Crystal structure of glycogen debranching enzyme and insights into its catalysis and disease-causing mutations

Abstract

Glycogen is a branched glucose polymer and serves as an important energy store. Its debranching is a critical step in its mobilization. In animals and fungi, the 170 kDa glycogen debranching enzyme (GDE) catalyses this reaction. GDE deficiencies in humans are associated with severe diseases collectively termed glycogen storage disease type III (GSDIII). We report crystal structures of GDE and its complex with oligosaccharides, and structure-guided mutagenesis and biochemical studies to assess the structural observations. These studies reveal that distinct domains in GDE catalyse sequential reactions in glycogen debranching, the mechanism of their catalysis and highly specific substrate recognition. The unique tertiary structure of GDE provides additional contacts to glycogen besides its active sites, and our biochemical experiments indicate that they mediate its recruitment to glycogen and regulate its activity. Combining the understanding of the GDE catalysis and functional characterizations of its disease-causing mutations provides molecular insights into GSDIII.

Figure 1. Structure of CgGDE.

(a) Domain arrangement of GDE. Domains M1, M2 and GC, and the GT subdomains A, B and C are colour-coded. This colouring scheme is used throughout the manuscript unless otherwise indicated. Domain boundaries for CgGDE are indicated. (b,c) Crystal structure of CgGDE. The views are related by an 180° rotation around the vertical axis. The red stars in b indicate the active sites. Bound oligosaccharides are shown in stick representation with their carbon atoms in black. Structural figures were prepared with pymol (http://www.pymol.org).

Figure 2. Structure and function of the GT domain.

(a) Structure of the GT domain. Structure of Taka-amylase A (right, PDB 7TAA) is shown for reference. CSRI–IV in both structures are coloured in blue and cyan, respectively. Catalytic residues are highlighted. Subdomain B and the equivalent region in Taka-amylase A (domain B) are omitted for clarity. (b) Structure of the GT domain active-site pocket. The −1 subsite in Taka-amylase A (grey for the carbon atoms) is superimposed for reference. Amino acid residue labels on the second lines are for Taka-amylase A. Catalytic residues are labelled in red. The B-1 residue of the bound oligosaccharide B in the maltopentaose complex structure and the −1 saccharide unit of acarbose in the Taka-amylase A structure are shown. (c) GT activity of CgGDE and its mutants. The reactions catalysed by CgGDE and its mutants with maltopentaose as the substrate were analysed with thin-layer chromatography. In the control experiment (lane Ctrl) no CgGDE were added to the reaction. Oligosaccharides with 2–7 residues (G2–G7) were used as standards.

Figure 3. Structure and function of the GC domain.

(a) Structure of the GC domain. Structure of the Aspergillus awamori glucoamylase catalytic domain (right, PDB 1GAH) is shown for reference. Catalytic residues are highlighted. (b) Structure of the GC domain active-site pocket. Structure of the Aspergillus awamori glucoamylase is superimposed for reference (grey for the carbon atoms). Amino acid residue labels on the second lines are for glucoamylase. Catalytic residues are labelled in red. The +1 and −1 saccharide units of acarbose in the glucoamylase structure are shown. They mimic the +1 and −1 residues in its substrate, the glycosidic bond between which gets hydrolysed. (c) Specific debranching activities of CgGDE and its mutants. The specific activity is defined as the debranching reaction rate at the substrate concentration of 13 mg ml⁻¹ (the reaction rate of the wild-type CgGDE plateaus at this substrate concentration, see Supplementary Fig. 4a), divided by the concentration of CgGDE. The error bars indicate standard deviations of triplicate experiments. The numbers above each bar indicate ratios to the wild-type value. ND indicates not detected. (d) Specific debranching activities of combinations of a GT-defective mutant and a GC-defective mutant. In reactions catalysed by a combination of mutants, they were added in a 1:1 ratio, and the concentration of one is used in calculating the specific activity. The activity of the wild-type CgGDE is shown for reference.

Figure 4. Substrate recognition by the GT domain active site.

(a) Difference electron-density map for the oligosaccharides bound at the GT domain active site. The map shown here and in Fig. 5a was calculated before oligosaccharides were incorporated in the atomic model, and contoured at 2 σ. (b) Accommodation of oligosaccharide M by the GT domain active site. (c) Accommodation of oligosaccharide B by the GT domain active site. Part of the nearby oligosaccharide M is also shown. The +1 and −1 saccharide units of acarbose in the Taka-amylase A structure (PDB 7TAA, grey for the carbon atoms) is shown in partial transparency. They mimic the +1 and −1 residues in the substrate. (d) Specific debranching activities of the W470A mutant and its combinations with mutants possessing only the GT or the GC activities.

Figure 5. Substrate recognition by the GC domain active site.

(a) Difference electron-density map for the oligosaccharide bound at the GC domain active site. (b) Accommodation of this oligosaccharide by the GC domain. (c) Specific debranching activities of CgGDE mutants R1123G and Y1407F, and their combinations with mutants possessing only the GT or GC activities.

Figure 6. Additional contacts of CgGDE with glycogen.

(a–c) Structures of the additional oligosaccharide-binding sites in GT subdomain B (a), domains M2 (b) and GC (c). (d) Mutations at the additional oligosaccharide-binding sites decrease the affinity to glycogen. CgGDE pulled-down by glycogen immobilized on concanavalin A agarose was analysed by SDS PAGE. (e) Specific debranching activities of CgGDE mutants Y408A, W958A, D1400A and their combinations with mutants possessing only the GT or GC activities.

Figure 7. Disease-causing mutations and sequence conservation of GDE.

(a) Disease-causing mutations of GDE. Missense mutations found in GSDIII patients are mapped onto the CgGDE structure, and represented by black spheres. (b) GDE residue conservation. The CgGDE is shown in surface representation, and coloured according to the conservation of individual residues. Bound oligosaccharides are shown in stick representation with their carbon atoms in white.