Protein Native-State Stabilization by Placing Aromatic Side Chains in N-Glycosylated Reverse Turns

Abstract

N-glycosylation of eukaryotic proteins helps them fold and traverse the cellular secretory pathway and can increase their stability, although the molecular basis for stabilization is poorly understood. Glycosylation of proteins at naïve sites (ones that normally are not glycosylated) could be useful for therapeutic and research applications, but currently results in unpredictable changes to protein stability. We show that placing a Phe residue two or three positions prior to a glycosylated Asn in distinct reverse turns facilitates stabilizing interactions between the aromatic side chain and the first N-acetylglucosamine (GlcNAc) of the glycan. Glycosylating this portable structural module, an “enhanced aromatic sequon”, in three different proteins stabilizes their native states by −0.7 to −2.0 kilocalories per mole and increases cellular glycosylation efficiency.

Specificity of O -glycosylation in enhancing the stability and cellulose binding affinity of Family 1 carbohydrate-binding modules

“…Additional attachment of mannose to the CBM only induces minor changes in T m . This finding is similar to the observations for studies of N-glycosylation, indicating that the large enhancements caused by O-mannosylation at Ser3 might be at least partially caused by interactions between the first two mannose units attached to Ser3 and its local amino acid residues (36,49).…”

Glycosylation of the enhanced aromatic sequon is similarly stabilizing in three distinct reverse turn contexts

“…N-glycosylation of proteins at naïve sites could be a useful strategy for stabilizing proteins in therapeutic and research applications, but without engineering guidelines, often results in unpredictable changes to protein energetics. We recently introduced the enhanced aromatic sequon as a family of portable structural motifs that are stabilized upon glycosylation in specific reverse turn contexts: a five-residue type I β-turn harboring a G1 β-bulge (using a Phe-Yyy-Asn-Xxx-Thr sequon) and a type II β-turn within a sixresidue loop (using a Phe-Yyy-Zzz-Asn-Xxx-Thr sequon) [Culyba EK, et al (2011) Science 331:571-575]. Here we show that glycosylating a new enhanced aromatic sequon, Phe-Asn-Xxx-Thr, in a type I′ β-turn stabilizes the Pin 1 WW domain.…”

“…We recently demonstrated that glycosylation of an Asn residue within the sequence Phe-Yyy-Asn-Xxx-Thr (where Yyy can likely be any amino acid and Xxx is any amino acid but Pro) stabilizes the glycosylation-naïve rat CD2 adhesion domain (RnCD2ad) and human muscle acylphosphatase (AcyP2) by approximately −2 kcal mol −1 , provided that Asn is located at the i þ 2 position of a type I β-turn with a G1 β-bulge (28, 29), hereafter called a type I β-bulge turn (30). Published structural data (31) from the human ortholog of RnCD2ad (HsCD2ad, Fig.…”

Communication: Folding of glycosylated proteins under confinement

“…new interactions between it and the protein 6 as from its bulkiness, which forces a less compact structure on the protein's unfolded state. 4 Another well-known mechanism for altering the biophysical and structural features of proteins is confinement.…”

“…In the bulk, glycosylation with the monosaccharide increases T F by 0.6%-1.2% and glycosylation with the penta-saccharide increases T F by 0.5%-1.9% compared to the T F of the unmodified protein. This increase in T F translates into an increase in the population of the folded state from 50% to 62% for SH3-(GlcNAc) 6 and to 70% for SH3-(Man 3 GlcNAc 2 ) 6 .…”