2ht4: Difference between revisions

Revision as of 18:45, 21 February 2008

Structure of the Escherichia coli ClC chloride channel Y445W mutant and Fab complex

OverviewOverview

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.

About this StructureAbout this Structure

2HT4 is a Single protein structure of sequence from Escherichia coli and Mus musculus with as ligand. Full crystallographic information is available from OCA.

ReferenceReference

Synergism between halide binding and proton transport in a CLC-type exchanger., Accardi A, Lobet S, Williams C, Miller C, Dutzler R, J Mol Biol. 2006 Sep 29;362(4):691-9. Epub 2006 Aug 2. PMID:16949616

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-[[Image:2ht4.gif|left|200px]]<br /><applet load="2ht4" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:2ht4.gif|left|200px]]<br /><applet load="2ht4" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="2ht4, resolution 3.20&Aring;" />
 '''Structure of the Escherichia coli ClC chloride channel Y445W mutant and Fab complex'''<br />
 ==Overview==
-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.
+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.
 ==About this Structure==
-HT4 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] and [http://en.wikipedia.org/wiki/Mus_musculus Mus musculus] with BR as [http://en.wikipedia.org/wiki/ligand ligand]. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=2HT4 OCA].
+HT4 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] and [http://en.wikipedia.org/wiki/Mus_musculus Mus musculus] with <scene name='pdbligand=BR:'>BR</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2HT4 OCA].
 ==Reference==
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 [[Category: membrane protein]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Wed Nov 21 11:58:00 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 17:45:24 2008''