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Comparative Study
. 2007 Jun 6;26(11):2797-807.
doi: 10.1038/sj.emboj.7601711. Epub 2007 May 10.

Noncovalent interaction between Ubc9 and SUMO promotes SUMO chain formation

Puck Knipscheer  1 , Willem J van Dijk, Jesper V Olsen, Matthias Mann, Titia K Sixma
Affiliations
Comparative Study

Noncovalent interaction between Ubc9 and SUMO promotes SUMO chain formation

Puck Knipscheer et al. EMBO J. .

Abstract

The ubiquitin-related modifier SUMO regulates a wide range of cellular processes by post-translational modification with one, or a chain of SUMO molecules. Sumoylation is achieved by the sequential action of several enzymes in which the E2, Ubc9, transfers SUMO from the E1 to the target mostly with the help of an E3 enzyme. In this process, Ubc9 not only forms a thioester bond with SUMO, but also interacts with SUMO noncovalently. Here, we show that this noncovalent interaction promotes the formation of short SUMO chains on targets such as Sp100 and HDAC4. We present a crystal structure of the noncovalent Ubc9-SUMO1 complex, showing that SUMO is located far from the E2 active site and resembles the noncovalent interaction site for ubiquitin on UbcH5c and Mms2. Structural comparison suggests a model for poly-sumoylation involving a mechanism analogous to Mms2-Ubc13-mediated ubiquitin chain formation.

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Figures

Figure 1
Figure 1
Structure of noncovalent Ubc9–SUMO1 complex. (A) Cartoon representation of the Ubc9–SUMO1 crystal structure, Ubc9 in blue and SUMO1 in yellow. The catalytic residue is shown in sticks. (B) Details of the interaction site. Residues of Ubc9 (upper panel) and SUMO1 (lower panel) involved in the interaction shown in sticks, counterpart shown as surface representation. (C) Close-up of Ubc9–SUMO1 interaction. (D) Superposition of the UbcH5c–Ubiquitin complex (purple and green, respectively) and the Ubc9–SUMO1 complex. Only UbcH5c and Ubc9 were used for the superposition, the angle between ubiquitin and SUMO is indicated. (E) Sequence alignment of Ubc9, UbcH5c and Mms2 showing secondary structure elements of Ubc9. Residues that loose at least 20% of their solvent accessible surface area upon complex formation with SUMO/ubiquitin are shown on a yellow background. (F) Sequence alignment of SUMO1, SUMO2 and ubiquitin with secondary structure elements of SUMO1 on top. Residues of SUMO1 involved in Ubc9 interaction (determined as in (E)) are shown on a blue background and homologous residues in SUMO2 are framed. Residues of ubiquitin involved in UbcH5c or Mms2 interaction have a purple background.
Figure 2
Figure 2
SUMO1 and SUMO2 have similar affinity for Ubc9. (A) Isothermal calorimetry data for noncovalent interaction between Ubc9 and SUMO1. Raw (upper panel) and processed (lower panel) data for 7 μM SUMO titrated with 12 μl injections of 70 μM Ubc9. Processed data points were fitted to a model describing a single set of binding sites. Thermodynamic parameters for the interaction are ΔH=−5.96±0.2 kcal/mol and −TΔS=−3.87 kcal/mol (B) Chromatograms of analytical gel filtration runs for Ubc9 with SUMO1 (upper panel) and Ubc9 with SUMO2 (lower panel). Runs of single proteins contained 60 μM Ubc9 or 300 μM SUMO1/2, complex runs contained 50 μM Ubc9 and 100 μM SUMO1/2. Ubc9–SUMO2 chromatogram has been scaled (see second y-axis) because SUMO2 contains few aromatic residues and therefore has low signals. (C) Gel-filtration-based shift assays visualized by Western blot analysis using anti-Ubc9. For SUMO2 (left panel), as well as for SUMO1 (right panel), several gel-filtration runs were performed with a constant Ubc9 and increasing SUMO concentrations (molar ratio is depicted on the left). Seven consecutive fractions ranging in elution volume from 1.15 to 1.4 ml are loaded on the gel for both SUMO1 and SUMO2.
Figure 3
Figure 3
Mutants that interrupt noncovalent Ubc9–SUMO binding but not thioester formation. (A) Thioester formation followed in time for Ubc9 and SUMO1 wild type and mutants. Concentrations were: 100 nM E1, 900 nM Ubc9 and 3 μM SUMO1. (B) Thioester formation assay comparing SUMOWT with SUMOE67R. Concentrations were: 200 nM E1, 1.4 μM Ubc9 and 15 μM SUMO1. (C) Thioester formation assay as in (B) comparing Ubc9WT with Ubc9H20D. (D) Noncovalent binding studied using analytical gel-filtration for Ubc9H20D with SUMO1. Curve indicated as ‘Ubc9+SUMO1' was 44 μM Ubc9H20D and 108 μM SUMO1, ‘Ubc9+more SUMO1' was 27 μM Ubc9H20D and 136 μM SUMO1. Free Ubc9H20D and the complex between Ubc9WT and SUMO are indicated for clarity.
Figure 4
Figure 4
Noncovalent Ubc9–SUMO interaction promotes SUMO chain formation. (A) Free SUMO chain formation for SUMO1, SUMO2 and SUMO2K11R with Ubc9WT and Ubc9H20D. Formation of SUMO chains is followed in time using SUMO1 or SUMO2 antibodies as indicated. Concentrations were: 100 nM E1, 400 nM Ubc9 and 20 μM SUMO. Note that Ubc9H20D is also strikingly different in forming higher order Uba2 conjugates that occur as a side effect of the reaction. Uba2 is a known target for sumoylation (Zhao et al, 2004; Hannich et al, 2005). The presence of SUMO-modified Uba2 was confirmed by mass spectrometry (data not shown). (B) Sumoylation of Sp100 with SUMO2 comparing Ubc9WT with Ubc9H20D. Concentrations were: 830 nM GST-Sp100, 175 nM E1, 400 nM Ubc9 and 20 μM SUMO2 and detection was with anti-GST. (C) SUMO chain formation on SP100 with SUMO2, comparing Ubc9WT with Ubc9H20D. Concentrations are identical to (B), except E1 was 150 nM, formation of GST-Sp100*SUMO2 conjugates is followed in time using either a GST antibody (upper panel) or a SUMO2 antibody (lower panel). (D) SUMO chain formation on Sp100 comparing several mutant proteins. Concentrations were: 1.3 μM GST-Sp100, 10 nM E1, 300 nM Ubc9 and 10 μM SUMO and detection was with anti-GST or anti-SUMO2. (E) Assay as in (C) but using GST-HDAC4 as a target and an HDAC4 antibody for the upper panel.
Figure 5
Figure 5
A model for SUMO chain formation. (A) K63 ubiquitin chain formation by the Mms2-Ubc13 heterodimer (Eddins et al, 2006)(PDB-code: 2GMI). Close-up shows Mms2 K63 in sticks and the Ubc13 thioester active site. (B) Structural model for SUMO chain formation. The noncovalent Ubc9–SUMO1 complex was superposed on Mms-Ub and another Ubc9 molecule was superposed on Ubc13 (A). Thioester SUMO was modeled by superposition on ubiquitin replacing the C-terminal tail with the one from ubiquitin. Close-up shows acceptor SUMO N-terminus and donor SUMO-Ubc9 thioester active site. Dotted line represents the N-terminal tail of SUMO that is not present in the structure.
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