Biochemical and structural insights into SUMO conjugation and recognition

The small ubiquitin-like modifier, SUMO is a ∼10 kDa polypeptide, which is covalently attached to lysine residues of target proteins in a manner mechanistically analogous to ubiquitylation. The enzymes involved in the SUMO modification pathway identified to date consist of a single E1 activating enzyme, a single E2 conjugating enzyme and two types of E3 ligases. A subset of SUMO modifications occur at lysine residues residing within a psi-K-X-E SUMO consensus motif (where psi represents a hydrophobic residue, K, the site of covalent attachment, X, any amino acid, and E, a residue with an acidic side-chain) that is recognized by the E2 Ubc9.;Phosphorylation of a serine residue in a SP-motif proximal to the SUMO consensus site by proline-directed kinases such as Cdk5 was recently reported to enhance SUMO conjugation to a number of target proteins. The stretch of amino acid residues comprising the SUMO consensus site and the SP motif (psi-K-X-E-X-X-S-P, where S represents the phosphorylated serine residue and P a proline residue) has been named the phosphorylation-dependent SUMO conjugation motif (PDSM). This extended motif couples two post-translational modifications to provide an additional regulatory element for SUMO modification. Using the transcription factor MEF2A as a model PDSM substrate, I attempted to discern the molecular basis for phosphorylation-dependent SUMO conjugation. Structural analysis revealed a previously uncharacterized basic surface on Ubc9 important for SUMO conjugation to phosphorylated MEF2A, but not non-phosphorylated MEF2A, and mutations within this surface abrogate phosphorylation-dependent SUMO conjugation in vitro and in vivo. Mutant Ubc9 isoforms defective for the ability to promote phosphorylation-dependent SUMO conjugation also impair postsynaptic differentiation in organotypic cerebellar slices.;In addition to covalent attachment, SUMO also non-covalently interacts with a number of proteins that contain a hydrophobic SUMO interacting motif (SIM). One such protein is the anti-recombinogenic helicase, Srs2, which also interacts with the eukaryotic processivity factory PCNA during S phase to prevent homologous recombination. Therefore, SUMO modification of PCNA enhances the affinity of PCNA for Srs2 and I attempted to characterize the interactions between these three proteins. Biochemical analysis showed that a (SIM) at the Srs2 C-terminus was both necessary and sufficient for interaction with SUMO and that the eleven C-terminal Srs2 residues were necessary but not sufficient to interact with PCNA. Using a catalytic domain of the SP-RING E3 ligase Siz1, preparative amounts of SUMO modified PCNA were obtained and purified to near homogeneity. In addition, crystal structures of trimeric and monomeric PCNA conjugated to SUMO were solved at resolutions of 2.3 A and 2.8 A respectively.;In the course of my doctoral research I was able to identify a surface on the SUMO E2 Ubc9 involved in MEF2A PDSM discrimination. Also, I biochemically characterized interactions between PCNA, SUMO, and Srs2 and purified preparative quantities of SUMO modified PCNA for crystallographic experiments, which produced two crystal structures of the PCNA SUMO adduct. The experiments outlined in these studies offer novel insights into conjugation and recognition by the small ubiquitin-like modifier, SUMO, which will provide a broader understanding of this post-translational modification pathway.