PDBsum entry 2esp

Ligase

PDB id: 2esp

Contents

Protein chain

149 a.a. *

Ligands

MPD

Metals

_CL

Waters ×137

* Residue conservation analysis

PDB id:

2esp

Name:

Ligase

Title:

Human ubiquitin-conjugating enzyme (e2) ubch5b mutant ile88ala

Structure:

Ubiquitin-conjugating enzyme e2 d2. Chain: a. Synonym: ubiquitin-protein ligase d2, ubiquitin carrier protein d2, ubiquitin-conjugating enzyme e2-17 kda 2, e217, kb 2. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: ube2d2, ubc4, ubch5b. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

1.52Å R-factor: 0.187 R-free: 0.233

Authors:

E.Ozkan,H.Yu,J.Deisenhofer

Key ref:

E.Ozkan et al. (2005). Mechanistic insight into the allosteric activation of a ubiquitin-conjugating enzyme by RING-type ubiquitin ligases. Proc Natl Acad Sci U S A, 102, 18890-18895. PubMed id: 16365295 DOI: 10.1073/pnas.0509418102

Date:

26-Oct-05 Release date: 06-Dec-05

Protein chain

P62837  (UB2D2_HUMAN) -  Ubiquitin-conjugating enzyme E2 D2 from Homo sapiens

Seq: 147 a.a.

Struc: 149 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class 2: 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: E.C.2.3.2.24 - (E3-independent) E2 ubiquitin-conjugating enzyme.
• Reaction: S-ubiquitinyl-[E1 ubiquitin-activating enzyme]-L-cysteine + [acceptor protein]-L-lysine = [E1 ubiquitin-activating enzyme]-L-cysteine + N₆- monoubiquitinyl-[acceptor protein]-L-lysine

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.0509418102

Proc Natl Acad Sci U S A 102:18890-18895 (2005)

PubMed id: 16365295

Mechanistic insight into the allosteric activation of a ubiquitin-conjugating enzyme by RING-type ubiquitin ligases.

E.Ozkan, H.Yu, J.Deisenhofer.

ABSTRACT

Ubiquitin-conjugating enzymes (E2s) collaborate with the ubiquitin-activating enzyme (E1) and ubiquitin ligases (E3s) to attach ubiquitin to target proteins. RING-containing E3s simultaneously bind to E2s and substrates, bringing them into close proximity and thus facilitating ubiquitination. We show herein that, although the E3-binding site on the human E2 UbcH5b is distant from its active site, two RING-type minimal E3 modules lacking substrate-binding functions greatly stimulate the rate of ubiquitin release from the UbcH5b-ubiquitin thioester. Using statistical coupling analysis and mutagenesis, we identify and characterize clusters of coevolving and functionally linked residues within UbcH5b that span its E3-binding and active sites. Several UbcH5b mutants are defective in their stimulation by E3s despite their abilities to bind to these E3s, to form ubiquitin thioesters, and to release ubiquitin at a basal rate. One such mutation, I37A, is distant from both the active site and the E3-binding site of UbcH5b. Our studies reveal structural determinants for communication between distal functional sites of E2s and suggest that RING-type E3s activate E2s allosterically.

Selected figure(s)

Figure 1.
Fig. 1. SCA of E2s. (A) Hierarchical clustering of a submatrix of pairwise statistical coupling energies of E2 residues, which are plotted as a color gradient, with blue and red representing the lowest (0 kT*, with kT* being an arbitrary energy-like unit) and highest (2 kT*) energies, respectively. Both columns and rows of the matrix represent residue positions in UbcH7 and UbcH5b (in parentheses). The two clusters of residues that exhibit similar coupling patterns are boxed with dashed lines. (B) The two clusters of residues in A are shown as space-filling models and mapped onto the structure of UbcH5b. Cluster I and II residues are colored green and cyan, respectively. E3-binding residues are shown in red. The active-site cysteine and asparagine are labeled.

Figure 2.
Fig. 2. E3-stimulated release of ubiquitin from the UbcH5b-ubiquitin thioester. (A) WT and 31 mutants of UbcH5b were tested for their ability to release ubiquitin from their thioesters in the presence of buffer (-) or Apc2/11 (+) with continuous E1-catalyzed ubiquitin charging. The bands corresponding to free UbcH5b, UbcH5b-ubiquitin thioester, and monoubiquitinated UbcH5b (with isopeptide-linked ubiquitin) are indicated. (B) Same as in A except that CNOT4N was used as the E3. (C) UbcH5b I88A was defective in supporting APC/C^Cdh1-mediated ubiquitination of cyclin B1. Xenopus egg APC/C was incubated with buffer or Cdh1 and assayed for its ability to ubiquitinate an N-terminal fragment (residues 1-102) of human cyclin B1 in the presence of the varying concentrations of WT or mutant UbcH5b. Cyclin B-ubiquitin conjugates are indicated.

Figures were selected by an automated process.