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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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The FRF Hairpin Drives UCH-L5 Inhibition(A) INO80GDEU differs from RPN13DEU mainly in the FRF hairpin and helix α6 (orange surfaces).(B) Helix α6 of INO80GDEU is dispensable for inhibition in Ub-AMC assays. Error bars, SD.(C) Inhibition is completely lost in the INO80GDEU F100A mutant (UIF100A) on Ub-AMC. Error bars, SD.(D) Mutant complex F100A gains Ub-GlySerThr-binding ability compared to WT INO80GDEU complex (from Figure 3A) in ITC.(E) Insertion of the FRF hairpin in RPN13DEUchimera abolishes the activation effect of WT RPN13DEU on UCH-L5 (UR and U from Figure 1B). Error bars, SD. See also Figure S3 and Tables S1–S3.
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fig5: The FRF Hairpin Drives UCH-L5 Inhibition(A) INO80GDEU differs from RPN13DEU mainly in the FRF hairpin and helix α6 (orange surfaces).(B) Helix α6 of INO80GDEU is dispensable for inhibition in Ub-AMC assays. Error bars, SD.(C) Inhibition is completely lost in the INO80GDEU F100A mutant (UIF100A) on Ub-AMC. Error bars, SD.(D) Mutant complex F100A gains Ub-GlySerThr-binding ability compared to WT INO80GDEU complex (from Figure 3A) in ITC.(E) Insertion of the FRF hairpin in RPN13DEUchimera abolishes the activation effect of WT RPN13DEU on UCH-L5 (UR and U from Figure 1B). Error bars, SD. See also Figure S3 and Tables S1–S3.

Mentions: To study what features in INO80GDEU are required to achieve UCH-L5 inhibition, we analyzed its differences with the activating DEUBAD domain of RPN13. The most striking difference between the DEUBAD domains of RPN13 and INO80G is the C-terminal part where three helices α6–α8 in RPN13DEU change to the extended α6 in INO80GDEU that packs against the CD (Figures 5A and S1D). Therefore, we created a shorter version of INO80GDEU, where helix α6 is removed (Figure 1A, residues 39–118, INO80GDEUΔα6). Surprisingly, the truncated protein INO80GDEUΔα6 still inhibited UCH-L5 (Figure 5B), demonstrating that helix α6 is not required for inhibition under these conditions.


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)

The FRF Hairpin Drives UCH-L5 Inhibition(A) INO80GDEU differs from RPN13DEU mainly in the FRF hairpin and helix α6 (orange surfaces).(B) Helix α6 of INO80GDEU is dispensable for inhibition in Ub-AMC assays. Error bars, SD.(C) Inhibition is completely lost in the INO80GDEU F100A mutant (UIF100A) on Ub-AMC. Error bars, SD.(D) Mutant complex F100A gains Ub-GlySerThr-binding ability compared to WT INO80GDEU complex (from Figure 3A) in ITC.(E) Insertion of the FRF hairpin in RPN13DEUchimera abolishes the activation effect of WT RPN13DEU on UCH-L5 (UR and U from Figure 1B). Error bars, SD. See also Figure S3 and Tables S1–S3.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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fig5: The FRF Hairpin Drives UCH-L5 Inhibition(A) INO80GDEU differs from RPN13DEU mainly in the FRF hairpin and helix α6 (orange surfaces).(B) Helix α6 of INO80GDEU is dispensable for inhibition in Ub-AMC assays. Error bars, SD.(C) Inhibition is completely lost in the INO80GDEU F100A mutant (UIF100A) on Ub-AMC. Error bars, SD.(D) Mutant complex F100A gains Ub-GlySerThr-binding ability compared to WT INO80GDEU complex (from Figure 3A) in ITC.(E) Insertion of the FRF hairpin in RPN13DEUchimera abolishes the activation effect of WT RPN13DEU on UCH-L5 (UR and U from Figure 1B). Error bars, SD. See also Figure S3 and Tables S1–S3.
Mentions: To study what features in INO80GDEU are required to achieve UCH-L5 inhibition, we analyzed its differences with the activating DEUBAD domain of RPN13. The most striking difference between the DEUBAD domains of RPN13 and INO80G is the C-terminal part where three helices α6–α8 in RPN13DEU change to the extended α6 in INO80GDEU that packs against the CD (Figures 5A and S1D). Therefore, we created a shorter version of INO80GDEU, where helix α6 is removed (Figure 1A, residues 39–118, INO80GDEUΔα6). Surprisingly, the truncated protein INO80GDEUΔα6 still inhibited UCH-L5 (Figure 5B), demonstrating that helix α6 is not required for inhibition under these conditions.

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