One-Step Sortase-Mediated Chemoenzymatic Semisynthesis of Deubiquitinase-Resistant Ub-Peptide Conjugates

[Image: see text] Post-translational modifications (PTMs) of proteins increase the functional diversity of the proteome and play crucial regulatory roles in cellular processes. Ubiquitination is a highly regulated and reversible PTM accomplished by a complex multistep process with the sequential action of several specific ubiquitinating (E1–E3) and deubiquitinating enzymes. The different types of ubiquitination (mono-, poly-mono-, and poly-) and the presence of several target sites in a single substrate add to its complexity, which makes the in vitro reconstitution of this ubiquitin (Ub) machinery a quite cumbersome process. Defects in components of the ubiquitination process also contribute to disease pathogenesis, especially cancer and neurodegeneration. This makes them of interest as potential therapeutic targets. Therefore, the development of efficient and reliable methods that will generate a highly homogeneous ubiquitinated peptide and protein conjugate is a topical subject area of research. In this report, we describe the development of a simple and efficient in vitro sortase-mediated chemoenzymatic strategy for semisynthesis of defined and homogeneous ubiquitin conjugates with more than 90% yield. This was achieved by engineering a sortase recognition motif in the dynamic C-terminus of ubiquitin and its conjugation to an isopeptide-linked di-Gly appended peptide LMFK(ε-GG)TEG corresponding to the ubiquitination site residues (383)LMFKTEG(389) of p53. The defined and homogeneous ubiquitin conjugates were also weighed for their recognition propensity by deubiquitinating enzymes. This facile semisynthesis of ubiquitin conjugates establishes a simple one-step sortase-mediated chemoenzymatic route for the synthesis of homogeneous and defined isopeptide-linked polypeptides and will help in understanding the complexity of the ubiquitination machinery as well as designing isopeptide drugs and therapeutics.