PDBsum entry 2bf8

Ligase

PDB id: 2bf8

Contents

Protein chains

154 a.a. *

77 a.a. *

Waters ×83

* Residue conservation analysis

PDB id:

2bf8

Name:

Ligase

Title:

Crystal structure of sumo modified ubiquitin conjugating enzyme e2- 25k

Structure:

Ubiquitin-conjugating enzyme e2-25 kda. Chain: a. Fragment: conserved core domain, residues 1-154. Synonym: e2-25k, ubiquitin-protein ligase, ubiquitin carrier protein, e2(25k), huntingtin interacting protein 2, hip-2. Engineered: yes. Other_details: covalent isopeptide link between e2-25k lysine 14 and sumo c-terminus. Ubiquitin-like protein smt3c.

Source:

Bos taurus. Bovine. Organism_taxid: 9913. Expressed in: escherichia coli. Expression_system_taxid: 469008. Homo sapiens. Human. Organism_taxid: 9606.

Resolution:

2.30Å R-factor: 0.213 R-free: 0.279

Authors:

A.Pichler,P.Knipscheer,E.Oberhofer,W.J.Van Dijk,R.Korner,J.Velgaard Olsen,S.Jentsch,F.Melchior,T.K.Sixma

Key ref:

A.Pichler et al. (2005). SUMO modification of the ubiquitin-conjugating enzyme E2-25K. Nat Struct Mol Biol, 12, 264-269. PubMed id: 15723079 DOI: 10.1038/nsmb903

Date:

06-Dec-04 Release date: 16-Feb-05

Protein chain

P61085  (UBE2K_BOVIN) -  Ubiquitin-conjugating enzyme E2 K from Bos taurus

Seq: 200 a.a.

Struc: 154 a.a.

Protein chain

P63165  (SUMO1_HUMAN) -  Small ubiquitin-related modifier 1 from Homo sapiens

Seq: 101 a.a.

Struc: 77 a.a.

Enzyme reactions

• Enzyme class 2: Chain A: 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 B: E.C.?

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: 10.1038/nsmb903

Nat Struct Mol Biol 12:264-269 (2005)

PubMed id: 15723079

SUMO modification of the ubiquitin-conjugating enzyme E2-25K.

A.Pichler, P.Knipscheer, E.Oberhofer, W.J.van Dijk, R.Körner, J.V.Olsen, S.Jentsch, F.Melchior, T.K.Sixma.

ABSTRACT

Post-translational modification with small ubiquitin-related modifier (SUMO) alters the function of many proteins, but the molecular mechanisms and consequences of this modification are still poorly defined. During a screen for novel SUMO1 targets, we identified the ubiquitin-conjugating enzyme E2-25K (Hip2). SUMO attachment severely impairs E2-25K ubiquitin thioester and unanchored ubiquitin chain formation in vitro. Crystal structures of E2-25K(1-155) and of the E2-25K(1-155)-SUMO conjugate (E2-25K(*)SUMO) indicate that SUMO attachment interferes with E1 interaction through its location on the N-terminal helix. The SUMO acceptor site in E2-25K, Lys14, does not conform to the consensus site found in most SUMO targets (PsiKXE), and functions only in the context of an alpha-helix. In contrast, adjacent SUMO consensus sites are modified only when in unstructured peptides. The demonstration that secondary structure elements are part of SUMO attachment signals could contribute to a better prediction of SUMO targets.

Selected figure(s)

Figure 2.
Figure 2. Sumoylation of E2-25K inhibits ubiquitin thioester formation. (a) Ubiquitin (Ub) chain formation. SUMO-modified (*S) or unmodified E2-25K (1.5 g; arrowheads), 8 g ubiquitin and 100 ng ubiquitin-E1 were incubated for indicated times with an ATP-regenerating system at 37 °C. Analysis was done by Coomassie blue staining. (b) Ubiquitin thioester formation. SUMO-modified or unmodified E2-25K (1 g), 2 g ubiquitin, 130 ng ubiquitin-E1 and ATP were incubated for 1 h at 30 °C. Analysis was done by immunoblotting. Left, nonreducing conditions allow detection of thioester; Right, reducing conditions. (c) Ubiquitin transfer. Equal amounts of E2-25K and E2-25K*SUMO ubiquitin thioesters were generated by incubating 10 g E2-25K, 10 g ubiquitin K48R and 1.25 g ubiquitin-E1 with ATP at 37 °C for 12 and 70 min, respectively. Ubiquitin-E1 was inhibited by EDTA, and wild-type ubiquitin was added to allow di-ubiquitin formation. Immunoblotting with anti-E2-25K (top) or anti-ubiquitin (bottom) followed. (d) Ubiquitin thioester formation of full-length and truncated E2-25K. 1 g of SUMOylated or unmodified full-length (top) or truncated E2-25K(1 -155) (bottom) was incubated with ATP, 1 g ubiquitin and indicated concentrations of ubiquitin-E1 for 30 min at 30 °C. Analysis under nonreducing conditions was done by immunoblotting. Asterisk, ATP-independent unspecific band. (e) Experiment was done as in d but in a time course using 6 ng E1 for E2-25K(1 -155) and 100 ng E1 for full-length E2-25K. Asterisk, isopeptide linked ubiquitin to E2-25K thioester; #, di-ubiquitin.

Figure 4.
Figure 4. SUMO target sites are defined by their structural context. (a) Position of four lysines in the N terminus of E2-25K. (b) In the folded protein, Lys14 is the preferred substrate. Wild type (WT) or indicated E2-25K mutant (500 ng), 1 g SUMO1, 300 ng Aos1 -Uba2, 500 ng Ubc9 and ATP were incubated for 30 min at 30 °C. (c) In an unfolded peptide, Lys10 is preferred. Indicated peptide (20 g) 1.5 g SUMO-E1, 340 ng Ubc9, 15 g SUMO and ATP were incubated for 0 -5 h. (d) In folded E2-25K protein, Lys14 is preferred. Wild type or indicated F2-25K mutant (7.5 g) 12 g SUMO-E1, 425 ng Ubc9, 20 g SUMO and ATP were incubated for 0 -2 h at 37 °C.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Struct Mol Biol (2005, 12, 264-269) copyright 2005.

Figures were selected by an automated process.