One-Pot, Photocontrolled Enzymatic Assembly of the Structure-Defined Heterotypic Polyubiquitin Chain

Heterotypic polyubiquitins are an emerging class of polyubiquitinsthat have attracted interest because of their potential diversityof structures and physiological functions. There is an increasingdemand for structure-defined synthesis of heterotypic chains to investigatethe topological factors underlying the intracellular signals thatare characteristically mediated by the heterotypic chain. However,the applicability of chemical and enzymatic polyubiquitin synthesisdeveloped to date has been limited by laborious rounds of ligationand purification or by a lack of modularity of the chain structurewith respect to the length and the branch position. Here, we establisheda one-pot, photocontrolled synthesis of structurally defined heterotypicpolyubiquitin chains. We designed ubiquitin derivatives with a photolabileprotecting group at a lysine residue used for polymerization. Repetitivecycles of linkage-specific enzymatic elongation and photoinduced deprotectionof the protected ubiquitin units enabled stepwise addition of ubiquitinswith appropriate functionalities to control the length and branchingpositions. The positional control of branching was achieved withoutisolation of intermediates, allowing one-pot synthesis of K63 triubiqutinchains and a K63/K48 heterotypic tetraubiquitin chain with definedbranching positions. The present study provides a chemical platformfor the efficient construction of long polyubiquitin chains with definedbranch structures that will facilitate the understanding of the essentialrelationships between functions and structures of the heterotypicchain that have hitherto been overlooked.

Automated summary

Heterotypic polyubiquitins, a newly emerging class of polyubiquitins, have attracted attention due to their diverse structures and physiological functions. However, the current methods for synthesizing heterotypic chains have limitations in terms of laborious processes and lack of modularity. In this study, a one-pot, photocontrolled synthesis method was established to construct structurally defined heterotypic polyubiquitin chains.