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PDBsum entry 3ea5
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Transport protein
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PDB id
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3ea5
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References listed in PDB file
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Key reference
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Title
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Kap95p binding induces the switch loops of rangdp to adopt the gtp-Bound conformation: implications for nuclear import complex assembly dynamics.
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Authors
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J.K.Forwood,
T.G.Lonhienne,
M.Marfori,
G.Robin,
W.Meng,
G.Guncar,
S.M.Liu,
M.Stewart,
B.J.Carroll,
B.Kobe.
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Ref.
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J Mol Biol, 2008,
383,
772-782.
[DOI no: ]
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PubMed id
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Abstract
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The asymmetric distribution of the nucleotide-bound state of Ran across the
nuclear envelope is crucial for determining the directionality of nuclear
transport. In the nucleus, Ran is primarily in the guanosine 5'-triphosphate
(GTP)-bound state, whereas in the cytoplasm, Ran is primarily guanosine
5'-diphosphate (GDP)-bound. Conformational changes within the Ran switch I and
switch II loops are thought to modulate its affinity for importin-beta. Here, we
show that RanGDP and importin-beta form a stable complex with a micromolar
dissociation constant. This complex can be dissociated by importin-beta binding
partners such as importin-alpha. Surprisingly, the crystal structure of the
Kap95p-RanGDP complex shows that Kap95p induces the switch I and II regions of
RanGDP to adopt a conformation that resembles that of the GTP-bound form. The
structure of the complex provides insights into the structural basis for the
gradation of affinities regulating nuclear protein transport.
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Figure 2.
Fig. 2. Nucleotide-bound state of Ran. (a) Identification of
nucleotide bound to Ran by reverse-phase HPLC confirmed the
presence of GDP. The stoichiometry of bound nucleotide was
assessed by titrating GDP and GTP standards (1.1, 0.58, 0.29,
0.14 nmol). This revealed that 0.71 nmol GDP bound to 0.86 nmol
Ran, which is consistent with 1:1 stoichiometry; GTP was not
detected. (b) Annealed omit F[o] − F[c] map near the
nucleotide bound to Ran (chain A) contoured at 5.0σ (left), and
anomalous map (right, contoured at 3.5σ) with a GDP molecule
and magnesium ion superimposed.
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Figure 3.
Fig. 3. Structure of Kap95p–RanGDP complex. (a) Two views
of the Kap95p–RanGDP structure, related by a 90° rotation
around the x-axis. Kap95p is shown in cyan, Ran is shown in
pink, and the basic patch region is highlighted in red. (b)
Superposition of free RanGDP (PDB ID 1BYU^18) and RanGDP in
complex with Kap95p. Switch regions are highlighted in light
blue (switch I), magenta (switch II), and pink (basic patch) for
free RanGDP, and in blue (switch I), purple (switch II), and red
(basic patch) for RanGDP in complex with Kap95p. (c)
Superposition of RanGDP and RanGTP (PDB ID 2BKU^5) in complex
with Kap95p; RanGDP switch regions are blue (switch I), purple
(switch II), and red (basic patch); RanGTP switch regions are
light blue (switch I), magenta (switch II), and pink (basic
patch). (d) Structural comparison of Kap95p–RanGTP,^5
Kap95p–RanGDP, and HEAT repeats 1–11 of importin-β in
complex with PTHrP.^27 The schematic diagrams summarize the
conformational differences. The conformational shift in the
basic patch region of Ran exposes a part of Kap95p HEAT repeat 7
that is important for binding NLS-containing importin-β cargoes
including PTHrP,^27 SREBP-2,^28 and importin α.^12 It is
unlikely that the observed structural differences between
Kap95p–RanGTP and Kap95p–RanGDP are the result of crystal
packing, because they both possessed an equivalent arrangement
of the protein molecules.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2008,
383,
772-782)
copyright 2008.
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