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

Crystal Structures UCH-L5/DEUBAD Complexes(A) Constructs used in this study.(B) RPN13DEU activates UCH-L5 (UR) while INO80GDEU inhibits UCH-L5 (UI) in Ub-AMC enzyme kinetics. The CD is slightly more active than FL UCH-L5 (U). See Figure 1A for naming codes. Error bars, SD.(C) Structure of apo UCH-L5 (3ihr). CD, blue; ULD domain, light blue.(D) Structure of UCH-L5/INO80GDEU (INO80GDEU, orange).(E) Structure of UCH-L5/RPN13DEU (RPN13DEU, green).(F) Structure of UCH-L5∼Ub-Prg/RPN13DEU (Ub-Prg, yellow).(G) The ULDs are found in different conformations across UCH-L5 structures. The CD is transparent for clarity.(H) RPN13DEU (green) changes toward an open state upon UCH-L5 complex formation compared to apo RPN13DEU (gray). Helices α1–4 and the α3-4 loop that undergo the largest changes are colored in darker shades.(I) Superposition of RPN13DEU and INO80GDEU. DEUBAD domains deviate most at FRF hairpin and helix α6. See also Tables 1, S1, S2, and S4 and Figure S1.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

fig1: Crystal Structures UCH-L5/DEUBAD Complexes(A) Constructs used in this study.(B) RPN13DEU activates UCH-L5 (UR) while INO80GDEU inhibits UCH-L5 (UI) in Ub-AMC enzyme kinetics. The CD is slightly more active than FL UCH-L5 (U). See Figure 1A for naming codes. Error bars, SD.(C) Structure of apo UCH-L5 (3ihr). CD, blue; ULD domain, light blue.(D) Structure of UCH-L5/INO80GDEU (INO80GDEU, orange).(E) Structure of UCH-L5/RPN13DEU (RPN13DEU, green).(F) Structure of UCH-L5∼Ub-Prg/RPN13DEU (Ub-Prg, yellow).(G) The ULDs are found in different conformations across UCH-L5 structures. The CD is transparent for clarity.(H) RPN13DEU (green) changes toward an open state upon UCH-L5 complex formation compared to apo RPN13DEU (gray). Helices α1–4 and the α3-4 loop that undergo the largest changes are colored in darker shades.(I) Superposition of RPN13DEU and INO80GDEU. DEUBAD domains deviate most at FRF hairpin and helix α6. See also Tables 1, S1, S2, and S4 and Figure S1.

Mentions: To study the regulation of UCH-L5 by DEUBAD domains, we purified human UCH-L5 in complex with the DEUBAD domains of RPN13 (amino acid [aa] 265–388, referred to as RPN13DEU) and INO80G (aa 39–170, referred to as INO80GDEU) (Figure 1A). We measured the catalytic activity of these complexes towards the minimal substrate ubiquitin-7-amido-4-methylcoumarin (Ub-AMC) (Dang et al., 1998; El Oualid et al., 2010) in comparison to full-length UCH-L5 alone (U) and its isolated CD. In line with previous data, we found that the DEUBAD domain of RPN13 activates UCH-L5 (UR) (Figure 1B; Hamazaki et al., 2006; Qiu et al., 2006; Yao et al., 2006). Since the UCH-L5 CD is more active than the full-length alone, the ULD domain partially inhibits activity (Yao et al., 2006). However, in the presence of RPN13DEU, UCH-L5 is significantly more active than the UCH-L5 CD, and, therefore, RPN13DEU does more than simply remove autoinhibition (Figure 1B). Strikingly, INO80GDEU severely inhibits activity under these conditions (UI) (Figure 1B).


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)

Crystal Structures UCH-L5/DEUBAD Complexes(A) Constructs used in this study.(B) RPN13DEU activates UCH-L5 (UR) while INO80GDEU inhibits UCH-L5 (UI) in Ub-AMC enzyme kinetics. The CD is slightly more active than FL UCH-L5 (U). See Figure 1A for naming codes. Error bars, SD.(C) Structure of apo UCH-L5 (3ihr). CD, blue; ULD domain, light blue.(D) Structure of UCH-L5/INO80GDEU (INO80GDEU, orange).(E) Structure of UCH-L5/RPN13DEU (RPN13DEU, green).(F) Structure of UCH-L5∼Ub-Prg/RPN13DEU (Ub-Prg, yellow).(G) The ULDs are found in different conformations across UCH-L5 structures. The CD is transparent for clarity.(H) RPN13DEU (green) changes toward an open state upon UCH-L5 complex formation compared to apo RPN13DEU (gray). Helices α1–4 and the α3-4 loop that undergo the largest changes are colored in darker shades.(I) Superposition of RPN13DEU and INO80GDEU. DEUBAD domains deviate most at FRF hairpin and helix α6. See also Tables 1, S1, S2, and S4 and Figure S1.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

fig1: Crystal Structures UCH-L5/DEUBAD Complexes(A) Constructs used in this study.(B) RPN13DEU activates UCH-L5 (UR) while INO80GDEU inhibits UCH-L5 (UI) in Ub-AMC enzyme kinetics. The CD is slightly more active than FL UCH-L5 (U). See Figure 1A for naming codes. Error bars, SD.(C) Structure of apo UCH-L5 (3ihr). CD, blue; ULD domain, light blue.(D) Structure of UCH-L5/INO80GDEU (INO80GDEU, orange).(E) Structure of UCH-L5/RPN13DEU (RPN13DEU, green).(F) Structure of UCH-L5∼Ub-Prg/RPN13DEU (Ub-Prg, yellow).(G) The ULDs are found in different conformations across UCH-L5 structures. The CD is transparent for clarity.(H) RPN13DEU (green) changes toward an open state upon UCH-L5 complex formation compared to apo RPN13DEU (gray). Helices α1–4 and the α3-4 loop that undergo the largest changes are colored in darker shades.(I) Superposition of RPN13DEU and INO80GDEU. DEUBAD domains deviate most at FRF hairpin and helix α6. See also Tables 1, S1, S2, and S4 and Figure S1.
Mentions: To study the regulation of UCH-L5 by DEUBAD domains, we purified human UCH-L5 in complex with the DEUBAD domains of RPN13 (amino acid [aa] 265–388, referred to as RPN13DEU) and INO80G (aa 39–170, referred to as INO80GDEU) (Figure 1A). We measured the catalytic activity of these complexes towards the minimal substrate ubiquitin-7-amido-4-methylcoumarin (Ub-AMC) (Dang et al., 1998; El Oualid et al., 2010) in comparison to full-length UCH-L5 alone (U) and its isolated CD. In line with previous data, we found that the DEUBAD domain of RPN13 activates UCH-L5 (UR) (Figure 1B; Hamazaki et al., 2006; Qiu et al., 2006; Yao et al., 2006). Since the UCH-L5 CD is more active than the full-length alone, the ULD domain partially inhibits activity (Yao et al., 2006). However, in the presence of RPN13DEU, UCH-L5 is significantly more active than the UCH-L5 CD, and, therefore, RPN13DEU does more than simply remove autoinhibition (Figure 1B). Strikingly, INO80GDEU severely inhibits activity under these conditions (UI) (Figure 1B).

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