PDBsum entry 2vrr

Cell cycle/ligase

PDB id: 2vrr

Contents

Protein chains

157 a.a.

79 a.a.

Ligands

FMT ×4

Metals

_NA

Waters ×192

References listed in PDB file

Key reference

Title

Ubc9 sumoylation regulates sumo target discrimination.

Authors

P.Knipscheer, A.Flotho, H.Klug, J.V.Olsen, W.J.Van dijk, A.Fish, E.S.Johnson, M.Mann, T.K.Sixma, A.Pichler.

Ref.

Mol Cell, 2008, 31, 371-382. [DOI no: 10.1016/j.molcel.2008.05.022]

PubMed id

18691969

Abstract

Posttranslational modification with small ubiquitin-related modifier, SUMO, is a widespread mechanism for rapid and reversible changes in protein function. Considering the large number of known targets, the number of enzymes involved in modification seems surprisingly low: a single E1, a single E2, and a few distinct E3 ligases. Here we show that autosumoylation of the mammalian E2-conjugating enzyme Ubc9 at Lys14 regulates target discrimination. While not altering its activity toward HDAC4, E2-25K, PML, or TDG, sumoylation of Ubc9 impairs its activity on RanGAP1 and strongly activates sumoylation of the transcriptional regulator Sp100. Enhancement depends on a SUMO-interacting motif (SIM) in Sp100 that creates an additional interface with the SUMO conjugated to the E2, a mechanism distinct from Ubc9 approximately SUMO thioester recruitment. The crystal structure of sumoylated Ubc9 demonstrates how the newly created binding interface can provide a gain in affinity otherwise provided by E3 ligases.

Figure 6.
Figure 6. Crystal Structure of Ubc9^*SUMO
(A) The Ubc9^*SUMO structure. Covalent linkage between Ubc9 Lys14 and the SUMO C terminus.
(B) The interface between Ubc9 and SUMO (H bonds: gray dotted lines, interface side chains as sticks).
(C) Superposition of Ubc9^*SUMO and E2-25K^*SUMO.
(D) Buried surface area between E2 and SUMO plotted against the sequences of Ubc9 and E2-25K.
(E) Model of the Ubc9^Δhairpin mutant. Deleted β-hairpin is in gray.
(F) Thioester formation for Ubc9^Δhairpin (670 nM) versus Ubc9^Δhairpin*SUMO (670 nM) assay as in Figure 3, with 260 nM Aos1-Uba2, 6.7 μM SUMO, and immunoblotting with α-Ubc9. Colors are as follows: blue Ubc9 (yellow catalytic Cys) with yellow SUMO, cyan E2-25K with pink SUMO.

Figure 7.
Figure 7. Model of Ubc9 Target Discrimination via Gain in Affinities
The increased affinity can be a result of an additional binding interface mediated by an E3 (right panel) or by direct interaction of the target with Ubc9 (either free, thioester, or conjugated to SUMO) (left panel). (Top) Cartoon of Ubc9 thioester-target interactions, mediated either through the target, conjugated SUMO, or an E3 ligase. (Bottom) Shown are the Ubc9 surfaces involved in these interactions; interaction between the catalytic cleft in Ubc9 with the SUMO consensus motif (light green). Additional binding interfaces (dark green) enhance affinity and hence modification. Sumoylation of Ubc9 increases the interaction of targets with a SIM (Song et al., 2005) at a defined distance as seen for Sp100 in this study (left panel). Alternatively, the target can recruit the SUMO vert, similar Ubc9 thioester (picture according to Reverter and Lima [2005]) in, e.g., Daxx and TDG (Figure S5 and ([Hochstrasser, 2007], [Lin et al., 2006] and [Takahashi et al., 2005]). RanGAP1 itself has two binding interfaces (Bernier-Villamor et al., 2002) (middle panel) and is an unusually efficient SUMO substrate. ELK1 contains a negatively charged amino acid-dependent sumoylation motif (Yang et al., 2006). Similarly, the SP-RING E3 ligases (model based on E2-RING complex in the ubiquitin SCF system [Zheng et al., 2000]) stabilize the interaction between the target and Ubc9, resulting in enhanced modification (right panel).

The above figures are reprinted by permission from Cell Press: Mol Cell (2008, 31, 371-382) copyright 2008.