Structural and Functional Characterization of Ubiquitin Variant Inhibitors of USP15

• Published in: Structure (London) 2019-04, Vol.27 (4), p.590-605.e5
• Main Authors: Teyra, Joan, Singer, Alex U., Schmitges, Frank W., Jaynes, Patrick, Kit Leng Lui, Sarah, Polyak, Maria J., Fodil, Nassima, Krieger, Jonathan R., Tong, Jiefei, Schwerdtfeger, Carsten, Brasher, Bradley B., Ceccarelli, Derek F.J., Moffat, Jason, Sicheri, Frank, Moran, Michael F., Gros, Philippe, Eichhorn, Pieter J.A., Lenter, Martin, Boehmelt, Guido, Sidhu, Sachdev S.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Catalytic Domain; Cloning, Molecular; Crystallography, X-Ray; deubiquitinase; DUSP; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Genetic Vectors - chemistry; Genetic Vectors - metabolism; HEK293 Cells; Humans; Models, Molecular; phage display; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Protein Multimerization; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity; TGF-β pathway; Transcription Factors - chemistry; Transcription Factors - genetics; Transcription Factors - metabolism; Transforming Growth Factor beta1 - chemistry; Transforming Growth Factor beta1 - genetics; Transforming Growth Factor beta1 - metabolism; Tripartite Motif Proteins - chemistry; Tripartite Motif Proteins - genetics; Tripartite Motif Proteins - metabolism; ubiquitin; Ubiquitin - chemistry; Ubiquitin - genetics; Ubiquitin - metabolism; ubiquitin-like; Ubiquitin-Protein Ligases - chemistry; Ubiquitin-Protein Ligases - genetics; Ubiquitin-Protein Ligases - metabolism; Ubiquitin-Specific Proteases - antagonists & inhibitors; Ubiquitin-Specific Proteases - chemistry; Ubiquitin-Specific Proteases - genetics; Ubiquitin-Specific Proteases - metabolism; Ubiquitination; UbV; USP15; USP4
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2019.01.002

The multi-domain deubiquitinase USP15 regulates diverse eukaryotic processes and has been implicated in numerous diseases. We developed ubiquitin variants (UbVs) that targeted either the catalytic domain or each of three adaptor domains in USP15, including the N-terminal DUSP domain. We also designed a linear dimer (diUbV), which targeted the DUSP and catalytic domains, and exhibited enhanced specificity and more potent inhibition of catalytic activity than either UbV alone. In cells, the UbVs inhibited the deubiquitination of two USP15 substrates, SMURF2 and TRIM25, and the diUbV inhibited the effects of USP15 on the transforming growth factor β pathway. Structural analyses revealed that three distinct UbVs bound to the catalytic domain and locked the active site in a closed, inactive conformation, and one UbV formed an unusual strand-swapped dimer and bound two DUSP domains simultaneously. These inhibitors will enable the study of USP15 function in oncology, neurology, immunology, and inflammation. [Display omitted] •Tight and selective UbVs target USP15 catalytic and adaptor domains•UbV inhibitors lock the USP15 active site in an inactive conformation•A strand-swapped UbV dimer binds two DUSP domains simultaneously•Linear UbV dimers are potent and specific USP15 inhibitors in cells Teyra et al. developed ubiquitin variants (UbVs) that targeted distinct domains of the deubiquitinase USP15 and revealed alternative inhibition and dimerization strategies. Linear UbV dimers exhibited improved potency and specificity of USP15 inhibition. UbVs can be used to study the catalytic mechanism and biological functions of USP15.