Abstract
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.
Highlights
RWD is a conserved domain in human proteome with unknown function
We found that the RWD domain is an E2-interacting module, and the mode of interaction observed here could represent how an RWD domain interacts with an E2 in ubiquitin-like modifications
Effect of the RWD Domain on SUMOylation—We investigated the effect of RWD on enhancing the formation of Ubc91⁄7SUMO-1 thioester conjugates (Fig. 1B), which is consistent with the previous report [2]
Summary
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 Ubc homodimer and conducted a biochemical investigation. Conclusion: The RWD domain binds to a Ubc surface that must interact with E1, E3, and SUMO. An RWD domain is a well conserved domain found through bioinformatic analysis of the human proteome sequence; its function has been unknown. 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 Ubc is located near the N terminus of Ubc 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. An RWD domain-containing protein, known as RSUME or RWDD3, was reported to affect post-translational modifications by the small ubiquitin-like modifiers (SUMO).. Tel.: 626-930-5408; Fax: 626-301-8186; E-mail: ychen@ coh.org. 2 The abbreviations used are: SUMO, small ubiquitin-like modifier; UFD, ubiquitin fold domain; HSQC, heteronuclear single quantum coherence; CSP, chemical shift perturbation
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