Structural Investigation of Processing α-Glucosidase I from Saccharomyces cerevisiae

• Main Author: Barker, Megan
• Other Authors: Rose, David
• Language: en_ca
• Published: 2010
• Subjects: Biomolecular structure; Eukaryotic glycosylation; glycosylation; N-glycan; N-linked glycosylation; Carbohydrate; biochemistry; Inhibitor; antiviral therapeutic; Structure-based drug design; glucosidase; glycosidase; glycoside hydrolase; post-translational modification; enzyme; enzyme mechanism; inverting mechanism; structural biology; protein structure; 6xHis tag; Crystal packing; sugar; substrate; X-ray crystallography; single anomalous dispersion; PHENIX; heavy-atom phasing; docking; autodock; autodock vina; biophysical methods; tryptophan fluorescence; glucose oxidase; activity assay; enzyme inhibition; Protein expression; Protein purification; Pichia pastoris; Saccharomyces cerevisiae; fondue; AOX1 promoter; glycoside hydrolysis; substrate binding model; molecular dynamics; Binding site; Active site cleft; Endoplasmic reticulum; Glycan processing; Glycan trimming; protein-protein interactions; cell-cell communication; X-ray diffraction; crystallization; secreted protein; substrate selectivity; kojibiose; glucotriose; miglitol; deoxynojirimycin; Glc3Man9GlcNAc2; free oligosaccharide species; GluI; Cwh41; Cwh41p; Cwht1p; 0306; 0307; 0760
• Online Access: http://hdl.handle.net/1807/32660

N-glycosylation is the most common eukaryotic post-translational modification, impacting on protein stability, folding, and protein-protein interactions. More broadly, N-glycans play biological roles in reaction kinetics modulation, intracellular protein trafficking, and cell-cell communications. The machinery responsible for the initial stages of N-glycan assembly and processing is found on the membrane of the endoplasmic reticulum. Following N-glycan transfer to a nascent glycoprotein, the enzyme Processing α-Glucosidase I (GluI) catalyzes the selective removal of the terminal glucose residue. GluI is a highly substrate-specific enzyme, requiring a minimum glucotriose for catalysis; this glycan is uniquely found in biology in this pathway. The structural basis of the high substrate selectivity and the details of the mechanism of hydrolysis of this reaction have not been characterized. Understanding the structural foundation of this unique relationship forms the major aim of this work. To approach this goal, the S. cerevisiae homolog soluble protein, Cwht1p, was investigated. Cwht1p was expressed and purified in the methyltrophic yeast P. pastoris, improving protein yield to be sufficient for crystallization screens. From Cwht1p crystals, the structure was solved using mercury SAD phasing at a resolution of 2 Å, and two catalytic residues were proposed based upon structural similarity with characterized enzymes. Subsequently, computational methods using a glucotriose ligand were applied to predict the mode of substrate binding. From these results, a proposed model of substrate binding has been formulated, which may be conserved in eukaryotic GluI homologs.