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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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Reactivation of the Inhibitor and Model for UCH-L5 Regulation(A) The crystal structure of UCH-L5∼Ub-Prg/INO80Gshort (middle) largely resembles the UCH-L5/RPN13DEU structures (left from Figure 1F), but not the UCH-L5/INO80GDEU structure (right from Figure 1D).(B) INO80Gshort (UIshort) can activate UCH-L5, albeit not to RPN13DEU levels (UR, UI, and U from Figure 1B), and depends on the ULD anchor for activation on Ub-AMC. Error bars, SD.(C) Activation by INO80Gshort correlates with enhanced Ub-GlySerThr binding in ITC compared to WT UCH-L5 (from Figure 3A).(D) The ULD anchors in the INO80Gshort (light blue) and RPN13DEU (gray) superimpose very well and stabilize the same intramolecular interaction.(E) Unmodulated UCH-L5 (middle) is characterized by CL and ULD flexibility that limits substrate binding and catalysis. Activation by RPN13DEU (left) limits flexibility by locking the ULD and CL in favorable conformations. INO80GDEU (right) locks the ULD in a conformation incompatible with substrate docking. See also Figure S4 and Tables S1–S3.
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fig6: Reactivation of the Inhibitor and Model for UCH-L5 Regulation(A) The crystal structure of UCH-L5∼Ub-Prg/INO80Gshort (middle) largely resembles the UCH-L5/RPN13DEU structures (left from Figure 1F), but not the UCH-L5/INO80GDEU structure (right from Figure 1D).(B) INO80Gshort (UIshort) can activate UCH-L5, albeit not to RPN13DEU levels (UR, UI, and U from Figure 1B), and depends on the ULD anchor for activation on Ub-AMC. Error bars, SD.(C) Activation by INO80Gshort correlates with enhanced Ub-GlySerThr binding in ITC compared to WT UCH-L5 (from Figure 3A).(D) The ULD anchors in the INO80Gshort (light blue) and RPN13DEU (gray) superimpose very well and stabilize the same intramolecular interaction.(E) Unmodulated UCH-L5 (middle) is characterized by CL and ULD flexibility that limits substrate binding and catalysis. Activation by RPN13DEU (left) limits flexibility by locking the ULD and CL in favorable conformations. INO80GDEU (right) locks the ULD in a conformation incompatible with substrate docking. See also Figure S4 and Tables S1–S3.

Mentions: Strikingly, the UCH-L5∼UbPrg/INO80Gshort crystal structure resembled the activated RPN13DEU complex rather than the inhibited state (Figures 6A, S4A, and S4B). In this complex, the ULD largely reverted to the conformation seen in the activated RPN13DEU complexes with an extended helix α10 (Figure S4C). All conformational changes in UCH-L5 required to create the canonical ubiquitin-binding mode were also in place (Figures S4D and S4E). The structure of INO80Gshort itself and its binding mode to UCH-L5 were moreover identical to INO80GDEU (apart from FRF hairpin and α5-6) and RPN13DEU (Figure S4F).


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)

Reactivation of the Inhibitor and Model for UCH-L5 Regulation(A) The crystal structure of UCH-L5∼Ub-Prg/INO80Gshort (middle) largely resembles the UCH-L5/RPN13DEU structures (left from Figure 1F), but not the UCH-L5/INO80GDEU structure (right from Figure 1D).(B) INO80Gshort (UIshort) can activate UCH-L5, albeit not to RPN13DEU levels (UR, UI, and U from Figure 1B), and depends on the ULD anchor for activation on Ub-AMC. Error bars, SD.(C) Activation by INO80Gshort correlates with enhanced Ub-GlySerThr binding in ITC compared to WT UCH-L5 (from Figure 3A).(D) The ULD anchors in the INO80Gshort (light blue) and RPN13DEU (gray) superimpose very well and stabilize the same intramolecular interaction.(E) Unmodulated UCH-L5 (middle) is characterized by CL and ULD flexibility that limits substrate binding and catalysis. Activation by RPN13DEU (left) limits flexibility by locking the ULD and CL in favorable conformations. INO80GDEU (right) locks the ULD in a conformation incompatible with substrate docking. See also Figure S4 and Tables S1–S3.
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fig6: Reactivation of the Inhibitor and Model for UCH-L5 Regulation(A) The crystal structure of UCH-L5∼Ub-Prg/INO80Gshort (middle) largely resembles the UCH-L5/RPN13DEU structures (left from Figure 1F), but not the UCH-L5/INO80GDEU structure (right from Figure 1D).(B) INO80Gshort (UIshort) can activate UCH-L5, albeit not to RPN13DEU levels (UR, UI, and U from Figure 1B), and depends on the ULD anchor for activation on Ub-AMC. Error bars, SD.(C) Activation by INO80Gshort correlates with enhanced Ub-GlySerThr binding in ITC compared to WT UCH-L5 (from Figure 3A).(D) The ULD anchors in the INO80Gshort (light blue) and RPN13DEU (gray) superimpose very well and stabilize the same intramolecular interaction.(E) Unmodulated UCH-L5 (middle) is characterized by CL and ULD flexibility that limits substrate binding and catalysis. Activation by RPN13DEU (left) limits flexibility by locking the ULD and CL in favorable conformations. INO80GDEU (right) locks the ULD in a conformation incompatible with substrate docking. See also Figure S4 and Tables S1–S3.
Mentions: Strikingly, the UCH-L5∼UbPrg/INO80Gshort crystal structure resembled the activated RPN13DEU complex rather than the inhibited state (Figures 6A, S4A, and S4B). In this complex, the ULD largely reverted to the conformation seen in the activated RPN13DEU complexes with an extended helix α10 (Figure S4C). All conformational changes in UCH-L5 required to create the canonical ubiquitin-binding mode were also in place (Figures S4D and S4E). The structure of INO80Gshort itself and its binding mode to UCH-L5 were moreover identical to INO80GDEU (apart from FRF hairpin and α5-6) and RPN13DEU (Figure S4F).

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