PDBsum entry 2bep

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Ligase PDB id
Protein chain
156 a.a. *
Waters ×260
* Residue conservation analysis
PDB id:
Name: Ligase
Title: Crystal structure of ubiquitin conjugating enzyme e2-25k
Structure: Ubiquitin-conjugating enzyme e2-25 kda. Chain: a. Fragment: conserved core domain, residues 1-155. Synonym: ubiquitin-protein ligase, ubiquitin carrier protei e2(25k), huntingtin interacting protein 2, hip-2, e2-25k. Engineered: yes
Source: Bos taurus. Bovine. Organism_taxid: 9913. Expressed in: escherichia coli. Expression_system_taxid: 469008.
1.80Å     R-factor:   0.169     R-free:   0.220
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
29-Nov-04     Release date:   16-Feb-05    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
P61085  (UBE2K_BOVIN) -  Ubiquitin-conjugating enzyme E2 K
200 a.a.
156 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

 Enzyme reactions 
   Enzyme class: E.C.  - Ubiquitin--protein ligase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: ATP + ubiquitin + protein lysine = AMP + diphosphate + protein N-ubiquityllysine
+ ubiquitin
+ protein lysine
+ diphosphate
+ protein N-ubiquityllysine
Molecule diagrams generated from .mol files obtained from the KEGG ftp site


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.
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.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
20584304 A.Solernou, and J.Fernandez-Recio (2010).
Protein docking by Rotation-Based Uniform Sampling (RotBUS) with fast computing of intermolecular contact distance and residue desolvation.
  BMC Bioinformatics, 11, 352.  
19889771 C.H.Tseng, T.S.Cheng, C.Y.Shu, K.S.Jeng, and M.M.Lai (2010).
Modification of small hepatitis delta virus antigen by SUMO protein.
  J Virol, 84, 918-927.  
21102611 J.R.Gareau, and C.D.Lima (2010).
The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition.
  Nat Rev Mol Cell Biol, 11, 861-871.  
20462400 K.A.Wilkinson, and J.M.Henley (2010).
Mechanisms, regulation and consequences of protein SUMOylation.
  Biochem J, 428, 133-145.  
20014027 T.Ju, W.Bocik, A.Majumdar, and J.R.Tolman (2010).
Solution structure and dynamics of human ubiquitin conjugating enzyme Ube2g2.
  Proteins, 78, 1291-1301.
PDB code: 2kly
19712108 D.E.Christensen, and R.E.Klevit (2009).
Dynamic interactions of proteins in complex networks: identifying the complete set of interacting E2s for functional investigation of E3-dependent protein ubiquitination.
  FEBS J, 276, 5381-5389.  
19744555 F.T.Kuo, I.K.Bentsi-Barnes, G.M.Barlow, J.Bae, and M.D.Pisarska (2009).
Sumoylation of forkhead L2 by Ubc9 is required for its activity as a transcriptional repressor of the Steroidogenic Acute Regulatory gene.
  Cell Signal, 21, 1935-1944.  
19240082 H.A.Blomster, V.Hietakangas, J.Wu, P.Kouvonen, S.Hautaniemi, and L.Sistonen (2009).
Novel proteomics strategy brings insight into the prevalence of SUMO-2 target sites.
  Mol Cell Proteomics, 8, 1382-1390.  
19721078 H.H.Hsiao, E.Meulmeester, B.T.Frank, F.Melchior, and H.Urlaub (2009).
"ChopNSpice," a mass spectrometric approach that allows identification of endogenous small ubiquitin-like modifier-conjugated peptides.
  Mol Cell Proteomics, 8, 2664-2675.  
19643976 L.Li, S.Liang, M.M.Pilcher, and S.O.Meroueh (2009).
Incorporating receptor flexibility in the molecular design of protein interfaces.
  Protein Eng Des Sel, 22, 575-586.  
19017645 Y.Tateishi, M.Ariyoshi, R.Igarashi, H.Hara, K.Mizuguchi, A.Seto, A.Nakai, T.Kokubo, H.Tochio, and M.Shirakawa (2009).
Molecular Basis for SUMOylation-dependent Regulation of DNA Binding Activity of Heat Shock Factor 2.
  J Biol Chem, 284, 2435-2447.  
18538659 E.Meulmeester, M.Kunze, H.H.Hsiao, H.Urlaub, and F.Melchior (2008).
Mechanism and consequences for paralog-specific sumoylation of ubiquitin-specific protease 25.
  Mol Cell, 30, 610-619.  
18155241 H.Windecker, and H.D.Ulrich (2008).
Architecture and assembly of poly-SUMO chains on PCNA in Saccharomyces cerevisiae.
  J Mol Biol, 376, 221-231.  
18403209 J.J.Perry, J.A.Tainer, and M.N.Boddy (2008).
A SIM-ultaneous role for SUMO and ubiquitin.
  Trends Biochem Sci, 33, 201-208.  
18344540 K.Schwamborn, P.Knipscheer, E.van Dijk, W.J.van Dijk, T.K.Sixma, R.H.Meloen, and J.P.Langedijk (2008).
SUMO assay with peptide arrays on solid support: insights into SUMO target sites.
  J Biochem, 144, 39-49.  
18331345 K.Thakar, R.Niedenthal, E.Okaz, S.Franken, A.Jakobs, S.Gupta, S.Kelm, and F.Dietz (2008).
SUMOylation of the hepatoma-derived growth factor negatively influences its binding to chromatin.
  FEBS J, 275, 1411-1426.  
18832349 N.Meednu, H.Hoops, S.D'Silva, L.Pogorzala, S.Wood, D.Farkas, M.Sorrentino, E.Sia, P.Meluh, and R.K.Miller (2008).
The Spindle Positioning Protein Kar9p Interacts With the Sumoylation Machinery in Saccharomyces cerevisiae.
  Genetics, 180, 2033-2055.  
18691969 P.Knipscheer, A.Flotho, H.Klug, J.V.Olsen, W.J.van Dijk, A.Fish, E.S.Johnson, M.Mann, T.K.Sixma, and A.Pichler (2008).
Ubc9 sumoylation regulates SUMO target discrimination.
  Mol Cell, 31, 371-382.
PDB code: 2vrr
18282128 V.G.Wilson, and P.R.Heaton (2008).
Ubiquitin proteolytic system: focus on SUMO.
  Expert Rev Proteomics, 5, 121-135.  
18492068 Z.Tang, C.M.Hecker, A.Scheschonka, and H.Betz (2008).
Protein interactions in the sumoylation cascade: lessons from X-ray structures.
  FEBS J, 275, 3003-3015.  
17803214 A.Heifetz, S.Pal, and G.R.Smith (2007).
Protein-protein docking: progress in CAPRI rounds 6-12 using a combination of methods: the introduction of steered solvated molecular dynamics.
  Proteins, 69, 816-822.  
17803217 A.May, and M.Zacharias (2007).
Protein-protein docking in CAPRI using ATTRACT to account for global and local flexibility.
  Proteins, 69, 774-780.  
17477837 B.T.Dye, and B.A.Schulman (2007).
Structural mechanisms underlying posttranslational modification by ubiquitin-like proteins.
  Annu Rev Biophys Biomol Struct, 36, 131-150.  
17671979 C.Wang, O.Schueler-Furman, I.Andre, N.London, S.J.Fleishman, P.Bradley, B.Qian, and D.Baker (2007).
RosettaDock in CAPRI rounds 6-12.
  Proteins, 69, 758-763.  
17803212 K.Wiehe, B.Pierce, W.W.Tong, H.Hwang, J.Mintseris, and Z.Weng (2007).
The performance of ZDOCK and ZRANK in rounds 6-11 of CAPRI.
  Proteins, 69, 719-725.  
17933515 P.Knipscheer, and T.K.Sixma (2007).
Protein-protein interactions regulate Ubl conjugation.
  Curr Opin Struct Biol, 17, 665-673.  
17491593 P.Knipscheer, W.J.van Dijk, J.V.Olsen, M.Mann, and T.K.Sixma (2007).
Noncovalent interaction between Ubc9 and SUMO promotes SUMO chain formation.
  EMBO J, 26, 2797-2807.
PDB code: 2uyz
18000527 R.Geiss-Friedlander, and F.Melchior (2007).
Concepts in sumoylation: a decade on.
  Nat Rev Mol Cell Biol, 8, 947-956.  
17894347 S.Chaudhury, A.Sircar, A.Sivasubramanian, M.Berrondo, and J.J.Gray (2007).
Incorporating biochemical information and backbone flexibility in RosettaDock for CAPRI rounds 6-12.
  Proteins, 69, 793-800.  
17876821 S.Grosdidier, C.Pons, A.Solernou, and J.Fernández-Recio (2007).
Prediction and scoring of docking poses with pyDock.
  Proteins, 69, 852-858.  
17803234 Vries, A.D.van Dijk, M.Krzeminski, M.van Dijk, A.Thureau, V.Hsu, T.Wassenaar, and A.M.Bonvin (2007).
HADDOCK versus HADDOCK: new features and performance of HADDOCK2.0 on the CAPRI targets.
  Proteins, 69, 726-733.  
17965193 S.Lorenzen, and Y.Zhang (2007).
Monte Carlo refinement of rigid-body protein docking structures with backbone displacement and side-chain optimization.
  Protein Sci, 16, 2716-2725.  
17803232 S.Qin, and H.X.Zhou (2007).
A holistic approach to protein docking.
  Proteins, 69, 743-749.  
18166654 S.W.Tait, Vries, C.Maas, A.M.Keller, C.S.D'Santos, and J.Borst (2007).
Apoptosis induction by Bid requires unconventional ubiquitination and degradation of its N-terminal fragment.
  J Cell Biol, 179, 1453-1466.  
17923699 V.Vethantham, N.Rao, and J.L.Manley (2007).
Sumoylation modulates the assembly and activity of the pre-mRNA 3' processing complex.
  Mol Cell Biol, 27, 8848-8858.  
17803223 X.Q.Gong, S.Chang, Q.H.Zhang, C.H.Li, L.Z.Shen, X.H.Ma, M.H.Wang, B.Liu, H.Q.He, W.Z.Chen, and C.X.Wang (2007).
A filter enhanced sampling and combinatorial scoring study for protein docking in CAPRI.
  Proteins, 69, 859-865.  
17853451 Y.Shen, R.Brenke, D.Kozakov, S.R.Comeau, D.Beglov, and S.Vajda (2007).
Docking with PIPER and refinement with SDU in rounds 6-11 of CAPRI.
  Proteins, 69, 734-742.  
16732283 A.A.Yunus, and C.D.Lima (2006).
Lysine activation and functional analysis of E2-mediated conjugation in the SUMO pathway.
  Nat Struct Mol Biol, 13, 491-499.
PDB codes: 2grn 2gro 2grp 2grq 2grr
17406544 A.Shevchenko, H.Tomas, J.Havlis, J.V.Olsen, and M.Mann (2006).
In-gel digestion for mass spectrometric characterization of proteins and proteomes.
  Nat Protoc, 1, 2856-2860.  
16710298 G.Buchwald, P.van der Stoop, O.Weichenrieder, A.Perrakis, M.van Lohuizen, and T.K.Sixma (2006).
Structure and E3-ligase activity of the Ring-Ring complex of polycomb proteins Bmi1 and Ring1b.
  EMBO J, 25, 2465-2474.
PDB code: 2ckl
17099698 L.Shen, M.H.Tatham, C.Dong, A.Zagórska, J.H.Naismith, and R.T.Hay (2006).
SUMO protease SENP1 induces isomerization of the scissile peptide bond.
  Nat Struct Mol Biol, 13, 1069-1077.
PDB codes: 2iy0 2iy1
16413479 M.Hochstrasser (2006).
Lingering mysteries of ubiquitin-chain assembly.
  Cell, 124, 27-34.  
16753028 O.Kerscher, R.Felberbaum, and M.Hochstrasser (2006).
Modification of proteins by ubiquitin and ubiquitin-like proteins.
  Annu Rev Cell Dev Biol, 22, 159-180.  
15882441 A.d'Azzo, A.Bongiovanni, and T.Nastasi (2005).
E3 ubiquitin ligases as regulators of membrane protein trafficking and degradation.
  Traffic, 6, 429-441.  
16125934 H.D.Ulrich (2005).
Mutual interactions between the SUMO and ubiquitin systems: a plea of no contest.
  Trends Cell Biol, 15, 525-532.  
16204249 J.Song, Z.Zhang, W.Hu, and Y.Chen (2005).
Small ubiquitin-like modifier (SUMO) recognition of a SUMO binding motif: a reversal of the bound orientation.
  J Biol Chem, 280, 40122-40129.
PDB code: 2asq
16007098 S.Raasi, R.Varadan, D.Fushman, and C.M.Pickart (2005).
Diverse polyubiquitin interaction properties of ubiquitin-associated domains.
  Nat Struct Mol Biol, 12, 708-714.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.