UBC9 Mutant Reveals the Impact of Protein Dynamics on Substrate Selectivity and SUMO Chain Linkages.

SUMO, a conserved ubiquitin-like protein, is conjugated to a multitude of cellular proteins to maintain genomic integrity and resist genotoxic stress. Studies of the SUMO E2 conjugating enzyme mutant, UBC9P123L, suggested that altered substrate specificity enhances cell sensitivity to DNA damaging agents. Using nuclear magnetic resonance chemical shift studies, we confirm that the mutation does not alter the core globular fold of UBC9, while 15N relaxation measurements demonstrate mutant-induced stabilization of distinct chemical states in residues near the active site cysteine and substrate recognition motifs. We further demonstrate that the P123L substitution induces a switch from the preferential addition of SUMO to lysine residues in unstructured sites to acceptor lysines embedded in secondary structures, thereby also inducing alterations in SUMO chain linkages. Our results provide new insights regarding the impact that structural dynamics of UBC9 have on substrate selection and specifically SUMO chain formation. These findings highlight the potential contribution of nonconsensus SUMO targets and/or alternative SUMO chain linkages on DNA damage response and chemotherapeutic sensitivity.

Structure of a SUMO-binding-motif mimic bound to Smt3p-Ubc9p: conservation of a non-covalent ubiquitin-like protein-E2 complex as a platform for selective interactions within a SUMO pathway.

The SUMO ubiquitin-like proteins play regulatory roles in cell division, transcription, DNA repair, and protein subcellular localization. Paralleling other ubiquitin-like proteins, SUMO proteins are proteolytically processed to maturity, conjugated to targets by E1-E2-E3 cascades, and subsequently recognized by specific downstream effectors containing a SUMO-binding motif (SBM). SUMO and its E2 from the budding yeast Saccharomyces cerevisiae, Smt3p and Ubc9p, are encoded by essential genes. Here we describe the 1.9 A resolution crystal structure of a non-covalent Smt3p-Ubc9p complex. Unexpectedly, a heterologous portion of the crystallized complex derived from the expression construct mimics an SBM, and binds Smt3p in a manner resembling SBM binding to human SUMO family members. In the complex, Smt3p binds a surface distal from Ubc9's catalytic cysteine. The structure implies that a single molecule of Smt3p cannot bind concurrently to both the non-covalent binding site and the catalytic cysteine of a single Ubc9p molecule. However, formation of higher-order complexes can occur, where a single Smt3p covalently linked to one Ubc9p's catalytic cysteine also binds non-covalently to another molecule of Ubc9p. Comparison with other structures from the SUMO pathway suggests that formation of the non-covalent Smt3p-Ubc9p complex occurs mutually exclusively with many other Smt3p and Ubc9p interactions in the conjugation cascade. By contrast, high-resolution insights into how Smt3p-Ubc9p can also interact with downstream recognition machineries come from contacts with the SBM mimic. Interestingly, the overall architecture of the Smt3p-Ubc9p complex is strikingly similar to recent structures from the ubiquitin pathway. The results imply that non-covalent ubiquitin-like protein-E2 complexes are conserved platforms, which function as parts of larger assemblies involved in many protein post-translational regulatory pathways.