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

Abstract

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.

Molecular basis of Lys11-polyubiquitin specificity in the deubiquitinase Cezanne

“…c , Kinetic parameters for all independently performed experiments of Lys11, Lys63 and Lys48 diUb cleavage by Cez WT. Values are in good agreement with previously published parameters derived from gel-based studies41. d , Summary of kinetic parameters for Lys11-, Lys63- and Lys48-linked diUb cleavage by Cez E157K.…”

Chemical ubiquitination for decrypting a cellular code

“…Recent biophysical analysis of thiol-ene produced Ub chains compared with native conjugates was carried out in order to ascertain the extent to which thiol-ene-generated Ub conjugates successfully mimic the native isopeptide bond. For the most part, high similarity between native and the non-native surrogates was found (by SAXS analysis) but DUB activity assays indicated that for OTUB1, the non-native surrogate was an unsuitable mimic of the native linkage [ 143 ].…”

Chemical Protein Synthesis Enabled Mechanistic Studies on the Molecular Recognition of K27‐linked Ubiquitin Chains

“…The final product 6 carrying a Lys27 mimic link (Lym27 diUb) was obtained on a 50 mg scale in a total yield of 6 % after folding (Figure B–D). To verify that the sulfur atom replacement did not affect the interaction, the binding affinity between Lym27 diUb and UCHL3 was determined ( K D =160±40 n m ; Figure E) and it is similar to that of wild‐type K27 diUb ( K D =190±30 n m ). Moreover, Lym27 diUb could be cleaved by a K27‐selective deubiquitinase, OTUD2, to a similar level as the wild‐type (Supporting Information, Figure S7).…”