Post-translational modification of proteins by the small ubiquitin-like modifier SUMO regulates nuclear transport, stress response, and signal transduction in eukaryotes. SUMO modification also appears to be essential for cell cycle progression in eukaryotes. Like ubiquitin modification, covalent attachment of SUMO to protein targets occurs on lysine residues.
SUMO modification is reversible, altering protein function through changes in cellular localization, biochemical activation, or through protection from other post-translational modifications. The observation that SUMO can be conjugated to proteins is a relatively new discovery (1996); and while several cellular proteins are known to be modified by SUMO, it remains a continued focus of study to establish SUMO's relevance in signal transduction, particularly in response to cellular stress and disease.
We are focusing on several SUMO processes that include its activation, conjugation, deconjugation, and recognition with various protein. We are utilizing these observations to uncover physiological targets for SUMO conjugation and to study the respective consequences for SUMO modification in vivo.
E1 activation in the SUMO pathway
E1 enzymes facilitate conjugation of ubiquitin and ubiquitin-like proteins through adenylation, thioester transfer within E1, and thioester transfer from E1 to E2 conjugating proteins. We determined structures of human heterodimeric Sae1/Sae2-Mg.ATP and Sae1/Sae2-SUMO-1-Mg.ATP complexes at atomic resolution, and despite the presence of Mg.ATP, the Sae1/Sae2-SUMO-1-Mg.ATP structure revealed a substrate complex insomuch as the SUMO C-terminus remains unmodified within the adenylation site and far from the catalytic cysteine.
This observation suggested that additional changes within the adenylation site may be required to facilitate chemistry prior to adenylation and thioester transfer. In addition, a mechanism for E2 recruitment to E1 was suggested by biochemical and genetic data, each of which supported a direct role for the E1 C-terminal ubiquitin-like domain for E2 recruitment during conjugation.
Substrate recognition in the SUMO pathway
The structural and biochemical basis for E2-dependent protein conjugation was uncovered by analysis of a complex between human Ubc9 and RanGAP1. These studies revealed structural determinants for recognition of consensus SUMO modification sequences found within SUMO conjugated proteins. Reconstitution of the sumoylation process in vitro enabled structural insights into the conjugation mechanism to be probed, and structure-based mutagenesis and biochemical analysis of Ubc9 and RanGAP1 reveal distinct motifs required for substrate binding and SUMO modification of substrates, such as p53, IkB, and RanGAP1. These studies were expanded to an in vivo screen in yeast for Ubc9-mediated conjugation, uncovering three critical E2 residues that were important for function in vivo and for conjugation in vitro. Interestingly, kinetic analysis of the mutant and wild-type Ubc9 suggested a catalytic mechanism for E2-mediated conjugation, at least during SUMO conjugation.
E3 ligase activity in the SUMO pathway
It is generally thought that unique combinations of E2s and E3s ensure specificity and enhance conjugation in the ubiquitin pathway. Since the SUMO pathway includes only one E2 and just a few E3s, it remains unclear how the cell achieves selective modification of a particular substrate. We have been employing both structural and biochemical studies to uncover the basis for substrate specificity using non-RING and RING E3s which differ in their ability to selectively target substrates for modification. One of these studies revealed the kinetic basis for E3 ligase domains within the Nup358/RanBP2 nucleoporin, and structural characterization of a complex between human Nup358/RanBP2, the SUMO E2 Ubc9, and SUMO-1 conjugated RanGAP1 revealed that the E3 catalyzed activity by organizing SUMO and Ubc9 into a more productive complex for conjugation.
Protease activity in the SUMO pathway
Equally important in the SUMO pathway is the process of deconjugation by the Ulp/Senp proteases. We have characterized several of these through the structure determination, biochemical, and genetic analysis of the complexes in yeast between S. cerevisiae Ulp1 and Smt3 and the biochemical and structural basis for SUMO-1, -2, -3 selectivity by the human Senp2 enzyme. In addition, we have also characterzed a 'ulp' type protease that is able to deconjugate Nedd8 rather than SUMO.