The essential mammalian enzyme O-GlcNAc Transferase (OGT) is uniquely responsible for transferring N-acetylglucosamine to over a thousand nuclear and cytoplasmic proteins, yet there is no known consensus sequence and it remains unclear how OGT recognizes its substrates. To address this question, we developed a protein microarray assay that chemoenzymatically labels de novo sites of glycosylation with biotin, allowing us to simultaneously assess OGT activity across >6000 human proteins. With this assay we examined the contribution to substrate selection of a conserved asparagine ladder within the lumen of OGT's superhelical tetratricopeptide repeat (TPR) domain. When five asparagines were mutated, OGT retained significant activity against short peptides, but showed limited limited glycosylation of protein substrates on the microarray. O-GlcNAcylation of protein substrates in cell extracts was also greatly attenuated. We conclude that OGT recognizes the majority of its substrates by binding them to the asparagine ladder in the TPR lumen proximal to the catalytic domain.
Despite significant progress in the design of receptors and sensors for simple polyols and monosaccharides, few synthetic receptors discriminate among multiple saccharide units simultaneously, especially under physiological conditions. Described here is the three-dimensional structure of a supramolecular complex-a β-peptide bundle-designed for the potential to interact simultaneously with as many as eight discrete monosaccharide units. The preliminary evaluation of this construct as a vehicle for polyol binding is also presented.
The selective recruitment of oligosaccharides, or even simple sugars, in water solvent is an unsolved molecular recognition problem. Structure-guided, electrostatic redesign led to a significant increase in the affinity of a β-peptide "borono-bundle" for simple sugars in neutral aqueous solution. The affinity for fructose (663 M(-1)) in water should allow its recruitment to the bundle surface for selective catalysis, and future work will focus in this direction.
Metal ion binding is exploited by
proteins in nature to catalyze
reactions, bind molecules, and favor discrete structures, but it has
not been demonstrated in β-peptides or their assemblies. Here
we report the design, synthesis, and characterization of a β-peptide
bundle that uniquely binds two Cd(II) ions in a distinct bicoordinate
array. The two Cd(II) ions bind with positive allosteric cooperativity
and increase the thermodynamic stability of the bundle by more than
50 °C. This system provides a unique, synthetic context to explore
allosteric regulation and should pave the way to sophisticated molecular
assemblies with catalytic and substrate-sensing functions that have
historically not been available to de novo designed synthetic proteomimetics
in water.
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