br STAR Methods br Author Contributions br Acknowledgments T

STAR★Methods
Author Contributions
Acknowledgments The authors thank Monika Kuhn for excellent technical assistance and Dr. Antje Schäfer for valuable scientific input. H.S. was supported by the DFG (FZ 82; Graduate School of Life Sciences and SCHI 425-6/1), and A.V.S. by Northwestern University and the Pew Charitable Trusts as Pew Scholar in the Biomedical Sciences. We thank Dr. Titia Sixma and Dr. Sonja Lorenz for kindly providing the plasmid constructs for human UBA1 and ubiquitin, respectively. We also thank BESSY and ESRF for synchrotron beamtime and support.
Introduction Aberrations in post-translational modifications by ubiquitin or ubiquitin-like citco (Ubl), such as the small ubiquitin-like modifiers (SUMO), are associated with the pathogenesis of life-threatening diseases, such as cancer (Sarge and Park-Sarge, 2011, Zhu et al., 2010), neurodegenerative disorders (Steffan et al., 2004, Subramaniam et al., 2009), and viral infection (Jaber et al., 2009, Kim et al., 2010). For example, multiple studies indicate that SUMOylation is dysregulated in many types of cancers and that the SUMO-activating enzyme (SAE, SUMO E1) could be a potential target to inhibit c-Myc- and KRas-dependent oncogenesis (He et al., 2017, Kessler et al., 2012, Luo et al., 2009, Yu et al., 2015) and reduce cancer cell stemness and resistance (Bogachek et al., 2016, Du et al., 2016). The activating enzyme catalyzing ubiquitin-like Atg8 and Atg12 modifications in autophagy, known as Atg7, has been shown as an indirect target for KRas-dependent oncogenesis (Guo et al., 2013, Rosenfeldt et al., 2013). Despite the importance of Ubl modifications in dysregulated signaling pathways in diseased cells, only a handful of U.S. Food and Drug Administration-approved drugs targeting this type of post-translational modifications have been developed. This deficiency illustrates knowledge gaps in targeting these enzymes by small molecules and underscores the need to discover novel chemotypes and mechanisms to inhibit Ubl modifications. An Ubl modification requires several steps that are catalyzed by three enzymes, referred to as E1 (activating enzyme), E2 (conjugation enzyme), and E3 (ligase). The SUMO E1 is a heterodimer of SAE1 and Uba2 (also known as SAE2). In brief, an Ubl is first activated by E1 through ATP hydrolysis and forms a thioester conjugate with E1. The Ubl is then transferred to E2, forming a thioester conjugate with E2. Finally, the Ubl is transferred to target proteins, a step usually catalyzed by an E3. Usually, Ubl modifications add new docking sites to target proteins. For example, SUMO modifications enables new protein-protein interactions through the SUMO-interacting motif in receptor proteins (Song et al., 2004, Song et al., 2005). At least three members of the SUMO family (SUMO1, 2, and 3) are ubiquitin-like proteins that can conjugate to other cellular proteins by a biochemical mechanism similar to ubiquitylation (Hay, 2005, Sarge and Park-Sarge, 2009, Yeh, 2009). Currently, the only known mechanism to inhibit the E1 enzymes targets their ATP-binding sites (Brownell et al., 2010, Soucy et al., 2009).