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Structure and catalytic regulatory function of ubiquitin specific protease 11 N-terminal and ubiquitin-like domains.

Harper S, Gratton HE, Cornaciu I, Oberer M, Scott DJ, Emsley J, Dreveny I - Biochemistry (2014)

Bottom Line: Crystal structures of the DU domains show a tandem arrangement with a shortened β-hairpin at the two-domain interface and altered surface characteristics compared to the homologues USP4 and USP15.Our data support a model whereby USP11 domains outside the catalytic core domain serve as protein interaction or trafficking modules rather than a direct regulatory function of the proteolytic activity.This highlights the diversity of USPs in substrate recognition and regulation of ubiquitin deconjugation.

View Article: PubMed Central - PubMed

Affiliation: Centre for Biomolecular Sciences, University of Nottingham, University Park Campus, Nottingham, NG7 2RD, United Kingdom.

ABSTRACT
The ubiquitin specific protease 11 (USP11) is implicated in DNA repair, viral RNA replication, and TGFβ signaling. We report the first characterization of the USP11 domain architecture and its role in regulating the enzymatic activity. USP11 consists of an N-terminal "domain present in USPs" (DUSP) and "ubiquitin-like" (UBL) domain, together referred to as DU domains, and the catalytic domain harboring a second UBL domain. Crystal structures of the DU domains show a tandem arrangement with a shortened β-hairpin at the two-domain interface and altered surface characteristics compared to the homologues USP4 and USP15. A conserved VEVY motif is a signature feature at the two-domain interface that shapes a potential protein interaction site. Small angle X-ray scattering and gel filtration experiments are consistent with the USP11DU domains and full-length USP11 being monomeric. Unexpectedly, we reveal, through kinetic assays of a series of deletion mutants, that the catalytic activity of USP11 is not regulated through intramolecular autoinhibition or activation by the N-terminal DU or UBL domains. Moreover, ubiquitin chain cleavage assays with all eight linkages reveal a preference for Lys(63)-, Lys(6)-, Lys(33)-, and Lys(11)-linked chains over Lys(27)-, Lys(29)-, and Lys(48)-linked and linear chains consistent with USP11's function in DNA repair pathways that is mediated by the protease domain. Our data support a model whereby USP11 domains outside the catalytic core domain serve as protein interaction or trafficking modules rather than a direct regulatory function of the proteolytic activity. This highlights the diversity of USPs in substrate recognition and regulation of ubiquitin deconjugation.

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Related in: MedlinePlus

Ubiquitin chain selectivity. (A) SDS-PAGE analysesof cleavageof diubiquitin to monoubiquitin by USP11 and deletion mutants withthe eight linkage types (as labeled). Assays were performed in duplicateand representative gels are shown. The lanes on each gel show a timecourse of a reaction with 75 nM USP11 and 5 μM substrate at0, 5, 10, 30, 60, 120, and 180 min with a final overnight sample takenafter 19 h. (B) Densitometric analysis of gels. XY scatter plots withdiubiquitin percentage plotted against time in minutes for each ofthe three hUSP11 constructs (as labeled) with each of the eight diubiquitinlinkage types (colors indicated in key). Diubiquitin percentage wascalculated from ImageJ analyses of SDS-PAGE gels by quantifying theamount of diubiquitin and monoubiquitin in each lane. The mean wascalculated from the duplicate assays, and nonlinear regression analyseswere performed on time points 0–180 min. Errors are given asstandard error mean. ON: overnight sample (not included in nonlinearregression analyses).
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fig5: Ubiquitin chain selectivity. (A) SDS-PAGE analysesof cleavageof diubiquitin to monoubiquitin by USP11 and deletion mutants withthe eight linkage types (as labeled). Assays were performed in duplicateand representative gels are shown. The lanes on each gel show a timecourse of a reaction with 75 nM USP11 and 5 μM substrate at0, 5, 10, 30, 60, 120, and 180 min with a final overnight sample takenafter 19 h. (B) Densitometric analysis of gels. XY scatter plots withdiubiquitin percentage plotted against time in minutes for each ofthe three hUSP11 constructs (as labeled) with each of the eight diubiquitinlinkage types (colors indicated in key). Diubiquitin percentage wascalculated from ImageJ analyses of SDS-PAGE gels by quantifying theamount of diubiquitin and monoubiquitin in each lane. The mean wascalculated from the duplicate assays, and nonlinear regression analyseswere performed on time points 0–180 min. Errors are given asstandard error mean. ON: overnight sample (not included in nonlinearregression analyses).

Mentions: We subsequentlyinvestigated the effect of the removal of ancillary USP11 domainson the specificity for diubiquitin substrates of all eight known linkagesoccurring in the cell. On the basis of diubiquitin chain cleavagetime-courses, FL-USP11 displayed little detectable activity for linearchains and highest activity toward Lys63- and Lys6-linked chains. Interestingly, FLΔUBL2 and CatΔUBL2 showedthese same preferences (Figure 5). On the basisof these six independent experiments, the eight different diubiquitinlinkages can broadly be split into three main groups by preferenceof all three proteins tested: Lys6- and Lys63-linked chains showed the highest levels of cleavage, Lys11- and Lys33-linked chains showed moderate cleavage, andLys27-, Lys29-, Lys48-linked andlinear chains demonstrated the lowest levels of cleavage. We conductedgel image analysis to further evaluate the data (Figures 5B and S5; Supporting Information). On the basis of the results for all three USP11 catalytic domainconstructs investigated, more than 65% of Lys63- and Lys6-linked diubiquitin was cleaved within 3 h and more than 85%after 19 h. FL-USP11 with Lys63-linked and FLΔUBL2with Lys6-linked chains as substrates showed complete cleavageafter 19 h. Lys33- and Lys11-linked chains werecleaved to more than 35% after 3 h and more than 45% after 19 h. Linear,Lys48-, Lys29-, and Lys27-linkeddiubiquitin chains were cleaved to less than 30% after 3 h and lessthan 50% even after 19 h.


Structure and catalytic regulatory function of ubiquitin specific protease 11 N-terminal and ubiquitin-like domains.

Harper S, Gratton HE, Cornaciu I, Oberer M, Scott DJ, Emsley J, Dreveny I - Biochemistry (2014)

Ubiquitin chain selectivity. (A) SDS-PAGE analysesof cleavageof diubiquitin to monoubiquitin by USP11 and deletion mutants withthe eight linkage types (as labeled). Assays were performed in duplicateand representative gels are shown. The lanes on each gel show a timecourse of a reaction with 75 nM USP11 and 5 μM substrate at0, 5, 10, 30, 60, 120, and 180 min with a final overnight sample takenafter 19 h. (B) Densitometric analysis of gels. XY scatter plots withdiubiquitin percentage plotted against time in minutes for each ofthe three hUSP11 constructs (as labeled) with each of the eight diubiquitinlinkage types (colors indicated in key). Diubiquitin percentage wascalculated from ImageJ analyses of SDS-PAGE gels by quantifying theamount of diubiquitin and monoubiquitin in each lane. The mean wascalculated from the duplicate assays, and nonlinear regression analyseswere performed on time points 0–180 min. Errors are given asstandard error mean. ON: overnight sample (not included in nonlinearregression analyses).
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Related In: Results  -  Collection

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fig5: Ubiquitin chain selectivity. (A) SDS-PAGE analysesof cleavageof diubiquitin to monoubiquitin by USP11 and deletion mutants withthe eight linkage types (as labeled). Assays were performed in duplicateand representative gels are shown. The lanes on each gel show a timecourse of a reaction with 75 nM USP11 and 5 μM substrate at0, 5, 10, 30, 60, 120, and 180 min with a final overnight sample takenafter 19 h. (B) Densitometric analysis of gels. XY scatter plots withdiubiquitin percentage plotted against time in minutes for each ofthe three hUSP11 constructs (as labeled) with each of the eight diubiquitinlinkage types (colors indicated in key). Diubiquitin percentage wascalculated from ImageJ analyses of SDS-PAGE gels by quantifying theamount of diubiquitin and monoubiquitin in each lane. The mean wascalculated from the duplicate assays, and nonlinear regression analyseswere performed on time points 0–180 min. Errors are given asstandard error mean. ON: overnight sample (not included in nonlinearregression analyses).
Mentions: We subsequentlyinvestigated the effect of the removal of ancillary USP11 domainson the specificity for diubiquitin substrates of all eight known linkagesoccurring in the cell. On the basis of diubiquitin chain cleavagetime-courses, FL-USP11 displayed little detectable activity for linearchains and highest activity toward Lys63- and Lys6-linked chains. Interestingly, FLΔUBL2 and CatΔUBL2 showedthese same preferences (Figure 5). On the basisof these six independent experiments, the eight different diubiquitinlinkages can broadly be split into three main groups by preferenceof all three proteins tested: Lys6- and Lys63-linked chains showed the highest levels of cleavage, Lys11- and Lys33-linked chains showed moderate cleavage, andLys27-, Lys29-, Lys48-linked andlinear chains demonstrated the lowest levels of cleavage. We conductedgel image analysis to further evaluate the data (Figures 5B and S5; Supporting Information). On the basis of the results for all three USP11 catalytic domainconstructs investigated, more than 65% of Lys63- and Lys6-linked diubiquitin was cleaved within 3 h and more than 85%after 19 h. FL-USP11 with Lys63-linked and FLΔUBL2with Lys6-linked chains as substrates showed complete cleavageafter 19 h. Lys33- and Lys11-linked chains werecleaved to more than 35% after 3 h and more than 45% after 19 h. Linear,Lys48-, Lys29-, and Lys27-linkeddiubiquitin chains were cleaved to less than 30% after 3 h and lessthan 50% even after 19 h.

Bottom Line: Crystal structures of the DU domains show a tandem arrangement with a shortened β-hairpin at the two-domain interface and altered surface characteristics compared to the homologues USP4 and USP15.Our data support a model whereby USP11 domains outside the catalytic core domain serve as protein interaction or trafficking modules rather than a direct regulatory function of the proteolytic activity.This highlights the diversity of USPs in substrate recognition and regulation of ubiquitin deconjugation.

View Article: PubMed Central - PubMed

Affiliation: Centre for Biomolecular Sciences, University of Nottingham, University Park Campus, Nottingham, NG7 2RD, United Kingdom.

ABSTRACT
The ubiquitin specific protease 11 (USP11) is implicated in DNA repair, viral RNA replication, and TGFβ signaling. We report the first characterization of the USP11 domain architecture and its role in regulating the enzymatic activity. USP11 consists of an N-terminal "domain present in USPs" (DUSP) and "ubiquitin-like" (UBL) domain, together referred to as DU domains, and the catalytic domain harboring a second UBL domain. Crystal structures of the DU domains show a tandem arrangement with a shortened β-hairpin at the two-domain interface and altered surface characteristics compared to the homologues USP4 and USP15. A conserved VEVY motif is a signature feature at the two-domain interface that shapes a potential protein interaction site. Small angle X-ray scattering and gel filtration experiments are consistent with the USP11DU domains and full-length USP11 being monomeric. Unexpectedly, we reveal, through kinetic assays of a series of deletion mutants, that the catalytic activity of USP11 is not regulated through intramolecular autoinhibition or activation by the N-terminal DU or UBL domains. Moreover, ubiquitin chain cleavage assays with all eight linkages reveal a preference for Lys(63)-, Lys(6)-, Lys(33)-, and Lys(11)-linked chains over Lys(27)-, Lys(29)-, and Lys(48)-linked and linear chains consistent with USP11's function in DNA repair pathways that is mediated by the protease domain. Our data support a model whereby USP11 domains outside the catalytic core domain serve as protein interaction or trafficking modules rather than a direct regulatory function of the proteolytic activity. This highlights the diversity of USPs in substrate recognition and regulation of ubiquitin deconjugation.

Show MeSH
Related in: MedlinePlus