Structural and biochemical studies of the SUMO conjugation pathway

SUMO (Small Ubiquitin-like Modifier), a member of the Ubiquitin (Ub) and Ubiquitin-like (Ubl) superfamily is conjugated to substrate proteins via a lysine residue on the substrate. Conjugation of SUMO requires a concerted action of the activating enzyme E1, conjugating enzyme E2, and E3 ligases. The E2 enzyme Ubc9, unique among conjugating E2s, exhibits specificity and interacts directly with many substrates containing the consensus motif (psi-K-x-D/E). E3 ligases, though dispensable in vitro, may serve to enhance conjugation and/or alter substrate specificity. Several studies of Ub and Ubl pathways have provided invaluable insights into mechanism of conjugation and substrate specificity in the pathways. However, the mechanism of the E2 enzymes and principles of substrate selection by the SP-RING family of SUMO E3 ligases have remained largely obscure.;To uncover the mode of action for the E2s, we utilized Ubc9, as a model E2 enzyme. Genetic complementation assays in combination with detailed kinetic analysis revealed three Ubc9 side chains that were critical for Ubc9 function in vivo and in vitro. X-ray structures for wild-type and mutant human Ubc9-RanGAP1 complexes showed partial loss of contacts to the substrate lysine in mutant complexes. Computational analysis predicted pK perturbations for the substrate lysine, and Ubc9 mutations weakened pK suppression through improper side chain coordination. Biochemical studies with various substrates confirmed both structural and computational predictions. The mechanism uncovered for lysine activation may be conserved among other E2 family members.;The SP-RING E3 ligase, Siz1 is required for SUMO modification of PCNA during S-phase on lysine 164, a non-consensus lysine residue. The X-ray structure of a catalytically active Siz1 at 2.6 A resolution in combination with biochemical studies revealed that the SP-RING and C-terminal domains are required for binding and activation of the E2∼SUMO thioester complex while the N-terminal PINIT domain is required for directing SUMO conjugation to lysine 164 whose modification depends on Siz1 E3 ligase. Mutational analysis revealed surfaces on Siz1 and PCNA required for efficient SUMO modification of PCNA in vitro and in vivo. This study demonstrated that the Siz1 binds PCNA in a unique configuration that enforces substrate specificity by positioning PCNA lysine 164 into the active site of the E2 Ubc9.