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: Our binding assays showed that INO80GDEU decreases the affinity of UCH-L5 for substrates (Figures 2A and 2B). To understand this effect, we analyzed how INO80GDEU affects UCH-L5 conformation (Figure 1) in more detail. INO80GDEU alters the ULD domain’s relative position and conformation in two specific ways. First, helix α9 and α10 are tilted by ∼30° compared to the active ULD conformation, and, second, the C-terminal end of helix α10 is bent toward the CD (Figure 4A). As a result, sections of the ULD and INO80GDEU occupy the canonical ubiquitin-binding exosites on UCH-L5 and thus prevent substrate docking (Figure 4A). The blockage of the exosites by the INO80GDEU complex presents a structural rationale for the decreased substrate binding that we observed, and provides a simple yet unexpectedly striking explanation of the INO80GDEU inhibition mechanism.
Affiliation: Division of Biochemistry and Cancer Genomics Center, The Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, the Netherlands.