PDBsum entry 1h8c

Apoptosis

PDB id: 1h8c

Contents

Protein chain

82 a.a. *

* Residue conservation analysis

PDB id:

1h8c

Name:

Apoptosis

Title:

Ubx domain from human faf1

Structure:

Fas-associated factor 1. Chain: a. Fragment: ubx domain. Synonym: faf1. Engineered: yes

Source:

Homo sapiens. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 562

NMR struc:

20 models

Authors:

M.M.Bycroft

Key ref:

A.Buchberger et al. (2001). The UBX domain: a widespread ubiquitin-like module. J Mol Biol, 307, 17-24. PubMed id: 11243799 DOI: 10.1006/jmbi.2000.4462

Date:

01-Feb-01 Release date: 13-Feb-01

Protein chain

Q9UNN5  (FAF1_HUMAN) -  FAS-associated factor 1 from Homo sapiens

Seq: 650 a.a.

Struc: 82 a.a.

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1006/jmbi.2000.4462

J Mol Biol 307:17-24 (2001)

PubMed id: 11243799

The UBX domain: a widespread ubiquitin-like module.

A.Buchberger, M.J.Howard, M.Proctor, M.Bycroft.

ABSTRACT

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.

Selected figure(s)

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.

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.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2001, 307, 17-24) copyright 2001.

Figures were selected by an automated process.