Limits...
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.

Show MeSH

Related in: MedlinePlus

Activation Mechanism RPN13DEU(A) Mutations in the ubiquitin interface severely compromise DUB activity irrespective of RPN13DEU (top). Location mutants (yellow sticks) on UCH-L5 (bottom).(B) Surface representation of UCH-L3 and UCH-L5 colored by conservation.(C) UCH-L5 helix α10 is anchored to the CD via an extensive polar network.(D and F) Compared to WT (UR and U from Figure 1B), UCH-L5 mutants E283A and M148A/F149A cannot be activated by RPN13DEU to the same extent on Ub-AMC. Error bars, SD.(E) The CL is positioned by RPN13DEU.(G) Activation of the combined CL and ubiquitin anchor mutants E283A/M148A/F149A is almost completely abrogated compared to WT (UR and U from Figure 1B). Error bars, SD. See also Figure S2 and Tables S1–S4.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4352763&req=5

fig3: Activation Mechanism RPN13DEU(A) Mutations in the ubiquitin interface severely compromise DUB activity irrespective of RPN13DEU (top). Location mutants (yellow sticks) on UCH-L5 (bottom).(B) Surface representation of UCH-L3 and UCH-L5 colored by conservation.(C) UCH-L5 helix α10 is anchored to the CD via an extensive polar network.(D and F) Compared to WT (UR and U from Figure 1B), UCH-L5 mutants E283A and M148A/F149A cannot be activated by RPN13DEU to the same extent on Ub-AMC. Error bars, SD.(E) The CL is positioned by RPN13DEU.(G) Activation of the combined CL and ubiquitin anchor mutants E283A/M148A/F149A is almost completely abrogated compared to WT (UR and U from Figure 1B). Error bars, SD. See also Figure S2 and Tables S1–S4.

Mentions: To investigate how RPN13DEU promotes enhanced substrate binding by UCH-L5, we tested the effect of mutations on activity. We first focused on the effect on activation of the ubiquitin-binding residues in UCH-L5. Mutations in these residues lowered the activity substantially, irrespective of the presence of RPN13DEU, indicating that they are primarily important for basic DUB function (Figure 3A). We then tested mutations of RPN13DEU located in the interface with ubiquitin, and found that these provided only a limited contribution to UCH-L5 activation (Figures 2D and S2C).


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)

Activation Mechanism RPN13DEU(A) Mutations in the ubiquitin interface severely compromise DUB activity irrespective of RPN13DEU (top). Location mutants (yellow sticks) on UCH-L5 (bottom).(B) Surface representation of UCH-L3 and UCH-L5 colored by conservation.(C) UCH-L5 helix α10 is anchored to the CD via an extensive polar network.(D and F) Compared to WT (UR and U from Figure 1B), UCH-L5 mutants E283A and M148A/F149A cannot be activated by RPN13DEU to the same extent on Ub-AMC. Error bars, SD.(E) The CL is positioned by RPN13DEU.(G) Activation of the combined CL and ubiquitin anchor mutants E283A/M148A/F149A is almost completely abrogated compared to WT (UR and U from Figure 1B). Error bars, SD. See also Figure S2 and Tables S1–S4.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

fig3: Activation Mechanism RPN13DEU(A) Mutations in the ubiquitin interface severely compromise DUB activity irrespective of RPN13DEU (top). Location mutants (yellow sticks) on UCH-L5 (bottom).(B) Surface representation of UCH-L3 and UCH-L5 colored by conservation.(C) UCH-L5 helix α10 is anchored to the CD via an extensive polar network.(D and F) Compared to WT (UR and U from Figure 1B), UCH-L5 mutants E283A and M148A/F149A cannot be activated by RPN13DEU to the same extent on Ub-AMC. Error bars, SD.(E) The CL is positioned by RPN13DEU.(G) Activation of the combined CL and ubiquitin anchor mutants E283A/M148A/F149A is almost completely abrogated compared to WT (UR and U from Figure 1B). Error bars, SD. See also Figure S2 and Tables S1–S4.
Mentions: To investigate how RPN13DEU promotes enhanced substrate binding by UCH-L5, we tested the effect of mutations on activity. We first focused on the effect on activation of the ubiquitin-binding residues in UCH-L5. Mutations in these residues lowered the activity substantially, irrespective of the presence of RPN13DEU, indicating that they are primarily important for basic DUB function (Figure 3A). We then tested mutations of RPN13DEU located in the interface with ubiquitin, and found that these provided only a limited contribution to UCH-L5 activation (Figures 2D and S2C).

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