Structural and functional characterization of ubiquitin variant inhibitors for the JAMM-family deubiquitinases STAMBP and STAMBPL1

J Biol Chem. 2021 Oct;297(4):101107. doi: 10.1016/j.jbc.2021.101107. Epub 2021 Aug 21.


Ubiquitination is a crucial posttranslational protein modification involved in a myriad of biological pathways. This modification is reversed by deubiquitinases (DUBs) that deconjugate the single ubiquitin (Ub) moiety or poly-Ub chains from substrates. In the past decade, tremendous efforts have been focused on targeting DUBs for drug discovery. However, most chemical compounds with inhibitory activity for DUBs suffer from mild potency and low selectivity. To overcome these obstacles, we developed a phage display-based protein engineering strategy for generating Ub variant (UbV) inhibitors, which was previously successfully applied to the Ub-specific protease (USP) family of cysteine proteases. In this work, we leveraged the UbV platform to selectively target STAMBP, a member of the JAB1/MPN/MOV34 (JAMM) metalloprotease family of DUB enzymes. We identified two UbVs (UbVSP.1 and UbVSP.3) that bind to STAMBP with high affinity but differ in their selectivity for the closely related paralog STAMBPL1. We determined the STAMBPL1-UbVSP.1 complex structure by X-ray crystallography, revealing hotspots of the JAMM-UbV interaction. Finally, we show that UbVSP.1 and UbVSP.3 are potent inhibitors of STAMBP isopeptidase activity, far exceeding the reported small-molecule inhibitor BC-1471. This work demonstrates that UbV technology is suitable to develop molecules as tools to target metalloproteases, which can be used to further understand the cellular function of JAMM family DUBs.

Keywords: JAMM domain; STAMBP; crystal structure; deubiquitinases; inhibitor; phage display; protein engineering; ubiquitin variants.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Crystallography, X-Ray
  • Endosomal Sorting Complexes Required for Transport* / antagonists & inhibitors
  • Endosomal Sorting Complexes Required for Transport* / chemistry
  • Humans
  • Peptide Hydrolases* / chemistry
  • Peptide Library*
  • Protease Inhibitors / chemistry*
  • Protein Structure, Quaternary
  • Ubiquitin Thiolesterase* / antagonists & inhibitors
  • Ubiquitin Thiolesterase* / chemistry
  • Ubiquitin* / chemistry
  • Ubiquitin* / genetics


  • Endosomal Sorting Complexes Required for Transport
  • Peptide Library
  • Protease Inhibitors
  • STAMBP protein, human
  • Ubiquitin
  • Peptide Hydrolases
  • STAMBPL1 protein, human
  • Ubiquitin Thiolesterase