PDBsum entry 3fsh

Ligase

PDB id: 3fsh

Contents

Protein chains

167 a.a. *

21 a.a. *

Waters ×40

* Residue conservation analysis

PDB id:

3fsh

Name:

Ligase

Title:

Crystal structure of the ubiquitin conjugating enzyme ube2g2 bound to the g2br domain of ubiquitin ligase gp78

Structure:

Ubiquitin-conjugating enzyme e2 g2. Chain: a, b. Synonym: ubiquitin-protein ligase g2, ubiquitin carrier protein g2. Engineered: yes. Autocrine motility factor receptor, isoform 2. Chain: c. Fragment: g2br. Synonym: amf receptor, isoform 2, gp78, ring finger protein 45. Engineered: yes

Source:

Mus musculus. Mouse. Organism_taxid: 10090. Gene: ube2g2. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes. Other_details: the peptide was chemically synthesized. This sequence occurs naturally in humans.

Resolution:

2.76Å R-factor: 0.216 R-free: 0.256

Authors:

D.Tu,A.T.Brunger

Key ref:

W.Li et al. (2009). Mechanistic insights into active site-associated polyubiquitination by the ubiquitin-conjugating enzyme Ube2g2. Proc Natl Acad Sci U S A, 106, 3722-3727. PubMed id: 19223579 DOI: 10.1073/pnas.0808564106

Date:

09-Jan-09 Release date: 10-Feb-09

Protein chains

P60605  (UB2G2_MOUSE) -  Ubiquitin-conjugating enzyme E2 G2 from Mus musculus

Seq: 165 a.a.

Struc: 167 a.a.

Protein chain

Q9UKV5  (AMFR_HUMAN) -  E3 ubiquitin-protein ligase AMFR from Homo sapiens

Seq: 643 a.a.

Struc: 21 a.a.

Enzyme reactions

• Enzyme class 2: Chains A, B: E.C.2.3.2.23 - E2 ubiquitin-conjugating enzyme.
• Reaction: S-ubiquitinyl-[E1 ubiquitin-activating enzyme]-L-cysteine + [E2 ubiquitin-conjugating enzyme]-L-cysteine = [E1 ubiquitin-activating enzyme]-L-cysteine + S-ubiquitinyl-[E2 ubiquitin-conjugating enzyme]-L- cysteine
• Enzyme class 3: Chain C: E.C.2.3.2.36 - RING-type E3 ubiquitin transferase (cysteine targeting).
• Reaction: [E2 ubiquitin-conjugating enzyme]-S-ubiquitinyl-L-cysteine + [acceptor protein]-L-cysteine = [E2 ubiquitin-conjugating enzyme]-L-cysteine + [acceptor protein]-S-ubiquitinyl-L-cysteine

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

DOI no: 10.1073/pnas.0808564106

Proc Natl Acad Sci U S A 106:3722-3727 (2009)

PubMed id: 19223579

Mechanistic insights into active site-associated polyubiquitination by the ubiquitin-conjugating enzyme Ube2g2.

W.Li, D.Tu, L.Li, T.Wollert, R.Ghirlando, A.T.Brunger, Y.Ye.

ABSTRACT

Lys-48-linked polyubiquitination regulates a variety of cellular processes by targeting ubiquitinated proteins to the proteasome for degradation. Although polyubiquitination had been presumed to occur by transferring ubiquitin molecules, one at a time, from an E2 active site to a substrate, we recently showed that the endoplasmic reticulum-associated RING finger ubiquitin ligase gp78 can mediate the preassembly of Lys-48-linked polyubiquitin chains on the catalytic cysteine of its cognate E2 Ube2g2 and subsequent transfer to a substrate. Active site-linked polyubiquitin chains are detected in cells on Ube2g2 and its yeast homolog Ubc7p, but how these chains are assembled is unclear. Here, we show that gp78 forms an oligomer via 2 oligomerization sites, one of which is a hydrophobic segment located in the gp78 cytosolic domain. We further demonstrate that a gp78 oligomer can simultaneously associate with multiple Ube2g2 molecules. This interaction is mediated by a novel Ube2g2 surface distinct from the predicted RING binding site. Our data suggest that a large gp78-Ube2g2 heterooligomer brings multiple Ube2g2 molecules into close proximity, allowing ubiquitin moieties to be transferred between neighboring Ube2g2s to form active site-linked polyubiquitin chains.

Selected figure(s)

Figure 1.
Oligomerization of gp78. (A) Schematic representation of the gp78 variants tested in B. (B) Both the cytosolic domain and the transmembrane segments of gp78 can interact with full-length gp78. Detergent extracts of 293T cells transfected with the indicated plasmids were subjected to immunoprecipitation (IP) followed by immunoblotting (IB) with the indicated antibodies. Note that the expressed gp78 proteins comigrate with IgG (*). WCE, whole cell extract. (C–F) Mapping the region in the gp78 cytosolic domain that is necessary for its self-association. (C) Schematic representation of the gp78 variants tested in D. (D) As in B, except that plasmids expressing the indicated gp78 variants were analyzed. (E) Schematic representation of the gp78 variants used in F. (F) As in B, except that plasmids expressing the indicated gp78 variants were analyzed. * indicates a gp78 degradation product.

Figure 5.
The structure of Ube2g2 bound to the gp78 G2BR domain. (A) Overall structure showing the Ube2g2–G2BR complex. Contoured at 3.5 σ around G2BR is a 2F[o] − F[c] σ[A]-weighted annealed omit map omitting G2BR. (B) The contacts between G2BR and Ube2g2. (C) A close-up view on the most critical contacts around A593 and F597 of G2BR. (D) The geometry of G2BR binding compared with RING binding and the active site. RING domain (blue) of the c-Cbl–UbcH7 complex (Protein Data Bank ID code 1FBV), and Ub (red) of the Mms2-Ubc13∼Ub covalent complex (Protein Data Bank ID code 2GMI) are docked onto the Ube2g2–G2BR complex based on E2 structural alignments. Overlay matrices were determined by DALI.

Figures were selected by an automated process.