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Structural basis of glycogen branching enzyme deficiency and pharmacologic rescue by rational peptide design.

Froese DS, Michaeli A, McCorvie TJ, Krojer T, Sasi M, Melaev E, Goldblum A, Zatsepin M, Lossos A, Álvarez R, Escribá PV, Minassian BA, von Delft F, Kakhlon O, Yue WW - Hum. Mol. Genet. (2015)

Bottom Line: Expression of recombinant GBE1-p.Y329S resulted in drastically reduced protein yield and solubility compared with wild type, suggesting this disease allele causes protein misfolding and may be amenable to small molecule stabilization.We demonstrate intracellular transport of this peptide, its binding and stabilization of GBE1-p.Y329S, and 2-fold increased mutant enzymatic activity compared with untreated patient cells.Together, our data provide the rationale and starting point for the screening of small molecule chaperones, which could become novel therapies for this disease.

View Article: PubMed Central - PubMed

Affiliation: Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, OX3 7DQ, UK.

No MeSH data available.


Related in: MedlinePlus

Oligosaccharide binding to hGBE1. (A) Chemical structures of acarbose (ACR) and (Glc7). (B) Surface representation of hGBE1 (Fig. 1A colouring) showing the bound oligosaccharides. (C) ACR binding cleft at the interface of the helical segment (orange), CBM48 (pink) and catalytic domain (green). Shown in sticks are ACR (yellow carbon atoms) and its contact protein residues (white carbon atoms). Inset, 2Fo-Fc electron density for the modelled ACR. (D) Sequence alignment of the ACR-binding residues of hGBE1 (underlined). Annotated branching enzyme sequences are from human (Uniprot ID Q04446), O. sativa SBE1 (Q01401), D. melanogaster (A1Z992), D. rerio (F8W5I0), M. tuberculosis (P9WN45) and E. coli (P07762). (E) Surface representation of the hGBE1–Glc7 complex to model the two GBE reaction steps. Left panel is overlayed with a decasaccharide ligand (blue and orange stick) and TIM barrel loops (green ribbon) from the B. amyloliquefaciens and B. licheniformis chimeric amylase structure (PDB code 1e3z) to highlight the broader active site cleft in hGBE1 owing to the absence of these amylase loops. Right panel is overlayed with maltotriose (cyan stick) from pig pancreatic α-amylase (PDB code 1ua3), as well as the β4-α4 loop from O. sativa SBE1 (purple) and M. tuberculosis GBE (yellow) structures, which is disordered in hGBE1. Superposition of hGBE1 with structural homologs is illustrated in Supplementary Material, Figure S3. (F) Close-up view of the hGBE1 active site barrel (cyan strands) that harbours the conserved residues (sticks) of the ‘−1’ subsite. Residues constituting the putative catalytic triad are coloured magenta.
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DDV280F2: Oligosaccharide binding to hGBE1. (A) Chemical structures of acarbose (ACR) and (Glc7). (B) Surface representation of hGBE1 (Fig. 1A colouring) showing the bound oligosaccharides. (C) ACR binding cleft at the interface of the helical segment (orange), CBM48 (pink) and catalytic domain (green). Shown in sticks are ACR (yellow carbon atoms) and its contact protein residues (white carbon atoms). Inset, 2Fo-Fc electron density for the modelled ACR. (D) Sequence alignment of the ACR-binding residues of hGBE1 (underlined). Annotated branching enzyme sequences are from human (Uniprot ID Q04446), O. sativa SBE1 (Q01401), D. melanogaster (A1Z992), D. rerio (F8W5I0), M. tuberculosis (P9WN45) and E. coli (P07762). (E) Surface representation of the hGBE1–Glc7 complex to model the two GBE reaction steps. Left panel is overlayed with a decasaccharide ligand (blue and orange stick) and TIM barrel loops (green ribbon) from the B. amyloliquefaciens and B. licheniformis chimeric amylase structure (PDB code 1e3z) to highlight the broader active site cleft in hGBE1 owing to the absence of these amylase loops. Right panel is overlayed with maltotriose (cyan stick) from pig pancreatic α-amylase (PDB code 1ua3), as well as the β4-α4 loop from O. sativa SBE1 (purple) and M. tuberculosis GBE (yellow) structures, which is disordered in hGBE1. Superposition of hGBE1 with structural homologs is illustrated in Supplementary Material, Figure S3. (F) Close-up view of the hGBE1 active site barrel (cyan strands) that harbours the conserved residues (sticks) of the ‘−1’ subsite. Residues constituting the putative catalytic triad are coloured magenta.

Mentions: To characterize the binding of oligosaccharides to branching enzymes, we co-crystallized hGBE1trunc with acarbose (ACR) or maltoheptaose (Glc7) (Fig. 2A). ACR is a pseudo-tetra-saccharide acting as active site inhibitor for certain GH13 amylases. In the hGBE1-ACR structure, acarbose is bound not at the expected active site but instead at the interface between the CBM48 and the catalytic domains (Fig. 2B). Within this oligosaccharide binding cleft (Fig. 2C), ACR interacts with protein residues from the N-terminal helical segment (Asn62 and Glu63), CBM48 domain (Trp91, Pro93, Tyr119, Gly120 and Lys121) as well as catalytic core (Trp332, Glu333 and Arg336). These interactions, likely to be conserved among species (Fig. 2D), include hydrogen bonds to the sugar hydroxyl groups as well as hydrophobic/aromatic interactions with the pyranose rings. The hGBE1-Glc7 structure reveals similar conformation and binding interactions of maltoheptaose for its first four 1,4-linked glucose units (Fig. 2B). The three following glucose units, however, extend away from the protomer surface and engage in interactions with a neighbouring non-crystallographic symmetry (NCS)-related protomer in the asymmetric unit (Supplementary Material, Fig. S2A). These artifactual interactions mediated by crystal packing are unlikely to be physiologically relevant.Figure 2.


Structural basis of glycogen branching enzyme deficiency and pharmacologic rescue by rational peptide design.

Froese DS, Michaeli A, McCorvie TJ, Krojer T, Sasi M, Melaev E, Goldblum A, Zatsepin M, Lossos A, Álvarez R, Escribá PV, Minassian BA, von Delft F, Kakhlon O, Yue WW - Hum. Mol. Genet. (2015)

Oligosaccharide binding to hGBE1. (A) Chemical structures of acarbose (ACR) and (Glc7). (B) Surface representation of hGBE1 (Fig. 1A colouring) showing the bound oligosaccharides. (C) ACR binding cleft at the interface of the helical segment (orange), CBM48 (pink) and catalytic domain (green). Shown in sticks are ACR (yellow carbon atoms) and its contact protein residues (white carbon atoms). Inset, 2Fo-Fc electron density for the modelled ACR. (D) Sequence alignment of the ACR-binding residues of hGBE1 (underlined). Annotated branching enzyme sequences are from human (Uniprot ID Q04446), O. sativa SBE1 (Q01401), D. melanogaster (A1Z992), D. rerio (F8W5I0), M. tuberculosis (P9WN45) and E. coli (P07762). (E) Surface representation of the hGBE1–Glc7 complex to model the two GBE reaction steps. Left panel is overlayed with a decasaccharide ligand (blue and orange stick) and TIM barrel loops (green ribbon) from the B. amyloliquefaciens and B. licheniformis chimeric amylase structure (PDB code 1e3z) to highlight the broader active site cleft in hGBE1 owing to the absence of these amylase loops. Right panel is overlayed with maltotriose (cyan stick) from pig pancreatic α-amylase (PDB code 1ua3), as well as the β4-α4 loop from O. sativa SBE1 (purple) and M. tuberculosis GBE (yellow) structures, which is disordered in hGBE1. Superposition of hGBE1 with structural homologs is illustrated in Supplementary Material, Figure S3. (F) Close-up view of the hGBE1 active site barrel (cyan strands) that harbours the conserved residues (sticks) of the ‘−1’ subsite. Residues constituting the putative catalytic triad are coloured magenta.
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DDV280F2: Oligosaccharide binding to hGBE1. (A) Chemical structures of acarbose (ACR) and (Glc7). (B) Surface representation of hGBE1 (Fig. 1A colouring) showing the bound oligosaccharides. (C) ACR binding cleft at the interface of the helical segment (orange), CBM48 (pink) and catalytic domain (green). Shown in sticks are ACR (yellow carbon atoms) and its contact protein residues (white carbon atoms). Inset, 2Fo-Fc electron density for the modelled ACR. (D) Sequence alignment of the ACR-binding residues of hGBE1 (underlined). Annotated branching enzyme sequences are from human (Uniprot ID Q04446), O. sativa SBE1 (Q01401), D. melanogaster (A1Z992), D. rerio (F8W5I0), M. tuberculosis (P9WN45) and E. coli (P07762). (E) Surface representation of the hGBE1–Glc7 complex to model the two GBE reaction steps. Left panel is overlayed with a decasaccharide ligand (blue and orange stick) and TIM barrel loops (green ribbon) from the B. amyloliquefaciens and B. licheniformis chimeric amylase structure (PDB code 1e3z) to highlight the broader active site cleft in hGBE1 owing to the absence of these amylase loops. Right panel is overlayed with maltotriose (cyan stick) from pig pancreatic α-amylase (PDB code 1ua3), as well as the β4-α4 loop from O. sativa SBE1 (purple) and M. tuberculosis GBE (yellow) structures, which is disordered in hGBE1. Superposition of hGBE1 with structural homologs is illustrated in Supplementary Material, Figure S3. (F) Close-up view of the hGBE1 active site barrel (cyan strands) that harbours the conserved residues (sticks) of the ‘−1’ subsite. Residues constituting the putative catalytic triad are coloured magenta.
Mentions: To characterize the binding of oligosaccharides to branching enzymes, we co-crystallized hGBE1trunc with acarbose (ACR) or maltoheptaose (Glc7) (Fig. 2A). ACR is a pseudo-tetra-saccharide acting as active site inhibitor for certain GH13 amylases. In the hGBE1-ACR structure, acarbose is bound not at the expected active site but instead at the interface between the CBM48 and the catalytic domains (Fig. 2B). Within this oligosaccharide binding cleft (Fig. 2C), ACR interacts with protein residues from the N-terminal helical segment (Asn62 and Glu63), CBM48 domain (Trp91, Pro93, Tyr119, Gly120 and Lys121) as well as catalytic core (Trp332, Glu333 and Arg336). These interactions, likely to be conserved among species (Fig. 2D), include hydrogen bonds to the sugar hydroxyl groups as well as hydrophobic/aromatic interactions with the pyranose rings. The hGBE1-Glc7 structure reveals similar conformation and binding interactions of maltoheptaose for its first four 1,4-linked glucose units (Fig. 2B). The three following glucose units, however, extend away from the protomer surface and engage in interactions with a neighbouring non-crystallographic symmetry (NCS)-related protomer in the asymmetric unit (Supplementary Material, Fig. S2A). These artifactual interactions mediated by crystal packing are unlikely to be physiologically relevant.Figure 2.

Bottom Line: Expression of recombinant GBE1-p.Y329S resulted in drastically reduced protein yield and solubility compared with wild type, suggesting this disease allele causes protein misfolding and may be amenable to small molecule stabilization.We demonstrate intracellular transport of this peptide, its binding and stabilization of GBE1-p.Y329S, and 2-fold increased mutant enzymatic activity compared with untreated patient cells.Together, our data provide the rationale and starting point for the screening of small molecule chaperones, which could become novel therapies for this disease.

View Article: PubMed Central - PubMed

Affiliation: Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, OX3 7DQ, UK.

No MeSH data available.


Related in: MedlinePlus