N-Glycosylation Enhances Conformational Flexibility of Protein Disulfide Isomerase Revealed by Microsecond Molecular Dynamics and Markov State Modeling

Abstract

Secreted proteins of eukaryotes are decorated with branched carbohydrate oligomers called glycans. This fact is only starting to be considered for in silico investigations of protein dynamics. Using all-atom molecular dynamics (MD) simulations and Markov state modeling (MSM), we unveil the influence of glycans on the conformational flexibility of the multidomain protein disulfide isomerase (PDI), which is a ubiquitous chaperone in the endoplasmic reticulum (ER). Yeast PDI (yPDI) from Saccharomyces cerevisiae is glycosylated at asparagine side chains and the knowledge of its five modified sites enables a realistic computational modeling. We compare simulations of glycosylated and unglycosylated yPDI and find that the presence of glycan–glycan and glycan–protein interactions influences the flexibility of PDI in different ways. For example, glycosylation reduces interdomain interactions, shifting the conformational ensemble toward more open, extended structures. In addition, we compare our results on yPDI with structural information of homologous proteins such as human PDI (hPDI), which is natively unglycosylated. Interestingly, hPDI lacks a surface recess that is present in yPDI. We find that glycosylation of yPDI facilitates its catalytic site to reach close to this surface recess. Hence, this might point to a possible functional relevance of glycosylation in yeast to act on substrates, while glycosylation seems redundant for the human homologous protein. We conclude that glycosylation is fundamental for protein dynamics, making it a necessity for a truthful representation of the flexibility and function in in silico studies of glycoproteins.

The Effects of N-linked Glycosylation on SLC6 Transporters

“…Glycosylation is an essential and universal post-translation modification for regulating protein function. The use of MD simulations enables an atomistic characterization of the structural and dynamic consequences of glycosylation 29,39,40 and other post-translational modifications. 14,41,42 Though, glycosylation has been widely understood to affect SLC6 transporter stability and trafficking, 1,2 our simulations show that N-glycosylation minimally affects overall transporter dynamics, but reduces the fluctuation of the extracellular loops.…”

“…With the surge in performance of graphical processing units and numerical algorithms, molecular dynamics (MD) simulations present a powerful approach to to characterize post-translational modifications and its effect on protein structure and dynamics. Recent applications of atomistic simulations to investigate post-translational modifications has identified how phosphorylation alters the hydrogen bonding network the serotonin transporter, 14 glycosylation induces open conformations of the yeast disulfide isomerase, 29 and nitration prevents ligand binding of a plant abscisic acid receptor. 30 Moreover, MD simulations provide a technique to probe the structural dynamics in a label-free, fully atomistic approach, ideal for addressing the differences in experimental setup.…”

Sequential in vitro enzymatic N-glycoprotein modification reveals site-specific rates of glycoenzyme processing

“…CD16a (Fc ᵧ receptor IIIa) is an IgG receptor that is known to have differential affinities to antibodies depending on its glycan presentation which impacts downstream signaling (22,33,38). Protein disulfide isomerase (PDI) is a resident ER glycoprotein that has been used as a model protein for studying N-glycan J o u r n a l P r e -p r o o f processing due to its ease of expression, analysis, and well-defined site-specific glycan heterogeneity (10,11,34,35,37). Etanercept is a bioengineered therapeutic fusion protein of a TNF⍺ receptor and an IgG1 Fc domain commonly used to help treat auto-immune disorders (39).…”

Sequential in vitro enzymatic N-glycoprotein modification reveals site-specific rates of glycoenzyme processing

“…CD16a (Fc ᵧ receptor IIIa) is an IgG receptor that is known to have differential affinities to antibodies depending on its glycan presentation which impacts downstream signaling (19,29,33). Protein disulfide isomerase (PDI) is a resident ER glycoprotein that has been used as a model protein for studying N-glycan processing due to its ease of expression, analysis, and well-defined site-specific glycan heterogeneity (9,10,(30)(31)(32). Etanercept is a bioengineered therapeutic fusion protein of a TNF⍺ receptor and an IgG1 Fc domain commonly used to help treat auto-immune disorders (34).…”

“…Some recent studies have taken a systems approach, often by monitoring in vivo processing of N-glycans in cell culture systems (9, 10). The involvement of the peptide-glycan interactions in affecting glycan conformation, and thus potentially N-glycan processing, has also been supported by molecular dynamics simulations using yeast protein disulfide isomerase (PDI) as a model reporter glycoprotein (30, 31). A recent study by Mathew et al studied early N-glycan processing steps through molecular dynamics simulations and in vitro processing of N-glycans on the yeast protein disulfide isomerase, a similar approach as this study (32).…”