role in histamine production

“…Despite the differences in the functions between the two types of CIC proteins, all members of this superfamily contain double-barreled architecture and possess voltage-dependent gating mechanism (Maduke et al 2000). EriC is a homodimeric transmembrane protein and exchanges protons with chlorides in a fixed 2:1 stoichiometry (Kieseritzky and Knapp 2010). Results from protein BLAST with L. reuteri 6475 EriC sequence revealed that proteins with EriC functional domains are ubiquitously present in bacteria including those that are members of the human intestinal microbiome.…”