spacer
spacer

PDBsum entry 2htk

Go to PDB code: 
protein metals Protein-protein interface(s) links
Membrane protein PDB id
2htk
Jmol PyMol
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    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chains
Pfam   ArchSchema ?
P37019  (CLCA_ECOLI) -  H(+)/Cl(-) exchange transporter ClcA
Seq:
Struc:
473 a.a.
444 a.a.*
Protein chains
Pfam   ArchSchema ?
Q4VBH1  (Q4VBH1_RAT) -  Ighg protein
Seq:
Struc:
467 a.a.
221 a.a.*
Protein chains
No UniProt id for this chain
Struc: 211 a.a.
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 61 residue positions (black crosses)

 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     membrane   4 terms 
  Biological process     transport   9 terms 
  Biochemical function     anion binding     7 terms  

 

 
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.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
22484316 A.Picollo, Y.Xu, N.Johner, S.Bernèche, and A.Accardi (2012).
Synergistic substrate binding determines the stoichiometry of transport of a prokaryotic H(+)/Cl(-) exchanger.
  Nat Struct Mol Biol, 19, 525.  
  20513761 A.Picollo, M.Malvezzi, and A.Accardi (2010).
Proton block of the CLC-5 Cl-/H+ exchanger.
  J Gen Physiol, 135, 653-659.  
19132363 G.Zifarelli, and M.Pusch (2010).
The role of protons in fast and slow gating of the Torpedo chloride channel ClC-0.
  Eur Biophys J, 39, 869-875.  
21048711 J.L.Robertson, L.Kolmakova-Partensky, and C.Miller (2010).
Design, function and structure of a monomeric ClC transporter.
  Nature, 468, 844-847.
PDB code: 3nmo
20929736 L.Feng, E.B.Campbell, Y.Hsiung, and R.MacKinnon (2010).
Structure of a eukaryotic CLC transporter defines an intermediate state in the transport cycle.
  Science, 330, 635-641.
PDB code: 3org
20204338 R.J.Naftalin (2010).
Reassessment of models of facilitated transport and cotransport.
  J Membr Biol, 234, 75.  
20598093 S.Wege, M.Jossier, S.Filleur, S.Thomine, H.Barbier-Brygoo, F.Gambale, and A.De Angeli (2010).
The proline 160 in the selectivity filter of the Arabidopsis NO(3)(-)/H(+) exchanger AtCLCa is essential for nitrate accumulation in planta.
  Plant J, 63, 861-869.  
20483324 Y.J.Ko, and W.H.Jo (2010).
Secondary water pore formation for proton transport in a ClC exchanger revealed by an atomistic molecular-dynamics simulation.
  Biophys J, 98, 2163-2169.  
  19364886 A.K.Alekov, and C.Fahlke (2009).
Channel-like slippage modes in the human anion/proton exchanger ClC-4.
  J Gen Physiol, 133, 485-496.  
19898476 A.Picollo, M.Malvezzi, J.C.Houtman, and A.Accardi (2009).
Basis of substrate binding and conservation of selectivity in the CLC family of channels and transporters.
  Nat Struct Mol Biol, 16, 1294-1301.  
18977737 C.Miller, and W.Nguitragool (2009).
A provisional transport mechanism for a chloride channel-type Cl-/H+ exchanger.
  Philos Trans R Soc Lond B Biol Sci, 364, 175-180.  
19580750 D.Wang, and G.A.Voth (2009).
Proton transport pathway in the ClC Cl-/H+ antiporter.
  Biophys J, 97, 121-131.  
19261613 E.Y.Bergsdorf, A.A.Zdebik, and T.J.Jentsch (2009).
Residues Important for Nitrate/Proton Coupling in Plant and Mammalian CLC Transporters.
  J Biol Chem, 284, 11184-11193.  
19131966 G.Zifarelli, and M.Pusch (2009).
Conversion of the 2 Cl(-)/1 H+ antiporter ClC-5 in a NO3(-)/H+ antiporter by a single point mutation.
  EMBO J, 28, 175-182.  
  19139174 H.H.Lim, and C.Miller (2009).
Intracellular proton-transfer mutants in a CLC Cl-/H+ exchanger.
  J Gen Physiol, 133, 131-138.
PDB codes: 3ejy 3ejz
19745816 S.M.Elvington, C.W.Liu, and M.C.Maduke (2009).
Substrate-driven conformational changes in ClC-ec1 observed by fluorine NMR.
  EMBO J, 28, 3090-3102.  
18063579 A.A.Zdebik, G.Zifarelli, E.Y.Bergsdorf, P.Soliani, O.Scheel, T.J.Jentsch, and M.Pusch (2008).
Determinants of anion-proton coupling in mammalian endosomal CLC proteins.
  J Biol Chem, 283, 4219-4227.  
18678918 H.Jayaram, A.Accardi, F.Wu, C.Williams, and C.Miller (2008).
Ion permeation through a Cl--selective channel designed from a CLC Cl-/H+ exchanger.
  Proc Natl Acad Sci U S A, 105, 11194-11199.
PDB code: 3det
18058905 Z.Kuang, A.Liu, and T.L.Beck (2008).
TransPath: a computational method for locating ion transit pathways through membrane proteins.
  Proteins, 71, 1349-1359.  
  17846164 A.M.Engh, J.D.Faraldo-Gómez, and M.Maduke (2007).
The mechanism of fast-gate opening in ClC-0.
  J Gen Physiol, 130, 335-349.  
  17389248 M.Walden, A.Accardi, F.Wu, C.Xu, C.Williams, and C.Miller (2007).
Uncoupling and turnover in a Cl-/H+ exchange transporter.
  J Gen Physiol, 129, 317-329.  
18093952 W.Nguitragool, and C.Miller (2007).
Inaugural Article: CLC Cl /H+ transporters constrained by covalent cross-linking.
  Proc Natl Acad Sci U S A, 104, 20659-20665.  
17410581 Z.Kuang, U.Mahankali, and T.L.Beck (2007).
Proton pathways and H+/Cl- stoichiometry in bacterial chloride transporters.
  Proteins, 68, 26-33.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.

 

spacer

spacer