title: Observation of an E2 (Ubc9)-homodimer by crystallography.
abstract: Post-translational modifications by the small ubiquitin-like modifiers (SUMO), in particular the formation of poly-SUMO-2 and -3 chains, regulates essential cellular functions and its aberration leads to life-threatening diseases (Geoffroy and Hay, 2009) [1]. It was shown previously that the non-covalent interaction between SUMO and the conjugating enzyme (E2) for SUMO, known as Ubc9, is required for poly-SUMO-2/3 chain formation (Knipscheer et al., 2007) [2]. However, the structure of SUMO-Ubc9 non-covalent complex, by itself, could not explain how the poly-SUMO-2/3 chain forms and consequently a Ubc9 homodimer, although never been observed, was proposed for poly-SUMO-2/3 chain formation (Knipscheer et al., 2007) [2]. Here, we solved the crystal structure of a heterotrimer containing a homodimer of Ubc9 and the RWD domain from RWDD3. The asymmetric Ubc9 homodimer is mediated by the N-terminal region of one Ubc9 molecule and a surface near the catalytic Cys of the second Ubc9 molecule (Fig. 1A). This N-terminal surface of Ubc9 that is involved in the homodimer formation also interacts with the RWD domain, the ubiquitin-fold domain of the SUMO activating enzyme (E1), SUMO, and the E3 ligase, RanBP2 (Knipscheer et al., 2007; Tong et al.. 1997; Tatham et al., 2005; Reverter and Lima, 2005; Capili and Lima, 2007; Wang et al., 2009, 2010; Wang and Chen, 2010; Alontaga et al., 2015) [2], [3], [4], [5], [6], [7], [8], [9], [10]. The existence of the Ubc9 homodimer in solution is supported by previously published solution NMR studies of rotational correlation time and chemical shift perturbation (Alontaga et al., 2015; Yuan et al., 1999) [10], [11]. Site-directed mutagenesis and biochemical analysis suggests that this dimeric arrangement of Ubc9 is likely important for poly-SUMO chain formation (Fig. 1B and C). The asymmetric Ubc9 homodimer described for the first time in this work could provide the critical missing link in the poly-SUMO chain formation mechanism. The data presented here are related to the research article entitled, "RWD domain as an E2 (Ubc9) interaction module" (Alontaga et al., 2015) [10]. The data of the crystal structure has been deposited to RCSB protein data bank with identifier: 4Y1L.
authors.affiliation: Department of Molecular Medicine, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, CA 91010, United States.
authors.forename: Aileen Y
authors.initials: AY
authors.lastname: Alontaga
authors.affiliation: Department of Molecular Medicine, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, CA 91010, United States.
authors.forename: Nigus D
authors.initials: ND
authors.lastname: Ambaye
authors.affiliation: Department of Molecular Medicine, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, CA 91010, United States.
authors.forename: Yi-Jia
authors.initials: YJ
authors.lastname: Li
authors.affiliation: Department of Molecular Medicine, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, CA 91010, United States.
authors.forename: Ramir
authors.initials: R
authors.lastname: Vega
authors.affiliation: Department of Molecular Medicine, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, CA 91010, United States.
authors.forename: Chih-Hong
authors.initials: CH
authors.lastname: Chen
authors.affiliation: Department of Molecular Medicine, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, CA 91010, United States.
authors.forename: Krzysztof P
authors.initials: KP
authors.lastname: Bzymek
authors.affiliation: Department of Molecular Medicine, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, CA 91010, United States.
authors.forename: John C
authors.initials: JC
authors.lastname: Williams
authors.affiliation: Department of Molecular Medicine, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, CA 91010, United States.
authors.forename: Weidong
authors.initials: W
authors.lastname: Hu
authors.affiliation: Department of Molecular Medicine, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, CA 91010, United States.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Chen
keywords: E1; Modifications; Poly-SUMO chain; RWD; SUMO; Ubc9; Ubiquitin; Ubiquitin-like