RWD Domain as an E2 (Ubc9)-Interaction Module.
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.
From the Department of Molecular Medicine and.
Aileen Y
AY
Alontaga
From the Department of Molecular Medicine and.
Nigus D
ND
Ambaye
From the Department of Molecular Medicine and.
Yi-Jia
YJ
Li
From the Department of Molecular Medicine and.
Ramir
R
Vega
From the Department of Molecular Medicine and.
Chih-Hong
CH
Chen
From the Department of Molecular Medicine and.
Krzysztof P
KP
Bzymek
From the Department of Molecular Medicine and.
John C
JC
Williams
the NMR Core Facility, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Weidong
W
Hu
From the Department of Molecular Medicine and ychen@coh.org.
Yuan
Y
Chen
D018360:Crystallography, X-Ray; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D025842:SUMO-1 Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D058207:Sumoylation; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D025842:SUMO-1 Protein; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D000098843:Ubiquitin-Conjugating Enzyme UBC9
E2; RWD; nuclear magnetic resonance (NMR); small ubiquitin-like modifier (SUMO); sumoylation; ubiquitylation (ubiquitination); x-ray crystallography