Conference 2021 Live Talk


Talk title

Synthesis of defined homogeneous site-specific ubiquitin conjugates by a chemo-enzymatic sortase mediated ligation strategy


Authors and Affiliations

Avinash Kumar Singh1,2

1. Department of Chemical Biology, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland.
2. Cell Biology Lab-2, National Institute of Immunology, New Delhi, India.




Ubiquitination, the covalent attachment of ubiquitin (Ub) to target proteins, represents one of the most versatile and common post-translational modifications (PTMs) in eukaryotic cells. It increases the functional diversity of the proteome and plays crucial regulatory roles in many fundamental cellular processes. Ubiquitination is a very complex post-translational event and involves the action of several ubiquitinating enzymes (E1-E3) as well as deubiquitinating enzymes (DUBs). The specific combinations of enzymes that are used to ubiquitylate and deubiquitylate target proteins are often unknown, making it challenging to understand the roles of Ub and Ubiquitin-like modifications (SUMOylation, Neddylation, ISGylation, Pupylation, etc.) and to decipher enzyme specificity. The availability of well-defined Ub-conjugates is necessary for untangling the mechanism of ubiquitination. However, assembly of homogeneous Ub-conjugates represents a challenge because of the multi-step chemical synthesis involved and the unwieldiness of the reconstituted biosynthetic systems. Here we report a simple chemo-enzymatic strategy for the synthesis of homogeneous and defined ubiquitin conjugates using Solid Phase Peptide Synthesis (SPPS) and Sortase Mediated Ligation (SML).


Sortase recognition motif 71LRLPXTGG78 was generated by engineering PXT substitution/insertion for R74 in the Ub C-terminal 71LRLRGG76. Three mutants having position X substituted with Leu, Asn and Glu, respectively, were prepared. A Gly-Gly appended peptide LMFK(ε-GG)TEG encompassing Ub target site of p53 corresponding to residues 383LMFKTEG389 was synthesized using Fmoc chemistry as a donor nucleophile. The peptide conjugation reactions were carried out using SaSrtA and SavSrtE and the deconjugation reactions were carried out with different DUBs.


Defined and homogeneous ubiquitin conjugates resistant to hydrolysis by deubiquitinating enzymes were successfully generated from recombinant ubiquitins and a Gly-Gly appended peptide derived from p53.


This facile semi-synthesis of ubiquitin conjugates establishes a general sortase-mediated route for the synthesis of isopeptide linked polypeptides which is E1/E2/E3-enzyme-independent and can open up new opportunities for the targeted analysis of ubiquitylation and SUMOylation regulatory processes.