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PDBsum entry 3kvt
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Potassium channel
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PDB id
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3kvt
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References listed in PDB file
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Key reference
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Title
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Zn2+-Binding and molecular determinants of tetramerization in voltage-Gated k+ channels.
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Authors
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K.A.Bixby,
M.H.Nanao,
N.V.Shen,
A.Kreusch,
H.Bellamy,
P.J.Pfaffinger,
S.Choe.
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Ref.
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Nat Struct Biol, 1999,
6,
38-43.
[DOI no: ]
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PubMed id
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Abstract
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The N-terminal, cytoplasmic tetramerization domain (T1) of voltage-gated K+
channels encodes molecular determinants for subfamily-specific assembly of
alpha-subunits into functional tetrameric channels. Crystal structures of T1
tetramers from Shaw and Shaker subfamilies reveal a common four-layered
scaffolding. Within layer 4, on the hypothetical membrane-facing side of the
tetramer, the Shaw T1 tetramer contains four zinc ions; each is coordinated by a
histidine and two cysteines from one monomer and by one cysteine from an
adjacent monomer. The amino acids involved in coordinating the Zn2+ ion occur in
a HX5CX20CC sequence motif that is highly conserved among all Shab, Shaw and
Shal subfamily members, but is not found in Shaker subfamily members. We
demonstrate by coimmunoprecipitation that a few characteristic residues in the
subunit interface are crucial for subfamily-specific tetramerization of the T1
domains.
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Figure 1.
Figure 1. a, The Shaw T1 tetramer drawn in MOLSCRIPT^37 and
rendered using POVRAY version 3.0. The subunit to the front
is not shown for clarity. The four-fold axis is vertical down
the central cavity. Layer 1 (residues 10−56) is colored in
green; layer 2 (residues 57−71) in blue; layer 3 (residues
72−94) in red; and layer 4 (95−111) in yellow. Zinc atoms
are shown as gray spheres. b, Stereodiagram of C  traces
of the superposition of the Shaw T1 (green) and Shaker T1
(magenta) structures (SETOR^ 38) in the same view as in Fig. 1a.
Secondary structure elements, N- and C- termini are labeled.
Arrows 1 and 2 denote two major variable regions between the two
structures. The vertical line represents the four-fold axis. c,
Stereodiagram of C traces
of the Shaw T1 (green) and Shaker T1 (magenta) to highlight the
difference in layer 4, viewed from the top of the teteramer in a
direction ~45^o tilted from the four-fold axis. The vertical
line in the background represents the four-fold axis.
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Figure 2.
Figure 2. a, The zinc binding in the Shaw T1 tetramer. His 75
(layer 3) is shown in red, Cys 102 and Cys 103 are shown in
yellow (layer 4). Cys 81 from the adjacent monomer layer 3, is
shown in black. Distances between Zn^2+ and coordinating atoms
are given.
b, X-ray fluorescence spectra were obtained from
single Shaw T1 crystals scanned from 9,200−9,800 eV (  E
= 0.8 eV) with a scintillation counter oriented at right angles
to the beam (BL1-5, Stanford Synchrotron Radiation Laboratory).
The fluorescence intensity was normalized to the flux of the
incident X-ray as measured by an ion chamber. The data were
processed with the program DISCO^39. The values of f" come from
fitting the data to the theoretical absorption curve for Zn in
the regions above and below the Zn K edge. The theoretical value
for the K edge of elemental Zn is 9,659 eV; the observed value
for two separate measurements was found to be about 2 eV higher.
Spectra taken on crystallization buffer and loop, cryoprotectant
and loop, and loop alone did not demonstrate any observable
signal for identical scans (data not shown). X-ray fluorescence
scanning of crystals at wavelengths around the Mn^2+ and Co^2+ K
edges also did not show any signal. Shaw T1 anomalous dispersion
data set was collected at 9,670 eV (1.28 Å). Overall
completeness was 99.9%, and Bijvoet pairs were 67% complete to
1.95 Å. An 8 peak
was located in an anomalous difference Patterson map which
corresponded to the site of the zinc in the monomer. c,
Stereodiagram of (2F[ o] - F[c]) electron density maps of Shaw
T1 superimposed with the final refined model for the region near
the layer 4 in a viewpoint similar to that in (a). Maps are
contoured at 1 .
d, Stereodiagram of (2F[o] - []F[c]) electron density maps of
Shaker T1 superimposed with the final refined model for the
region showing the layers 3 and 4 in the region near the
C-terminus of layer 4. Maps are contoured at 1  .
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nat Struct Biol
(1999,
6,
38-43)
copyright 1999.
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