Chemical Protein Synthesis Enabled Mechanistic Studies on the Molecular Recognition of K27-linked Ubiquitin Chains.

New synthetic strategies that exploited the strengths of both chemoselective ligation and recombinant protein expression were developed to prepare K27 di-ubiquitins (diUb), which enabled mechanistic studies on the molecular recognition of K27-linked Ubs by single-molecule Förster resonance energy transfer (smFRET) and X-ray crystallography. The results revealed that free K27 diUb adopted a compact conformation, whereas upon binding to UCHL3, K27 diUb was remodeled to an open conformation. The K27 isopeptide bond remained rigidly buried inside the diUb moiety during binding, an interesting unique structural feature that may explain the distinctive biological function of K27 Ub chains.

Small molecule inhibitors reveal allosteric regulation of USP14 via steric blockade.

The ubiquitin system is important for drug discovery, and the discovery of selective small-molecule inhibitors of deubiquitinating enzymes (DUBs) remains an active yet extremely challenging task. With a few exceptions, previously developed inhibitors have been found to bind the evolutionarily conserved catalytic centers of DUBs, resulting in poor selectivity. The small molecule IU1 was the first-ever specific inhibitor identified and exhibited surprisingly excellent selectivity for USP14 over other DUBs. However, the molecular mechanism for this selectivity was elusive. Herein, we report the high-resolution co-crystal structures of the catalytic domain of USP14 bound to IU1 and three IU1 derivatives. All the structures of these complexes indicate that IU1 and its analogs bind to a previously unknown steric binding site in USP14, thus blocking the access of the C-terminus of ubiquitin to the active site of USP14 and abrogating USP14 activity. Importantly, this steric site in USP14 is very unique, as suggested by structural alignments of USP14 with several known DUB X-ray structures. These results, in conjunction with biochemical characterization, indicate a coherent steric blockade mechanism for USP14 inhibition by compounds of the IU series. In light of the recent report of steric blockade of USP7 by FT671, this work suggests a potential generally applicable allosteric mechanism for the regulation of DUBs via steric blockade, as showcased by our discovery of IU1-248 which is 10-fold more potent than IU1.

Productive Amyrin Synthases for Efficient α-Amyrin Synthesis in Engineered Saccharomyces cerevisiae.

α-Amyrin is a plant-derived pentacyclic triterpenoid, with a lot of important physiological and pharmacological activities. The formation of α-amyrin from (3 S)-2,3-oxidosqualene is catalyzed by α-amyrin synthase (α-AS), a member of the oxidosqualene cyclase (OSC) protein family. However, α-amyrin is not yet commercially developed due to its extremely low productivity in plants. The engineered Saccharomyces cerevisiae with efficient α-amyrin production pathway could be used as an alternative and sustainable solution to produce α-amyrin from renewable raw materials. To efficiently improve α-amyrin production in S. cerevisiae, we identified two α-ASs, EjAS and MdOSC1 from Eriobotrya japonica and Malus × domestica, respectively, through strict bioinformatics screening criteria and phylogenetic analysis. The specific activities of purified EjAS and MdOSC1 were 0.0032 and 0.0293 µmol/min/mg, respectively. EjAS produced α-amyrin and ß-amyrin at a ratio of 17:3, MdOSC1 produced α-amyrin, ß-amyrin and lupeol at a ratio of 86:13:1, indicating MdOSC1 had significantly higher specific activity and higher ratio of α-amyrin than EjAS. Furthermore, MdOSC1 was introduced into S. cerevisiae combining with the increased supply of (3 S)-2,3-oxidosqualene to achieve the encouraging α-amyrin production, and the titer of α-amyrin achieved 11.97 ± 0.61 mg/L, 5.8 folds of the maximum production reported.