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PDBsum entry 2khh
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Transport protein
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PDB id
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2khh
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References listed in PDB file
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Key reference
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Title
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Structural requirements for the ubiquitin-Associated domain of the mRNA export factor mex67 to bind its specific targets, The transcription elongation tho complex component hpr1 and nucleoporin fxfg repeats.
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Authors
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M.Hobeika,
C.Brockmann,
F.Gruessing,
D.Neuhaus,
G.Divita,
M.Stewart,
C.Dargemont.
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Ref.
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J Biol Chem, 2009,
284,
17575-17583.
[DOI no: ]
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PubMed id
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Abstract
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The ubiquitin-associated (UBA) domain of the principal Saccharomyces cerevisiae
mRNA nuclear export factor, Mex67, can bind both nuclear pore protein
(nucleoporin) FG repeats and Hpr1, a component of the TREX.THO complex that
functions to link transcription and export. Using fluorescence resonance energy
transfer-based assays, we show here that Hpr1 and the FG repeats interact with
overlapping binding sites on the Mex67 UBA domain. We present the solution
structure of the Mex67 UBA domain (UBA-Mex67) complexed with a FXFG nucleoporin
peptide and define residues engaged in the interaction and those involved in the
FXFG-induced conformational change. We show by NMR titration that the binding of
Hpr1 produces analogous changes in chemical shifts in similar regions of the UBA
domain. Together the data presented here indicate that both Hpr1 and FXFG
nucleoporins may bind in a similar way to the UBA-Mex67 domain. However, whereas
binding of Hpr1 allows UBA-Mex67 to interact with tetra-ubiquitin, the complex
between UBA-Mex67 and FXFG is unable to bind mono- or tetra-ubiquitin,
suggesting that both substrate binding and also the nature of the substrate may
influence the affinity of the UBA-Mex67 domain for ubiquitin.
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Figure 1.
Hpr1 and FXFG repeats have comparable affinity for the UBA
domain of Mex67.A, binding of FXFG to UBA-Mex67 (400 nm) was
followed by measuring tryptophan emission at 340 nm upon
excitation at 290 nm. B, binding of FXFG, FF18-Nsp1, and Hpr1-C
to UBA-Mex67 was monitored by adding increasing concentrations
of FITC-labeled proteins (UBA-Mex67 partners) to dansyl-labeled
UBA-Mex67 (400 nm) and measuring the quenching of dansyl
fluorescence at 440 nm, upon excitation at 350 nm. The titration
curves were fitted according to a quadratic equation using
Grafit Software (Erithacus Software Ltd.) (40).
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Figure 4.
Chemical shift mapping of UBA-Mex67 residues upon binding of
FXFG and Hpr1. Normalized combined chemical shift difference
Δδ upon binding of Hpr1-C (A) and the FXFG peptide (B) is
mapped onto the structure of the UBA-Mex67 domain. Residues
showing a normalized Δδ more than 0.6 upon binding of Hpr1 are
shown in dark green, whereas residues that show a normalized
Δδ more than 0.3 are shown in light green. Corresponding
residues in the FXFG titrations are shown in orange and yellow.
The position of the FXFG peptide is shown in blue. C, the same
data mapped onto the sequence to enable direct comparison.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2009,
284,
17575-17583)
copyright 2009.
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