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

Abstract

Cotranslational N-glycosylation can accelerate protein folding, slow protein unfolding, and increase protein stability, but the molecular basis for these energetic effects is incompletely understood. 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 six-residue 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. Comparing the energetic effects of glycosylating these three enhanced aromatic sequons in the same host WW domain revealed that the glycosylation-mediated stabilization is greatest for the enhanced aromatic sequon complementary to the type I β-turn with a G1 β-bulge. However, the portion of the stabilization from the tripartite interaction between Phe, Asn(GlcNAc), and Thr is similar for each enhanced aromatic sequon in its respective reverse turn context. Adding the Phe-Asn-Xxx-Thr motif (in a type I' β-turn) to the enhanced aromatic sequon family doubles the number of proteins that can be stabilized by glycosylation without having to alter the native reverse turn type.

Fig. 1.

Matching enhanced aromatic sequons with reverse turn conformations that can facilitate stabilizing interactions among Phe, Asn(GlcNAc1), and Thr. (A) Glycosylated five-residue type I β-bulge turn from the adhesion domain of the human protein CD2 (PDB accession code 1GYA; ref. 31). (Left) CPK representation; (Right) stick representation. (B) Type II β-turn in a six-residue loop (PDB accession code 1PIN; ref. 34) (C) five-residue type I β-bulge turn (PDB accession code 2F21; ref. 36), and (D) four-residue type I′ β-turn (PDB accession code 1ZCN; ref. 36) from variants of the WW domain of human protein Pin1. Structures are rendered in Pymol, with dotted lines depicting hydrogen bonds. Amino acid positions within the WW domain where we incorporated components of the enhanced aromatic sequon are highlighted in yellow. Interatomic distances between the side-chain beta carbons (Cβ’s) at these positions are indicated with black lines, the distances in Å are depicted in black font.

Fig. 2.

Triple mutant cycle cubes formed by protein 4, glycoprotein 4g, and their derivatives (A); protein 5, glycoprotein 5g, and their derivatives (B); and protein 6, glycoprotein 6g, and their derivatives (C).

Fig. 3.

Origin of the increase in stability of 4-F,T, 5-F,T, and 6-F,T upon glycosylation. ΔΔG_f,total (yellow bars) is the sum of the energetic effects of (1) the Asn19 to Asn(GlcNAc)19 mutation (, blue bars); (2) the two-way interaction between Phe16 and Asn(GlcNAc)19 (, red bars); (3) the two-way interaction between Asn(GlcNAc)19 and Thr21 (, green bars); and (4) the three-way interaction between Phe16, Asn(GlcNAc)19, and Thr21 (, purple bars). , , , and , are parameters obtained from least-squares regression of Eq. 1; error bars represent the corresponding standard errors.