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PDBsum entry 1h8c
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* Residue conservation analysis
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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 ubx domain: a widespread ubiquitin-Like module.
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Authors
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A.Buchberger,
M.J.Howard,
M.Proctor,
M.Bycroft.
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Ref.
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J Mol Biol, 2001,
307,
17-24.
[DOI no: ]
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PubMed id
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Abstract
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The UBX domain is an 80 amino acid residue module that is present typically at
the carboxyl terminus of a variety of eukaryotic proteins. In an effort to
elucidate the function of UBX domains, we solved the three-dimensional structure
of the UBX domain of human Fas-associated factor-1 (FAF1) by NMR spectroscopy.
The structure has a beta-Grasp fold characterised by a
beta-beta-alpha-beta-beta-alpha-beta secondary-structure organisation. The five
beta strands are arranged into a mixed sheet in the order 21534. The longer
first helix packs across the first three strands of the sheet, and a second
shorter 3(10) helix is located in an extended loop connecting strands 4 and 5.
In the absence of significant sequence similarity, the UBX domain can be
superimposed with ubiquitin with an r.m.s.d. of 1.9 A, suggesting that the two
structures share the same superfold, and an evolutionary relationship. However,
the absence of a carboxyl-terminal extension containing a double glycine motif
and of suitably positioned lysine side-chains makes it highly unlikely that UBX
domains are either conjugated to other proteins or part of mixed UBX-ubiquitin
chains. Database searches revealed that most UBX domain-containing proteins
belong to one of four evolutionarily conserved families represented by the human
FAF1, p47, Y33K, and Rep8 proteins. A role of the UBX domain in
ubiquitin-related processes is suggested.
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Figure 1.
Figure 1. Three-dimensional structure of the UBX domain of
human FAF1. (a) Stereo view of an overlay of the 20 best NMR
structures. (b) Ribbon representation in the same orientation as
in (a), highlighting the structural elements. (c) Conserved
putative interaction site. The side-chains of the exposed and
conserved residues R11, F51, P52, and R53 are depicted as
ball-and-stick models. (b) and (c) were prepared using MOLSCRIPT
[Kraulis 1991]. The DNA sequence encoding the carboxyl-terminal
100 residues of human FAF1 was cloned into the pRSET-derived
pHisGro vector (M. P. & A. R. Fersht, unpublished results)
allowing for expression of the FAF1 UBX domain as a
hexahistidine-tagged fusion protein with the apical domain of
GroEL. Expression of isotopically labelled protein in E. coli
C41 (DE3) cells, purification, and proteolytic removal of the
fusion moiety were performed as described [Buchberger et al
2000]. NMR samples were 5 mM protein in 20 mM phosphate buffer
(pH 5.8). NMR spectra were recorded at 37°C on a Bruker AMX
500 spectrometer equipped with a pulsed field gradient, triple
resonance probe. Resonance assignments were obtained using
standard double and triple resonance experiments [Bax and
Grzesiek 1993]. Briefly, backbone assignments were obtained
using 3D 1H,15N-edited TOCSY, HBHACONH, CBCACONH and HNCACB
experiments. Side-chain assignments were obtained from HCCONH
and CCONH experiments recorded on a Bruker DRX 600 spectrometer.
Additional side-chain assignments were obtained using 2D 1H,1H
DQF-COSY, 2D 1H,1H TOCSY and 3D HCCH-TOCSY experiments.
Stereospecific assignments for the methyl groups of valine and
leucine residues were obtained as described [Neri et al 1989]
using a 10 % fractionally 13C-labelled sample. The assignments
have been deposited in the BioMagResBank under accession number
4952. Torsional restraints for 43 f and 43 q angles were
obtained from an analysis of C', N, C^a, Ha, and C^b chemical
shifts with TALOS [Cornilescu et al 1999a] and from experimental
3J[HNHa] coupling constants measured using an HNHA experiment
[Kuboniwa et al 1994]. x1 side-chain restraints for 30 residues
were determined from J[ab] coupling constants measured by a HNHB
experiment and from NOE data. Distance restraints were obtained
from the analysis of 2D 1H,1H NOESY, 3D 1H,15N-edited NOESY and
3D 1H,13C-edited NOESY experiments all recorded with 100 ms
mixing time. Peak volumes were converted into inter-proton
distance restraints based on known distances. The distance
restraints were then classified as strong, medium or weak,
corresponding to upper distance bounds of 2.7 Å, 3.3
Å, and 5.0 Å, respectively. An additional 0.5
Å was added to upper distance bounds for atoms involving
methyl protons. A lower distance bound of 1.8 Å was used
for all NOE-derived distance restraints. Structures were
calculated using 207 intra-residue, 186 sequential, 92
medium-range and 322 long-range NOE restraints after elimination
of redundant distance restraints using the program AQUA
[Laskowski et al 1996]. In addition, 33 hydrogen bond restraints
were used in the calculations. These were based on the
observation of J connectivity between amide protons and hydrogen
bond-accepting carbonyl carbon atoms [Cornilescu et al 1999b],
the identification of potential hydrogen bond donors from a
1H,15N HSQC spectrum recorded immediately after dissolving a
protein sample in 100 % 2H[2]O, and the characteristic NOE
patterns that are observed for residues in regular secondary
structure [Wuthrich 1986]. Structures were calculated using
simulated annealing from random starting structures using the
program X-PLOR 3.8 [Brunger 1992]. The coordinates have been
deposited in the PDB, entry 1H8C.
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Figure 3.
Figure 3. Structural alignment of the UBX domain with
ubiquitin. (a) Stereoview of the superimposed backbone traces of
the FAF1 UBX domain (yellow) and human ubiquitin (magenta; PDB
entry 1aar). The superimposition was done using Insight (MSI).
The orientation is the same as in Figure 1(a). (b) Sequence
alignment based on the structure comparison. Elements of
secondary structure are indicated above and below the sequences
of the FAF1 UBX domain and human ubiquitin, respectively.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2001,
307,
17-24)
copyright 2001.
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Secondary reference #1
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Title
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Identification and characterization of human fas associated factor 1, Hfaf1.
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Authors
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S.W.Ryu,
S.K.Chae,
K.J.Lee,
E.Kim.
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Ref.
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Biochem Biophys Res Commun, 1999,
262,
388-394.
[DOI no: ]
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PubMed id
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