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PDBsum entry 3fhc
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Transport protein/hydrolase
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PDB id
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3fhc
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References listed in PDB file
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Key reference
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Title
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The mRNA export protein dbp5 binds RNA and the cytoplasmic nucleoporin nup214 in a mutually exclusive manner.
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Authors
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H.Von moeller,
C.Basquin,
E.Conti.
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Ref.
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Nat Struct Biol, 2009,
16,
247-254.
[DOI no: ]
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PubMed id
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Abstract
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The DEAD-box protein DBP5 is essential for mRNA export in both yeast and humans.
It binds RNA and is concentrated and locally activated at the cytoplasmic side
of the nuclear pore complex. We have determined the crystal structures of human
DBP5 bound to RNA and AMPPNP, and bound to the cytoplasmic nucleoporin NUP214.
The structures reveal that binding of DBP5 to nucleic acid and to NUP214 is
mutually exclusive. Using in vitro assays, we demonstrate that NUP214 decreases
both the RNA binding and ATPase activities of DBP5. The interactions are
mediated by conserved residues, implying a conserved recognition mechanism.
These results suggest a framework for the consecutive steps leading to the
release of mRNA at the final stages of nuclear export. More generally, they
provide a paradigm for how binding of regulators can specifically inhibit
DEAD-box proteins.
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Figure 1.
(a) Schematic representation of the domain organization of
DBP5 with the domain boundaries of the protein constructs used
in this study. The RecA-like domains are colored blue and the
N-terminal region is shown in orange. (b) Protein precipitations
with biotinylated single-stranded RNA identify the RNA binding
region of human DBP5 for structural studies. Purified proteins
were mixed with 5' end–biotinylated 20-mer single-stranded RNA
and incubated with or without AMPPNP or ADP, as indicated.
Proteins mixtures before (input, 17% of the total) and after
coprecipitation (precipitate) were separated on a 15% (w/v)
acrylamide SDS-PAGE and visualized using Coomassie stain. The
molecular weight standards are shown in lane 1. DBP5 constructs
are described in a. NUP214   C
corresponds to residues 1–405 (human sequence). (c) Structure
of DBP5 N
(light blue) bound to AMPPNP (yellow) and single-stranded RNA
(black). The magnesium ion at the ATP binding site is shown in
magenta. Six ordered nucleotides of a single-stranded poly-U RNA
are present in the 2.2-Å resolution structure. The N- and
C-terminal residues of DBP5 visible in the electron density are
indicated. The N-terminal region of DBP5 (residues 75–92,
orange) folds into a short  -helix.
This and all other ribbon diagrams in the manuscript were
generated using PyMol (http://www.pymol.org).
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Figure 3.
(a) Close-up of the region of interaction between the
N-terminal RecA-like domain of DBP5 (in blue) and NUP214 (in
green) in the DBP5 N
C–NUP214
C
structure. The residues involved in hydrophobic and
electrostatic interactions are highlighted. (b) Protein
precipitations by GST pull-downs with DBP5 and NUP214 mutant
proteins. The pull-down assays were carried out as described in
Figure 2a. The DBP5 D223R, I258A, R259D or R262A mutations of
DBP5 impair its interaction with NUP214. NUP214 V353A shows
reduced binding to DBP5, whereas NUP214 D359R impairs the
interaction.
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nat Struct Biol
(2009,
16,
247-254)
copyright 2009.
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