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PDBsum entry 2atk
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Immune system/ion transport
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PDB id
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2atk
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Contents |
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219 a.a.
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212 a.a.
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103 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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Molecular determinants of gating at the potassium-Channel selectivity filter.
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Authors
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J.F.Cordero-Morales,
L.G.Cuello,
Y.Zhao,
V.Jogini,
D.M.Cortes,
B.Roux,
E.Perozo.
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Ref.
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Nat Struct Mol Biol, 2006,
13,
311-318.
[DOI no: ]
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PubMed id
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Abstract
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We show that in the potassium channel KcsA, proton-dependent activation is
followed by an inactivation process similar to C-type inactivation, and this
process is suppressed by an E71A mutation in the pore helix. EPR spectroscopy
demonstrates that the inner gate opens maximally at low pH regardless of the
magnitude of the single-channel-open probability, implying that stationary
gating originates mostly from rearrangements at the selectivity filter. Two E71A
crystal structures obtained at 2.5 A reveal large structural excursions of the
selectivity filter during ion conduction and provide a glimpse of the range of
conformations available to this region of the channel during gating. These data
establish a mechanistic basis for the role of the selectivity filter during
channel activation and inactivation.
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Figure 4.
Figure 4. Two crystal structures of the E71A mutant. (a)
Electron density map of residues 60–84 from two diagonally
symmetric subunits for the crystal form of E71A with residue
Asp80 flipped upward (flipped E71A). Sticks, polypeptide chain;
blue mesh, 1-  contour
of the 2F[o] – F[c] electron density map for the protein;
magenta mesh, 2- contour
of the ions; black dotted ovals, cavities created by the absence
of the Glu71 side chain. (b) Single-subunit line representation
of the P-loop of the flipped (red) and nonflipped (black) E71A
structures overlaid onto the wild-type structure^32 (PDB entry
1K4C; gray) highlights the conformational rearrangements in the
backbone of the selectivity filter (residues 75–79). Insets,
the side chain conformational changes in Asp80 and Trp67 as
fitted to the 2- contour
of the simulated annealing omit map. The omit maps were
calculated for residues 79–84 and 67, respectively; atoms
within 3.5 Å of selected residues were also omitted in the
calculation. The density attributed to the alternate rotamer of
the Trp67 side chain in the flipped X-ray structure is clearly
distinct from the density of the lipid observed near this
position in the WT X-ray structure^33. (c) One-dimensional
electron density profiles for the two crystal conformations of
the E71A mutant (flipped and nonflipped). Top, F[o] – F[c]
omit maps of K^+ ions in the selectivity filter shown relative
to the protein model. The electron density maps are shown as a
6- contour
for the flipped E71A conformer and as 7- (blue)
and 4- (cyan)
contours for the nonflipped E71A conformer. Different contour
levels were chosen for the purpose of visual clarity. Bottom,
one-dimensional electron density profile along the central
symmetry (z) axis is shown using the ion in the cavity as z = 0.
Gray-shaded peaks represent the profile for the wild-type
channel (PDB entry 1K4C at 2.0 Å). Numbers and E at top
denote the K^+-binding sites (S0–S4 and S[ext],
respectively).(d) Comparison of crystallographic B-factors for
P-loop residues (63–83) from the WT KcsA structure (bottom
chart) and the nonflipped E71A mutant (E71A-NF, top chart).
Black dotted line represents the mean value for all atoms in the
P-loop. Vertical capped lines represent the  3
values
for each data set.
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Figure 6.
Figure 6. A mechanistic interpretation of KcsA gating. (a)
Possible mechanism of action of the E71A mutation in stabilizing
the open state. A single-subunit P-loop is shown with positions
67, 71 and 80 in stick representation. In the wild-type channel
(left), the interaction between Asp80 and Trp67 destabilizes the
conductive conformation of the filter and promotes inactivation
through an as yet unknown mechanism. Eliminating the Asp80-Glu71
carboxyl-carboxylate (E71A, right) disrupts the hydrogen bonding
network between the signature sequence (Gly-Tyr-Gly-Asp) and the
pore helix, causing an increase in Asp80 dynamics and perturbing
the Asp80-Trp67 interaction. This sharply decreases entry into
the inactivated state, stabilizing the open state. (b) Top,
cartoon representation of the structural conformation associated
with each kinetic state. Bottom, correlation of specific kinetic
transitions with KcsA single-channel behavior. Because
stationary gating is dominated by the deeply inactivated state,
single-channel openings occur mainly as a result of rare returns
from the inactivated state owing to conformational changes in
the selectivity filter while the lower gate remains open.
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nat Struct Mol Biol
(2006,
13,
311-318)
copyright 2006.
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