Predictions for α-Helical Glycopeptide Design from Structural Bioinformatics Analysis

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Glycosylation not only impacts the functions of glycoproteins but can also improve glycoprotein stability and folding efficiency—characteristics that are desirable for protein engineering and therapeutic design. To further elucidate the effects of N-glycosylation on protein structure and to provide principles useful for the rational design of α-helical glycopeptides, we investigate stabilizing protein–sugar interactions in α-helical glycosylation sites using an integrated structural bioinformatics analysis and molecular dynamics simulation approach. We identify two glycan conformations with an Asn χ₁ of 180° or 300° that are amenable to α-helical structure in natural α-helical glycosylation sites in the Protein Data Bank. A combination of sterics and favorable intraglycopeptide enthalpy explains the existence of only these two conformations. Furthermore, we catalog all known protein–sugar interactions that utilize these conformational modes. The most common interactions involve either a Glu residue at the −4 position interacting with the GlcNAc whose Asn has χ₁ = 300° or a Glu residue at the +4 position interacting with the GlcNAc whose Asn has χ₁ = 180°. Via metadynamics simulations of model α-helical glycopeptides with each of these two interactions, we find that both interactions are stabilizing as a result of favorable electrostatic intraglycopeptide interactions. Thus, we suggest that incorporating a Glu at either the −4 or +4 position relative to an N-linked glycan may be a useful strategy for engineering stable α-helical glycoproteins.