Comparison of native and non-native ubiquitin oligomers reveals analogous structures and reactivities

Protein Sci. 2016 Feb;25(2):456-71. doi: 10.1002/pro.2834. Epub 2016 Jan 12.


Ubiquitin (Ub) chains regulate a wide range of biological processes, and Ub chain connectivity is a critical determinant of the many regulatory roles that this post-translational modification plays in cells. To understand how distinct Ub chains orchestrate different biochemical events, we and other investigators have developed enzymatic and non-enzymatic methods to synthesize Ub chains of well-defined length and connectivity. A number of chemical approaches have been used to generate Ub oligomers connected by non-native linkages; however, few studies have examined the extent to which non-native linkages recapitulate the structural and functional properties associated with native isopeptide bonds. Here, we compare the structure and function of Ub dimers bearing native and non-native linkages. Using small-angle X-ray scattering (SAXS) analysis, we show that scattering profiles for the two types of dimers are similar. Moreover, using an experimental structural library and atomistic simulations to fit the experimental SAXS profiles, we find that the two types of Ub dimers can be matched to analogous structures. An important application of non-native Ub oligomers is to probe the activity and selectivity of deubiquitinases. Through steady-state kinetic analyses, we demonstrate that different families of deubiquitinases hydrolyze native and non-native isopeptide linkages with comparable efficiency and selectivity. Considering the significant challenges associated with building topologically diverse native Ub chains, our results illustrate that chains harboring non-native linkages can serve as surrogate substrates for explorations of Ub function.

Keywords: protein conjugation; protein structures and functions; small angle x-ray scattering; ubiquitin signaling.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Databases, Protein
  • Humans
  • Hydrolysis
  • Models, Molecular
  • Protein Conformation
  • Protein Multimerization
  • Scattering, Small Angle
  • Ubiquitin / chemistry*
  • Ubiquitin / metabolism
  • Ubiquitin-Specific Proteases / metabolism
  • X-Ray Diffraction


  • Ubiquitin
  • Ubiquitin-Specific Proteases