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PDBsum entry 2htk
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Membrane protein
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PDB id
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2htk
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Contents |
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444 a.a.
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221 a.a.
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211 a.a.
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References listed in PDB file
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Key reference
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Title
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Synergism between halide binding and proton transport in a clc-Type exchanger.
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Authors
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A.Accardi,
S.Lobet,
C.Williams,
C.Miller,
R.Dutzler.
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Ref.
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J Mol Biol, 2006,
362,
691-699.
[DOI no: ]
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PubMed id
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Abstract
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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.
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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.
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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.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2006,
362,
691-699)
copyright 2006.
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