NMR Studies of Ubc9 Mutant Identify Structural Basis for SUMO Target Selection

Abstract

BackgroundThe regulated biosynthesis, trafficking, interaction, and degradation of proteins are essential for normal cell growth. SUMO is a conserved ubiquitin‐like protein that is conjugated to a multitude of cellular proteins and mediates these functions. The transient SUMO modification (SUMOylation) of proteins affects protein function, allowing cells to respond to environmental stress without the need for de novo protein synthesis. Not surprisingly, dysregulation of SUMO conjugation has been directly implicated in the pathogenesis of human cancer and in modulating cellular responses to chemotherapeutics. Yet, the underlying determinants of SUMO target specificity.Materials and MethodsSUMO conjugation is accomplished by an ATP‐dependent cascade of enzymes, which includes a single activating enzyme (SAE1/2 heterodimer), SUMO‐1 or SUMO‐2/3, and a single conjugating enzyme (Ubc9). We used yeast genetics, biochemical SUMO conjugation assays, and NMR spectroscopy to define the basis for alterations in substrate selection and SUMO chain formation.ResultsOur studies suggest that the altered substrate specificity induced by the Ubc9P123L mutant enhances cell sensitivity to a wide range of DNA damaging agents. NMR analyses of wild‐type and mutant Ubc9 enzymes have identified critical flexibility in Ubc9 necessary for SUMO target selection. In parallel, biochemical and genetic studies demonstrate the impact this dysregulation of Ubc9 function has on SUMO conjugation and cell viability.ConclusionsOur results provide new insights into the structural features of Ubc9 that underlie substrate selection and the impact of SUMO chain linkages on cell viability. These findings constitute a paradigm shift in our understanding of SUMOylation and the potential contribution of SUMO chain linkages to tumorigenesis and chemotherapeutic drug response.