Abstract
Post-translational modification of proteins by ubiquitin and ubiquitin-like modifiers, such as SUMO, are key events in protein homeostasis or DNA damage response. Smc5/6 is a nuclear multi-subunit complex that participates in the recombinational DNA repair processes and is required in the maintenance of chromosome integrity. Nse2 is a subunit of the Smc5/6 complex that possesses SUMO E3 ligase activity by the presence of a SP-RING domain that activates the E2~SUMO thioester for discharge on the substrate. Here we present the crystal structure of the SUMO E3 ligase Nse2 in complex with an E2-SUMO thioester mimetic. In addition to the interface between the SP-RING domain and the E2, the complex reveals how two SIM (SUMO-Interacting Motif) -like motifs in Nse2 are restructured upon binding the donor and E2-backside SUMO during the E3-dependent discharge reaction. Both SIM interfaces are essential in the activity of Nse2 and are required to cope with DNA damage.
Highlights
Post-translational modification of proteins by ubiquitin and ubiquitin-like modifiers, such as SUMO, are key events in protein homeostasis or DNA damage response
To gain insights into the mechanism of SUMO E3 ligases, we have reconstituted the complex between yeast Smc5/Nse[2] with an E2-SUMO thioester mimetic, which contains a stable peptide bond between E2 and SUMO, instead of the labile natural thioester bond
We will use SUMO to refer to yeast Smt[3] and E2 to yeast Ubc[9] throughout the text. Such E2-SUMO thioester mimetic has been engineered based on previous work[17,43], by the substitution of Ala[129] to lysine in a location next to the active site Cys[93] in Ubc[9], and Lys[153] for arginine to prevent unwanted E2 SUMOylation[29,44]
Summary
Post-translational modification of proteins by ubiquitin and ubiquitin-like modifiers, such as SUMO, are key events in protein homeostasis or DNA damage response. In addition to the interface between the SP-RING domain and the E2, the complex reveals how two SIM (SUMO-Interacting Motif) -like motifs in Nse[2] are restructured upon binding the donor and E2-backside SUMO during the E3-dependent discharge reaction Both SIM interfaces are essential in the activity of Nse[2] and are required to cope with DNA damage. The SUMO transfer from E2 to the substrate can be enhanced by the action of E3 ligases, which facilitate substrate binding while increasing the catalytic rate for the E2~SUMO discharge on the substrate Such E3-dependent stimulation of the E2~SUMO thioester discharge is mechanistically conducted by the stabilization of a closed or active conformation of the E2~SUMO when binding the E3 ligase[6]. E2s can employ a similar mechanism to stabilize a closed conformation in the absence of E39
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