Biocompatible Lysine Protecting Groups for the Chemoenzymatic Synthesis of K48/K63 Heterotypic and Branched Ubiquitin Chains

Abstract

The elucidation of emerging biological functions of heterotypic and branched ubiquitin (Ub) chains requires new strategies for their preparation with defined lengths and connectivity. While in vitro enzymatic assembly using expressed E1-activating and E2-conjugating enzymes can deliver homotypic chains, the synthesis of branched chains typically requires extensive mutations of lysines or other sequence modifications. The combination of K48- and K63-biased E2-conjugating enzymes and two new carbamate protecting groupspyridoxal 5′-phosphate (PLP)-cleavable aminobutanamide carbamate (Abac group) and periodate-cleavable aminobutanol carbamate (Aboc group)provides a strategy for the synthesis of heterotypic and branched Ub trimers, tetramers, and pentamers. The Abac- and Aboc-protected lysines are readily prepared and incorporated into synthetic ubiquitin monomers. As these masking groups contain a basic amine, they preserve the overall charge and properties of the Ub structure, facilitating folding and enzymatic conjugations. These protecting groups can be chemoselectively removed from folded Ub chains and monomers by buffered solutions of PLP or NaIO4. Through the incorporation of a cleavable C-terminal His-tag on the Ub acceptor, the entire process of chain building, iterative Abac deprotections, and global Aboc cleavage can be conducted on a resin support, obviating the need for handling and purification of the intermediate oligomers. Simple modulation of the Ub monomers affords various K48/K63 branched chains, including tetramers and pentamers not previously accessible by synthetic or biochemical methods.

Atomic Tailoring of Ubiquitin Side Chains Influences E2‐Mediated Ubiquitin Chain Formation

“…Using this strategy, the chain linkage would be determined by pre-designed Ub, regardless of the specificity of E2 for specific lysine residues. [21] Securing the necessary Ub monomers requires chemical synthesis to access proteins with at least 6 non-canonical amino acid residues. Full-length Ub can be prepared by solid phase peptide synthesis (SPPS) in a linear manner [22] -an approach used to access some of the Ub monomers used in this work, but which requires numerous pseudoproline building blocks and multiple purifications.…”