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

Abstract

Heterotypic polyubiquitins are an emerging class of polyubiquitins that have attracted interest because of their potential diversity of structures and physiological functions. There is an increasing demand for structure-defined synthesis of heterotypic chains to investigate the topological factors underlying the intracellular signals that are characteristically mediated by the heterotypic chain. However, the applicability of chemical and enzymatic polyubiquitin synthesis developed to date has been limited by laborious rounds of ligation and purification or by a lack of modularity of the chain structure with respect to the length and the branch position. Here, we established a one-pot, photocontrolled synthesis of structurally defined heterotypic polyubiquitin chains. We designed ubiquitin derivatives with a photolabile protecting group at a lysine residue used for polymerization. Repetitive cycles of linkage-specific enzymatic elongation and photoinduced deprotection of the protected ubiquitin units enabled stepwise addition of ubiquitins with appropriate functionalities to control the length and branching positions. The positional control of branching was achieved without isolation of intermediates, allowing one-pot synthesis of K63 triubiqutin chains and a K63/K48 heterotypic tetraubiquitin chain with defined branching positions. The present study provides a chemical platform for the efficient construction of long polyubiquitin chains with defined branch structures that will facilitate the understanding of the essential relationships between functions and structures of the heterotypic chain that have hitherto been overlooked.

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

“…The finding that a non-Lys mutation can prevent or deter continued chain formation may be of relevance to ongoing efforts in chemoenzymatic preparation of branched Ub chains. [40] Although KAHA ligation to form Ub with Ala46Hse and AcÀ Met1Nle provides high quality material in larger quantities, the detrimental effect of these mutation on the activity of Ubc13/Mms2 mediated chain formation renders the previously reported linear synthesis the currently preferred method for access to synthetic Ub derivatives, an approach we have used in our recent research efforts. The folding and activity are also influenced heavily by the length of the Lys side chains, impeding the acceptor ability greatly.…”

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

“… , Enzyme-based construction of branched Ub chains can be achieved by mixing two linkage-selective E2s and mutating Ub lysines to arginines to obtain V-shaped K11/K48 or K48/K63 branched trimers with extensive Lys to Arg mutations. , Strieter et al successfully synthesized branched triubiquitin activity-based probes by a combination of enzymatic conjugation and a Cys modification reaction . During the revision of this manuscript, Okamoto and co-workers published an approach to ubiquitin chains using a photocleavable lysine protecting group . Lang et al have prepared heterotypic and branched Ub trimers without Lys to Arg mutations by sortase-mediated conjugations of Ub monomers containing Gly-Gly-Lys isopeptides .…”

“… 26 During the revision of this manuscript, Okamoto and co-workers published an approach to ubiquitin chains using a photocleavable lysine protecting group. 27 Lang et al have prepared heterotypic and branched Ub trimers without Lys to Arg mutations by sortase-mediated conjugations of Ub monomers containing Gly-Gly-Lys isopeptides. 28 Fushman and co-workers have incorporated a Boc-protected lysine at K48 by genetic code expansion and enzymatically conjugated the protected Ub to an acceptor Ub with C-terminal blocking such as Ub (1–77) or truncated Ub (1-74) to selectively obtain a Ub dimer.…”