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Mechanism of UCH-L5 activation and inhibition by DEUBAD domains in RPN13 and INO80G.

Sahtoe DD, van Dijk WJ, El Oualid F, Ekkebus R, Ovaa H, Sixma TK - Mol. Cell (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry and Cancer Genomics Center, The Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, the Netherlands.

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Inhibition Mechanism INO80GDEU(A) The ubiquitin-docking site on UCH-L5 is blocked as a result of INO80GDEU-induced conformational changes of the ULD. RPN13DEU is removed for clarity.(B and C) The FRF hairpin mimics the ubiquitin β1-β2 hairpin to bind the Leu38 pocket.(D) In contrast to the activated state, helix α8 in UCH-L5 adopts an extended state in the INO80GDEU complex to bury Ile216.(E) The ULD anchor interaction is disrupted in the INO80GDEU complex due to ULD tilting and helix α10 bending, establishing intramolecular stacking of Arg287 on Trp36.
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fig4: Inhibition Mechanism INO80GDEU(A) The ubiquitin-docking site on UCH-L5 is blocked as a result of INO80GDEU-induced conformational changes of the ULD. RPN13DEU is removed for clarity.(B and C) The FRF hairpin mimics the ubiquitin β1-β2 hairpin to bind the Leu38 pocket.(D) In contrast to the activated state, helix α8 in UCH-L5 adopts an extended state in the INO80GDEU complex to bury Ile216.(E) The ULD anchor interaction is disrupted in the INO80GDEU complex due to ULD tilting and helix α10 bending, establishing intramolecular stacking of Arg287 on Trp36.

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.


Mechanism of UCH-L5 activation and inhibition by DEUBAD domains in RPN13 and INO80G.

Sahtoe DD, van Dijk WJ, El Oualid F, Ekkebus R, Ovaa H, Sixma TK - Mol. Cell (2015)

Inhibition Mechanism INO80GDEU(A) The ubiquitin-docking site on UCH-L5 is blocked as a result of INO80GDEU-induced conformational changes of the ULD. RPN13DEU is removed for clarity.(B and C) The FRF hairpin mimics the ubiquitin β1-β2 hairpin to bind the Leu38 pocket.(D) In contrast to the activated state, helix α8 in UCH-L5 adopts an extended state in the INO80GDEU complex to bury Ile216.(E) The ULD anchor interaction is disrupted in the INO80GDEU complex due to ULD tilting and helix α10 bending, establishing intramolecular stacking of Arg287 on Trp36.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4352763&req=5

fig4: Inhibition Mechanism INO80GDEU(A) The ubiquitin-docking site on UCH-L5 is blocked as a result of INO80GDEU-induced conformational changes of the ULD. RPN13DEU is removed for clarity.(B and C) The FRF hairpin mimics the ubiquitin β1-β2 hairpin to bind the Leu38 pocket.(D) In contrast to the activated state, helix α8 in UCH-L5 adopts an extended state in the INO80GDEU complex to bury Ile216.(E) The ULD anchor interaction is disrupted in the INO80GDEU complex due to ULD tilting and helix α10 bending, establishing intramolecular stacking of Arg287 on Trp36.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry and Cancer Genomics Center, The Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, the Netherlands.

Show MeSH
Related in: MedlinePlus