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Glycosylation is a key mechanism for orchestrating the bioactivity, metabolism and location of small molecules in living cells. In plants, a large multigene family of glycosyltransferases is involved in these processes, conjugating hormones, secondary metabolites, biotic and abiotic environmental toxins, to impact directly on cellular homeostasis. The red grape enzyme UDP‐glucose:flavonoid 3‐ O ‐glycosyltransferase ( Vv GT1) is responsible for the formation of anthocyanins, the health‐promoting compounds which, in planta , function as colourants determining flower and fruit colour and are precursors for the formation of pigmented polymers in red wine. We show that Vv GT1 is active, in vitro , on a range of flavonoids. Vv GT1 is somewhat promiscuous with respect to donor sugar specificity as dissected through full kinetics on a panel of nine sugar donors. The three‐dimensional structure of Vv GT1 has also been determined, both in its ‘Michaelis’ complex with a UDP‐glucose‐derived donor and the acceptor kaempferol and in complex with UDP and quercetin. These structures, in tandem with kinetic dissection of activity, provide the foundation for understanding the mechanism of these enzymes in small molecule homeostasis.

Tumour necrosis factor (TNF) is a trimeric protein which signals through two membrane receptors, TNFR1 and TNFR2. Previously, we identified small molecules that inhibit human TNF by stabilising a distorted trimer and reduce the number of receptors bound to TNF from three to two. Here we present a biochemical and structural characterisation of the small molecule-stabilised TNF-TNFR1 complex, providing insights into how a distorted TNF trimer can alter signalling function. We demonstrate that the inhibitors reduce the binding affinity of TNF to the third TNFR1 molecule. In support of this, we show by X-ray crystallography that the inhibitor-bound, distorted, TNF trimer forms a complex with a dimer of TNFR1 molecules. This observation, along with data from a solution-based network assembly assay, leads us to suggest a model for TNF signalling based on TNF-TNFR1 clusters, which are disrupted by small molecule inhibitors. Small molecules stabilising a distorted TNF trimer can inhibit TNF signaling, but the underlying mechanism is unclear. Here, the authors characterize the inhibitor-bound TNF-receptor complex structurally and biochemically, showing that the inhibitors alter TNF-receptor binding stoichiometry and cluster formation.

Eukaryotic initiation factor 4E (eIF4E) serves as a regulatory hub for oncogene-driven protein synthesis and is considered a promising anticancer target. Here we screen a fragment library against eIF4E and identify a ligand-binding site with previously unknown function. Follow-up structure-based design yields a low nM tool compound ( 4 , K d  = 0.09 µM; LE 0.38), which disrupts the eIF4E:eIF4G interaction, inhibits translation in cell lysates, and demonstrates target engagement with eIF4E in intact cells (EC 50  = 2 µM). By coupling targeted protein degradation with genetic rescue using eIF4E mutants, we show that disruption of both the canonical eIF4G and non-canonical binding sites is likely required to drive a strong cellular effect. This work highlights the power of fragment-based drug discovery to identify pockets in difficult-to-drug proteins and how this approach can be combined with genetic characterization and degrader technology to probe protein function in complex biological systems. A structure-guided fragment screen identified a compound, which interacts with a ligand-binding site of unknown function in eukaryotic initiation factor 4E (eIF4E). X-ray crystallography was used to characterise binding and target engagment was shown in cells.

Glycosylation of macrolide antibiotics confers host cell immunity from endogenous and exogenous agents. The Streptomyces antibioticus glycosyltransferases, OleI and OleD, glycosylate and inactivate oleandomycin and diverse macrolides including erythromycin, respectively. The structure of these enzyme-ligand complexes, in tandem with kinetic analysis of site-directed variants, provide insight into the interaction of macrolides with their synthetic apparatus. Erythromycin binds to OleD and the 23S RNA of its target ribosome in the same conformation and, although the antibiotic contains a large number of polar groups, its interaction with these macromolecules is primarily through hydrophobic contacts. Erythromycin and oleandomycin, when bound to OleD and OleI, respectively, adopt different conformations, reflecting a subtle effect on sugar positioning by virtue of a single change in the macrolide backbone. The data reported here provide structural insight into the mechanism of resistance to both endogenous and exogenous antibiotics, and will provide a platform for the future redesign of these catalysts for antibiotic remodelling.

The enzymatic transfer of activated mannose yields mannosides in glycoconjugates and oligo- and polysaccharides. Yet, despite its biological necessity, the mechanism by which glycosyltransferases recognize mannose and catalyze its transfer to acceptor molecules is poorly understood. Here, we report broad high-throughput screening and kinetic analyses of both natural and synthetic substrates of Rhodothermus marinus mannosylglycerate synthase (MGS), which catalyzes the formation of the stress protectant 2- O -α- D -mannosyl glycerate. The sequence of MGS indicates that it is at the cusp of inverting and retaining transferases. The structures of apo MGS and complexes with donor and acceptor molecules, including GDP-mannose, combined with mutagenesis of the binding and catalytic sites, unveil the mannosyl transfer center. Nucleotide specificity is as important in GDP- D -mannose recognition as the nature of the donor sugar.

Cytoplasmic O -GlcNac modification of proteins is thought to have dynamic interplay with phosphorylation and thus be involved in regulation of signaling processes. The complete structure of an OGT homolog is now presented, suggesting how diverse ligands can be presented to the active site of the enzyme. N -Acetylglucosamine ( O -GlcNAc) modification of proteins provides a mechanism for the control of diverse cellular processes through a dynamic interplay with phosphorylation. UDP-GlcNAc:polypeptidyl transferase (OGT) catalyzes O -GlcNAc addition. The structure of an intact OGT homolog and kinetic analysis of human OGT variants reveal a contiguous superhelical groove that directs substrates to the active site.

Immunomodulatory imide drugs (IMiDs) including thalidomide, lenalidomide, and pomalidomide, can be used to induce degradation of a protein of interest that is fused to a short zinc finger (ZF) degron motif. These IMiDs, however, also induce degradation of endogenous neosubstrates, including IKZF1 and IKZF3. To improve degradation selectivity, we took a bump-and-hole approach to design and screen bumped IMiD analogs against 8380 ZF mutants. This yielded a bumped IMiD analog that induces efficient degradation of a mutant ZF degron, while not affecting other cellular proteins, including IKZF1 and IKZF3. In proof-of-concept studies, this system was applied to induce efficient degradation of TRIM28, a disease-relevant protein with no known small molecule binders. We anticipate that this system will make a valuable addition to the current arsenal of degron systems for use in target validation.