Reply to Function and Structure of the RWD Domain
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This is a response to a letter by Paez-Pereda and Arzt (1)
The Letter to the Editor by Paez-Pereda and Arzt (1) suggested that our recent findings (2) are consistent with their previous findings (3). We disagree.
The cited publication (3) proposed that RSUME interacts with both Ubc9 and SUMO-1 simultaneously, although the binding affinity was not measured, and promoted noncovalent interactions between Ubc9 and SUMO-1 (“RSUME Physically Interacts with Ubc9 and SUMO-1” on page 313, Fig. 4, C and D, and Fig. 7A of Ref. 3). In contrast, our NMR chemical shift perturbation data show that Ubc9 cannot bind to SUMO and RWD simultaneously (2).
In addition, our NMR data support only the mode of interaction observed in the x-ray structure of the RWD-Ubc9 complex (Fig. 2 in Ref. 2) and does not suggest other modes of interaction. In addition, we showed that Ubc9-RWD interaction is not similar to the Ubc13-MMS2 interaction (Fig. 6 of Ref. 2); therefore, we conclude that E2-UEV interactions are not conserved.
Furthermore, we showed that the in vitro biochemical “enhancement effects” of RWD are due to RWD's inhibition of E1-E2 interaction to alleviate the E2 inhibitory effect on E1 catalysis. We could not observe SUMOylation enhancement effects in cells using the constructs obtained from Dr. Arzt's laboratory and the same conditions as described previously (3). In fact, a search of the literature showed that there have been no publications to independently verify their findings published 8 years ago (3).
In conclusion, our study does not support the previous finding (3). Instead, our finding has provided the basis for future studies of the functions of RWD-containing proteins (2).
[1] M. Páez-Pereda,et al. Function and Structure of the RWD Domain , 2015, The Journal of Biological Chemistry.
[2] Weidong Hu,et al. RWD Domain as an E2 (Ubc9)-Interaction Module* , 2015, The Journal of Biological Chemistry.
[3] F. Holsboer,et al. RSUME, a Small RWD-Containing Protein, Enhances SUMO Conjugation and Stabilizes HIF-1α during Hypoxia , 2007, Cell.