Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Jun 8;369(3):608-18.
doi: 10.1016/j.jmb.2007.04.006. Epub 2007 Apr 6.

Structure and analysis of a complex between SUMO and Ubc9 illustrates features of a conserved E2-Ubl interaction

Allan D Capili  1 , Christopher D Lima
Affiliations

Structure and analysis of a complex between SUMO and Ubc9 illustrates features of a conserved E2-Ubl interaction

Allan D Capili et al. J Mol Biol. .

Abstract

The SUMO E2 Ubc9 serves as a lynchpin in the SUMO conjugation pathway, interacting with the SUMO E1 during activation, with thioester linked SUMO after E1 transfer and with the substrate and SUMO E3 ligases during conjugation. Here, we describe the structure determination of a non-covalent complex between human Ubc9 and SUMO-1 at 2.4 A resolution. Non-covalent interactions between Ubc9 and SUMO are conserved in human and yeast insomuch as human Ubc9 interacts with each of the human SUMO isoforms, and yeast Ubc9 interacts with Smt3, the yeast SUMO ortholog. Structural comparisons reveal similarities to several other non-covalent complexes in the ubiquitin pathway, suggesting that the non-covalent Ubc9-SUMO interface may be important for poly-SUMO chain formation, for E2 recruitment to SUMO conjugated substrates, or for mediating E2 interactions with either E1 or E3 ligases. Biochemical analysis suggests that this surface is less important for E1 activation or di-SUMO-2 formation, but more important for E3 interactions and for poly-SUMO chain formation when the chain exceeds more than two SUMO proteins.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Characterization of the Ubc9-SUMO interaction
(A) Gel filtration analysis for interactions between human Ubc9 and SUMO-1 (left), human Ubc9 and either SUMO-2 or SUMO-3 (middle), or yeast Ubc9 and Smt3 (right). UV chromatograms are shown in the top panel, color coded for the particular protein in question. SDS-PAGE analysis for the resulting fractions stained with Coomassie Blue with indicated proteins labeled on the right. Apparent molecular weight markers and indicated molecular weights are shown to the left of each gel inset. The elution volume and gel filtration molecular weight standards are indicated for each panel. (B) Ribbon representation of the Ubc9-SUMO-1 structure with Ubc9 in blue and SUMO-1 in yellow. The Ubc9 catalytic cysteine is labeled (C93). Respective N- and C-termini are labeled. All graphics prepared using PYMOL. (C) Close-up of the Ubc9-SUMO-1 interface with residues labeled and shown in stick representation. Dashed lines indicate potential hydrogen bond interactions. (D) Superposition of human and yeast Ubc9 (PDB 2GJD) highlighting the conserved residues involved in the interaction with SUMO-1 color coded and labeled. (E) Superposition of human SUMO-1, SUMO-2 (PDB 2IO0), SUMO-3 (PDB 2IO1) and yeast Smt3 (PDB 1EUV) highlighting the conserved residues involved in the interaction with Ubc9 color coded and labeled. (F) Sequence alignment for SUMO and Ubc9 isoforms. Top panel includes alignment of human SUMO isoforms, yeast Smt3, and human ubiquitin. Amino acid numbering for SUMO-1 is shown above the alignment. Conserved residues shaded yellow, identical residues shaded orange. The bottom panel includes an alignment between yeast and human Ubc9 with similar and identical residues shaded yellow. Amino acid numbering is shown above the alignment. Black or red dots above either alignment indicate side chain or main chain contacts, respectively. Recombinant human Ubc9, SUMO-1, SUMO-2, SUMO-3, yeast Ubc9 and yeast Smt3 were prepared as previously described.,, Gel filtration was performed with an analytical Superose 12 column (GE Healthcare Bio-Sciences) using individual proteins at 100 μM and complexes at equimolar concentrations (100 μM). 200 μL were injected in each case and fractions analyzed by SDS-PAGE and Coomassie blue staining. The buffer used for Ubc9 and SUMO-1/2/3 analysis consisted of 20 mM Tris, pH 8.0, 50 mM NaCl, 1 mM βME. The buffer used for yeast Ubc9 and yeast Smt3 consisted of 20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM βME.
Figure 2
Figure 2. Structural comparisons between Ubc9-SUMO-1 and other E2 complexes
In each instance, the respective E2 molecules were superimposed to generate the resulting composite models. (A) Two orientations of the non-covalent Ubc9-SUMO-1 complex (blue and yellow) in which the SUMO-1 molecule (magenta) from the SUMO-1-RanGAP1/Ubc9/Nup358 structure (PDB 1Z5S) was modeled to illustrate differences in respective SUMO conformations. (B) Two orientations of the same comparison to panel A in which the Nup358/RanBP2 E3 is included to illustrate the overlap between the non-covalent SUMO interaction and a domain within the E3. (C) Structure of the UbcH5-ubiquitin non-covalent complex (PDB 2FUH). (D) Structure of the non-covalent interaction between Mms2 and ubiquitin (PDB 2GMI). (E) Structure of the Ubc12 interaction with the Nedd8 E1 ubiquitin-like domain (PDB 1Y8X). (F) Model of the SUMO E1 ubiquitin-like domain in complex with the non-covalent complex between Ubc9-SUMO-1 to illustrate that the two interactions may partially overlap.
Figure 3
Figure 3. Biochemical and mutational analysis
(A) Gel filtration and SDS-PAGE analysis of Ubc9 mutants to assess non-covalent complex formation with SUMO-2. R13A and R13E (left), R17A and R17E (middle), and F22A (right) were combined with equimolar quantities of SUMO-2 and analyzed by gel filtration. UV chromatograms are shown (top), color coded for the particular protein in question. SDS-PAGE analysis (bottom) for the resulting fractions stained with Coomassie Blue. Indicated proteins labeled (right) with apparent molecular weight markers and molecular weights (left). For comparison, each panel includes chromatogram profiles and SDS-PAGE analysis for wild-type Ubc9, SUMO-2, and wild-type Ubc9-SUMO-2 complex. Gel filtration was conducted similar to Figure 1A, with running buffer consisting of 20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM βME. (B) Time-course for E2-thioester formation and di-SUMO-2 conjugation for Ubc9 wild-type and mutants. Non-reducing lanes indicate formation of thioester-linked SUMO-2 to Ubc9. Reduced lanes indicate conjugated SUMO-2 species. Reactions contained 10 μM SUMO-2, 1 μM Ubc9, 100 nM E1 in reaction buffer (20 mM Tris, pH 8.0, 50 mM NaCl, 1 mM ATP, 5 mM MgCl2, and 0.01% Tween-20) incubated at 37 °C. Samples were taken at indicated time points and stopped with sample loading buffer containing SDS and UREA. Reduced samples contained 100 mM BME. Samples were resolved on SDS-PAGE and blotted for SUMO-2 using SUMO-2 antibody (Invitrogen). (C) Effects of increasing concentrations of Ubc9 on the formation of di-SUMO-2 or poly-SUMO-2 conjugated chains. Reactions contained 100 μM SUMO-2, 100 nM E1, and Ubc9 concentrations ranging from 1–100 μM, in reaction buffer and incubated at 37 °C for 2 hrs. Samples were resolved on SDS-PAGE and blotted for SUMO-2. (D) Time-course for di-SUMO-2 and poly-SUMO-2 formation. Reactions containing 100 μM SUMO-2, 10 μM Ubc9, and 100 nM E1 in reaction buffer were incubated at 37 °C. Samples were taken at indicated time points, resolved on SDS-PAGE and blotted for SUMO-2. SUMO-2 species are indicated for mono-, di-, and poly-SUMO-2 forms. (E) Ability of an E3 ligase, the Nup358/RanBP2 IR1* domain, to catalyze SUMO-2 chain formation. Reactions contained 100 nM Ubc9, 100 nM E1, 400 nM IR1* domain, with SUMO-2 titrated from 10 nM – 10 μM. Samples were incubated at 37 °C in reaction buffer for 180 min, resolved on SDS-PAGE and blotted for SUMO-2. (F) Model for the interactions between Ubc9 and SUMO described in the text. The model depicts Ubc9 thioester-linked to an activated SUMO (SD for donor SUMO) in a non-covalent complex with SUMO (S) able to interact with another molecule of SUMO (SA for acceptor SUMO). This non-covalent SUMO (S) does not interfere with thioester formation or substrate binding; however, it may play a role in SUMO chain extension from a di-SUMO conjugated species.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Kerscher O, Felberbaum R, Hochstrasser M. Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu Rev Cell Dev Biol. 2006;22:159–80. - PubMed
    1. Hershko A, Ciechanover A. The ubiquitin system. Annu Rev Biochem. 1998;67:425–79. - PubMed
    1. Hochstrasser M. There’s the rub: a novel ubiquitin-like modification linked to cell cycle regulation. Genes Dev. 1998;12:901–7. - PubMed
    1. Laney JD, Hochstrasser M. Substrate targeting in the ubiquitin system. Cell. 1999;97:427–30. - PubMed
    1. Schwartz DC, Hochstrasser M. A superfamily of protein tags: ubiquitin, SUMO and related modifiers. Trends Biochem Sci. 2003;28:321–8. - PubMed

Publication types

Associated data