PDBsum entry 2htk

Membrane protein

PDB id: 2htk

Contents

Protein chains

444 a.a. *

221 a.a. *

211 a.a. *

Metals

_BR ×2

* Residue conservation analysis

PDB id:

2htk

Name:

Membrane protein

Title:

Structure of the escherichia coli clc chloride channel y445a mutant and fab complex

Structure:

H(+)/cl(-) exchange transporter clca. Chain: a, b. Synonym: 1ecclc h+/cl- antiporter. Clc-ec1. Engineered: yes. Mutation: yes. Fab fragment, heavy chain. Chain: c, e. Fab fragment, light chain. Chain: d, f

Source:

Escherichia coli. Organism_taxid: 562. Gene: clca, eric. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008. Mus musculus. House mouse. Organism_taxid: 10090. Cell_line: hybridoma cell line.

Biol. unit:

Hexamer (from PQS)

Resolution:

3.41Å R-factor: 0.266 R-free: 0.276

Authors:

A.Accardi,S.Lobet,C.Williams,C.Miller,R.Dutzler

Key ref:

A.Accardi et al. (2006). Synergism between halide binding and proton transport in a CLC-type exchanger. J Mol Biol, 362, 691-699. PubMed id: 16949616 DOI: 10.1016/j.jmb.2006.07.081

Date:

26-Jul-06 Release date: 19-Sep-06

Protein chains

P37019  (CLCA_ECOLI) -  H(+)/Cl(-) exchange transporter ClcA from Escherichia coli (strain K12)

Seq: 473 a.a.

Struc: 444 a.a.*

Protein chains

Q4VBH1  (Q4VBH1_RAT) -  Ighg protein from Rattus norvegicus

Seq: 467 a.a.

Struc: 221 a.a.*

Protein chains

No UniProt id for this chain

Struc: 211 a.a.

* PDB and UniProt seqs differ at 61 residue positions (black crosses)

DOI no: 10.1016/j.jmb.2006.07.081

J Mol Biol 362:691-699 (2006)

PubMed id: 16949616

Synergism between halide binding and proton transport in a CLC-type exchanger.

A.Accardi, S.Lobet, C.Williams, C.Miller, R.Dutzler.

ABSTRACT

The Cl-/H+ exchange-transporter CLC-ec1 mediates stoichiometric transmembrane exchange of two Cl- ions for one proton. A conserved tyrosine residue, Y445, coordinates one of the bound Cl- ions visible in the structure of this protein and is located near the intersection of the Cl- and H+ pathways. Mutants of this tyrosine were scrutinized for effects on the coupled transport of Cl- and H+ determined electrophysiologically and on protein structure determined crystallographically. Despite the strong conservation of Y445 in the CLC family, substitution of F or W at this position preserves wild-type transport behavior. Substitution by A, E, or H, however, produces uncoupled proteins with robust Cl- transport but greatly impaired movement of H+. The obligatory 2 Cl-/1 H+ stoichiometry is thus lost in these mutants. The structures of all the mutants are essentially identical to wild-type, but apparent anion occupancy in the Cl- binding region correlates with functional H+ coupling. In particular, as determined by anomalous diffraction in crystals grown in Br-, an electrophysiologically competent Cl- analogue, the well-coupled transporters show strong Br- electron density at the "inner" and "central" Cl- binding sites. However, in the uncoupled mutants, Br- density is absent at the central site, while still present at the inner site. An additional mutant, Y445L, is intermediate in both functional and structural features. This mutant clearly exchanges H+ for Cl-, but at a reduced H+-to-Cl- ratio; likewise, both the central and inner sites are occupied by Br-, but the central site shows lower Br- density than in wild-type (or in Y445F,W). The correlation between proton coupling and central-site occupancy argues that halide binding to the central transport site somehow facilitates movement of H+, a synergism that is not readily understood in terms of alternating-site antiport schemes.

Selected figure(s)

Figure 1.
Figure 1. Structure of CLC-ec1. (a) Ribbon representation of the homodimer, with the two subunits colored in red and gray and the extracellular side on top. Green spheres represent Cl^− ions at the inner and central positions. (b) The Cl^− binding region in a view similar to (a). Atoms of the protein backbone (N, C, CA) and selected side-chains are shown. The two H^+-transfer glutamate side-chains are highlighted in yellow. The Cl^− ions bound to the inner and central' site are shown as green spheres. The three ion binding sites are labeled. Putative trajectories for Cl^− (green) and H^+ (red) are shown as broken curves. Figure 1. Structure of CLC-ec1. (a) Ribbon representation of the homodimer, with the two subunits colored in red and gray and the extracellular side on top. Green spheres represent Cl^− ions at the inner and central positions. (b) The Cl^− binding region in a view similar to (a). Atoms of the protein backbone (N, C, CA) and selected side-chains are shown. The two H^+-transfer glutamate side-chains are highlighted in yellow. The Cl^− ions bound to the inner and central' site are shown as green spheres. The three ion binding sites are labeled. Putative trajectories for Cl^− (green) and H^+ (red) are shown as broken curves.

Figure 2.
Figure 2. Proton coupling and Br^− binding for wild-type and Y445F. (a) Raw traces of CLC-ec1 currents in response to 3 s voltage pulses (− 100 to + 100 mV in 10 mV increments) in the presence of a four-unit pH gradient (pH[cis] 3/pH[trans] 7). (b) I–V curves of WT (open circles) and Y445F (filled circles) in a four-unit pH gradient. (c) Reversal potential variation with pH (left) or Cl^− gradients (right). Reversal potentials, V[rev], were determined for wild-type (open circles; data from Accardi and Miller^3) or Y445F (filled circles) from I–V curves as in (b), under varying pH or Cl^− gradients. The cis solution, which contained 300 mM Cl^− at pH 3, was kept constant, while the trans solution was varied. Each point represents the average of data from at least three separate bilayers. (e) View of the anion binding region for wild-type and Y445F from within the membrane with the extracellular side on top. The anomalous Br^- electron density maps (red) were contoured at 7σ. Figure 2. Proton coupling and Br^− binding for wild-type and Y445F. (a) Raw traces of CLC-ec1 currents in response to 3 s voltage pulses (− 100 to + 100 mV in 10 mV increments) in the presence of a four-unit pH gradient (pH[cis] 3/pH[trans] 7). (b) I–V curves of WT (open circles) and Y445F (filled circles) in a four-unit pH gradient. (c) Reversal potential variation with pH (left) or Cl^− gradients (right). Reversal potentials, V[rev], were determined for wild-type (open circles; data from Accardi and Miller[3]^3) or Y445F (filled circles) from I–V curves as in (b), under varying pH or Cl^− gradients. The cis solution, which contained 300 mM Cl^− at pH 3, was kept constant, while the trans solution was varied. Each point represents the average of data from at least three separate bilayers. (e) View of the anion binding region for wild-type and Y445F from within the membrane with the extracellular side on top. The anomalous Br^- electron density maps (red) were contoured at 7σ.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2006, 362, 691-699) copyright 2006.

Figures were selected by an automated process.