RWD Domain as an E2 (Ubc9)-Interaction Module

• Published in: The Journal of biological chemistry Vol. 290; no. 27; pp. 16550 - 16559
• Main Authors: Alontaga, Aileen Y., Ambaye, Nigus D., Li, Yi-Jia, Vega, Ramir, Chen, Chih-Hong, Bzymek, Krzysztof P., Williams, John C., Hu, Weidong, Chen, Yuan
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 03.07.2015; American Society for Biochemistry and Molecular Biology
• Subjects: Crystallography, X-Ray; Enzymology; Humans; Kinetics; Models, Molecular; nuclear magnetic resonance (NMR); Protein Binding; Protein Structure, Tertiary; RWD; small ubiquitin-like modifier (SUMO); SUMO-1 Protein - chemistry; SUMO-1 Protein - genetics; SUMO-1 Protein - metabolism; Sumoylation; Ubiquitin-Conjugating Enzymes - chemistry; Ubiquitin-Conjugating Enzymes - genetics; Ubiquitin-Conjugating Enzymes - metabolism; Ubiquitin-Protein Ligases - chemistry; Ubiquitin-Protein Ligases - genetics; Ubiquitin-Protein Ligases - metabolism; ubiquitylation (ubiquitination); x-ray crystallography; ubiquitylation (ubiquitination); x-ray crystallography; sumoylation; nuclear magnetic resonance (NMR); small ubiquitin-like modifier (SUMO); E2; RWD
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1074/jbc.M115.644047

An RWD domain is a well conserved domain found through bioinformatic analysis of the human proteome sequence; however, its function has been unknown. Ubiquitin-like modifications require the catalysis of three enzymes generally known as E1, E2, and E3. We solved the crystal structure of the E2 for the small ubiquitin-like modifiers (SUMO) in complex with an RWD domain and confirmed the structure using solution NMR analysis. The binding surface of RWD on Ubc9 is located near the N terminus of Ubc9 that is known to be involved in noncovalent binding of the proteins in the conjugation machinery, including a domain of E1, SUMO, and an E3 ligase. NMR data indicate that the RWD domain does not bind to SUMO and E1. The interaction between RWD and Ubc9 has a Kd of 32 ± 4 μm. Consistent with the structure and binding affinity and in contrast to a previous report, the RWD domain and RWDD3 have minimal effects on global SUMOylation. The structural and biochemical information presented here forms the basis for further investigation of the functions of RWD-containing proteins. Background: RWD is a conserved domain in human proteome with unknown function. Results: We solved the crystal structure of an RWD domain in complex with a Ubc9 homodimer and conducted a biochemical investigation. Conclusion: The RWD domain binds to a Ubc9 surface that also must interact with E1, E3, and SUMO. Significance: This study establishes a function for the evolutionary conserved RWD domain.