Mechanism of UCH-L5 activation and inhibition by DEUBAD domains in RPN13 and INO80G.
Bottom Line: In this process, large conformational changes create small but highly specific interfaces that mediate activity modulation of UCH-L5 by altering the affinity for substrates.Our results establish how related domains can exploit enzyme conformational plasticity to allosterically regulate DUB activity.These allosteric sites may present novel insights for pharmaceutical intervention in DUB activity.
Affiliation: Division of Biochemistry and Cancer Genomics Center, The Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, the Netherlands.Show MeSH
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Mentions: To study the regulation of UCH-L5 by DEUBAD domains, we purified human UCH-L5 in complex with the DEUBAD domains of RPN13 (amino acid [aa] 265–388, referred to as RPN13DEU) and INO80G (aa 39–170, referred to as INO80GDEU) (Figure 1A). We measured the catalytic activity of these complexes towards the minimal substrate ubiquitin-7-amido-4-methylcoumarin (Ub-AMC) (Dang et al., 1998; El Oualid et al., 2010) in comparison to full-length UCH-L5 alone (U) and its isolated CD. In line with previous data, we found that the DEUBAD domain of RPN13 activates UCH-L5 (UR) (Figure 1B; Hamazaki et al., 2006; Qiu et al., 2006; Yao et al., 2006). Since the UCH-L5 CD is more active than the full-length alone, the ULD domain partially inhibits activity (Yao et al., 2006). However, in the presence of RPN13DEU, UCH-L5 is significantly more active than the UCH-L5 CD, and, therefore, RPN13DEU does more than simply remove autoinhibition (Figure 1B). Strikingly, INO80GDEU severely inhibits activity under these conditions (UI) (Figure 1B).
Affiliation: Division of Biochemistry and Cancer Genomics Center, The Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, the Netherlands.