Nonenzymatic Assembly of Natural Polyubiquitin Chains of Any Linkage Composition and Isotopic Labeling Scheme

Abstract

Polymeric chains made of a small protein ubiquitin act as molecular signals regulating a variety of cellular processes controlling essentially all aspects of eukaryotic biology. Uncovering the mechanisms that allow differently linked polyubiquitin chains to serve as distinct molecular signals requires the ability to make these chains with the native connectivity, defined length, linkage composition, and in sufficient quantities. This however has been a major impediment in the ubiquitin field. Here we present a robust, efficient, and widely accessible method for controlled iterative non-enzymatic assembly of polyubiquitin chains using recombinant ubiquitin monomers as the primary building blocks. This method uses silver-mediated condensation reaction between the C-terminal thioester of one ubiquitin and the ε-amine of a specific lysine on the other ubiquitin. We augment the non-enzymatic approaches developed recently by using removable orthogonal amine-protecting groups, Alloc and Boc. The use of bacterially expressed ubiquitins allows cost-effective isotopic enrichment of any individual monomer in the chain. We demonstrate that our method yields completely natural polyubiquitin chains (free of mutations and linked through native isopeptide bonds) of essentially any desired length, linkage composition, and isotopic labeling scheme, and in milligram quantities. Specifically, we successfully made Lys11-linked di-, tri-, and tetra-ubiquitins, Lys33-linked di-ubiquitin, and a mixed-linkage Lys33,Lys11-linked tri-ubiquitin. We also demonstrate the ability to obtain, by high-resolution NMR, residue-specific information on ubiquitin units at any desired position in such chains. This method opens up essentially endless possibilities for rigorous structural and functional studies of polyubiquitin signals.

Linkage-specific conformational ensembles of non-canonical polyubiquitin chains

“…K6, K27, K29, and K33-linked chains that are free of any mutations were made using the nonenzymatic assembly approach. 20 Ub 2 s containing K11, K48, and K63 linkages were made enzymatically employing chain-terminating mutations. 4,31,50 We applied NMR spectroscopy to attain atomic-level resolution into structural and dynamical properties of each Ub 2 in solution.…”

“…With recent advances in chemical biology techniques employing native isopeptide linkages (e.g. 20,21 ) and recent discoveries of several linkage-specific deubiquitinating enzymes 5 , crystal structures have been obtained for the unbound versions of some of these chains (Figure 1). 4,5,22–26 At the time of this writing, K27 remains the only lysine linkage for which there is no crystal structure.…”

Synthetic polyubiquitinated α-Synuclein reveals important insights into the roles of the ubiquitin chain in regulating its pathophysiology

“…These advances provided access to new structural information about K33-and K6-linked di-Ub chains using NMR and X-ray crystallography, respectively (11,12). More recently, tetra-Ub chains in a free form or anchored to a tripeptide were also synthesized chemically (13)(14)(15).…”

Peeling away the layers of ubiquitin signaling complexities with synthetic ubiquitin–protein conjugates

“…Depending on the size of the polypeptide substrate, the auxiliary can be introduced using solid-phase peptide synthesis [19••] or the nonsense-suppression strategy involving an orthogonal transfer RNA synthetase and tRNA CUA pair (where CUA is the anticodon of the tRNA) [20]. Nonsense-suppression technology can also be used to incorporate orthogonal amine-protecting groups for site-specific isopeptide bond formation (Figure 2B) [21,22•]. …”