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PDBsum entry 2aef
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Metal transport, membrane protein
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PDB id
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2aef
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References listed in PDB file
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Key reference
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Title
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Structures of the mthk rck domain and the effect of ca2+ on gating ring stability.
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Authors
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J.Dong,
N.Shi,
I.Berke,
L.Chen,
Y.Jiang.
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Ref.
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J Biol Chem, 2005,
280,
41716-41724.
[DOI no: ]
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PubMed id
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Abstract
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MthK is a Ca2+-gated K+ channel from Methanobacterium autotrophicum. The crystal
structure of the MthK channel in a Ca2+-bound open state was previously
determined at 3.3 A and revealed an octameric gating ring composed of eight
intracellular ligand-binding RCK (regulate the conductance of K+) domains. It
was suggested that Ca2+ binding regulates the gating ring conformation, which in
turn leads to the opening and closing of the channel. However, at 3.3 AA
resolution, the molecular details of the structure are not well defined, and
many of the conclusions drawn from that structure were hypothetical. Here we
have presented high resolution structures of the MthK RCK domain with and
without Ca2+ bound from three different crystals. These structures revealed a
dimeric architecture of the RCK domain and allowed us to visualize the Ca2+
binding and protein-protein contacts at atomic detail. The dimerization of RCK
domains is also conserved in other RCK-regulated K+ channels and transporters,
suggesting that the RCK dimer serves as a basic unit in the gating ring
assembly. A comparison of these dimer structures confirmed that the dimer
interface is indeed flexible as suggested previously. However, the
conformational change at the flexible interface is of an extent smaller than the
previously hypothesized gating ring movement, and a reconstruction of these
dimers into octamers by applying protein-protein contacts at the fixed interface
did not generate enclosed gating rings. This indicated that there is a high
probability that the previously defined fixed interface may not be fixed during
channel gating. In addition to the structural studies, we have also carried out
biochemical analyses and have shown that near physiological pH, isolated RCK
domains form a stable octamer in solution, supporting the notion that the
formation of octameric gating ring is a functionally relevant event in MthK
gating. Additionally, our stability studies indicated that Ca2+ binding
stabilizes the RCK domains in this octameric state.
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Figure 1.
FIGURE 1. Structures of RCK domains. A, a single subunit of
the MthK RCK. B, a homodimer of the MthK RCK domain in a
Ca^2+(gold sphere)-bound state. The two subunits are colored red
and cyan. C, a homodimer of the E. coli RCK domain that has no
C-terminal subdomain (Protein Data Bank code 1ID1). D, a
homodimer of the KTN domain from M. jannaschii KtrA in a
NAD-bound state (Protein Data Bank code 1LSS). Each subunit
(ribbon style) and its bound NAD (ball-and-stick) are of the
same color. The C-terminal region right after helix  F was
removed in the construct for crystallization.
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Figure 3.
FIGURE 3. Superimposition of different MthK RCK structures.
A, superimposition of two P2[1] crystal structures with (red)
and without (black) Ca^2+. B, superimposition of the gating
ring-forming RCK (black) with the RCK from the P2[1] crystal
(red). C, superimposition of the gating ring-forming RCK (black)
with RCK from the R32 crystal (red).
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2005,
280,
41716-41724)
copyright 2005.
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