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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

Crystal structure of hGBE1. (A and B) Orthogonal views of hGBE1 showing the N-terminal helical segment (orange), CBM48 (pink), central catalytic domain (green) and C-terminal domain (blue). The catalytic triad Asp357-Glu412-Asp481 is shown as red sticks. Numbers refer to domain boundaries. N- and C-termini are labelled as grey spheres. (C) Superposition of branching enzyme structures from human (hGBE1, this study), O. sativa SBE1 and M. tuberculosis GBE, highlighting the conserved domain architecture and three regions of structural variation. (D) Domain organization of hGBE1, O. sativa SBE1 and M. tuberculosis GBE revealing differences in the N-terminus between prokaryotic and eukaryotic polypeptides. Prokaryotic GBEs contain two N-terminal carbohydrate-binding domains (N1, N2) whereas eukaryotes contain only one (CBM48) and replace the prokaryotic N1 domain with a helical extension.
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DDV280F1: Crystal structure of hGBE1. (A and B) Orthogonal views of hGBE1 showing the N-terminal helical segment (orange), CBM48 (pink), central catalytic domain (green) and C-terminal domain (blue). The catalytic triad Asp357-Glu412-Asp481 is shown as red sticks. Numbers refer to domain boundaries. N- and C-termini are labelled as grey spheres. (C) Superposition of branching enzyme structures from human (hGBE1, this study), O. sativa SBE1 and M. tuberculosis GBE, highlighting the conserved domain architecture and three regions of structural variation. (D) Domain organization of hGBE1, O. sativa SBE1 and M. tuberculosis GBE revealing differences in the N-terminus between prokaryotic and eukaryotic polypeptides. Prokaryotic GBEs contain two N-terminal carbohydrate-binding domains (N1, N2) whereas eukaryotes contain only one (CBM48) and replace the prokaryotic N1 domain with a helical extension.

Mentions: hGBE1 is an elongated molecule (longest dimension >85 Å) composed of four structural regions (Fig. 1A and B): the N-terminal helical segment (aa 43–75), a carbohydrate-binding module 48 (CBM48; aa 76–183), a central catalytic core (aa 184–600) and the C-terminal amylase-like barrel domain (aa 601–702). A structural overlay of hGBE1 with reported branching enzyme structures from O. sativa SBE1 (17) (PDB: 3AMK, Cα-RMSD: 1.4 Å, sequence identity: 54%) and M. tuberculosis GBE (19) (3K1D, 2.1 Å, 28%) (Fig. 1C) highlights the conserved catalytic core housing the active site within a canonical (βα)6 barrel (16). Nevertheless, the different branching enzymes show greater structural variability in the N-terminal region preceding the catalytic core, as well as in two surface-exposed loops of the TIM barrel (Fig. 1C). For example, in O. sativa SBE1 and human GBE1 structures, the helical segment precedes the CBM48 module, whereas in M. tuberculosis GBE, the helical segment is replaced by an additional β-sandwich module (N1 in Fig. 1C and D). The closer homology of hGBE1 with O. sativa SBE1, whose substrate is starch, than with the bacterial paralog M. tuberculosis GBE, suggests a similar evolutionary conservation in the branching enzyme mechanism for glycogen and starch, both involving a growing linear α1,4-linked glucan chain as substrate.Figure 1.


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)

Crystal structure of hGBE1. (A and B) Orthogonal views of hGBE1 showing the N-terminal helical segment (orange), CBM48 (pink), central catalytic domain (green) and C-terminal domain (blue). The catalytic triad Asp357-Glu412-Asp481 is shown as red sticks. Numbers refer to domain boundaries. N- and C-termini are labelled as grey spheres. (C) Superposition of branching enzyme structures from human (hGBE1, this study), O. sativa SBE1 and M. tuberculosis GBE, highlighting the conserved domain architecture and three regions of structural variation. (D) Domain organization of hGBE1, O. sativa SBE1 and M. tuberculosis GBE revealing differences in the N-terminus between prokaryotic and eukaryotic polypeptides. Prokaryotic GBEs contain two N-terminal carbohydrate-binding domains (N1, N2) whereas eukaryotes contain only one (CBM48) and replace the prokaryotic N1 domain with a helical extension.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581599&req=5

DDV280F1: Crystal structure of hGBE1. (A and B) Orthogonal views of hGBE1 showing the N-terminal helical segment (orange), CBM48 (pink), central catalytic domain (green) and C-terminal domain (blue). The catalytic triad Asp357-Glu412-Asp481 is shown as red sticks. Numbers refer to domain boundaries. N- and C-termini are labelled as grey spheres. (C) Superposition of branching enzyme structures from human (hGBE1, this study), O. sativa SBE1 and M. tuberculosis GBE, highlighting the conserved domain architecture and three regions of structural variation. (D) Domain organization of hGBE1, O. sativa SBE1 and M. tuberculosis GBE revealing differences in the N-terminus between prokaryotic and eukaryotic polypeptides. Prokaryotic GBEs contain two N-terminal carbohydrate-binding domains (N1, N2) whereas eukaryotes contain only one (CBM48) and replace the prokaryotic N1 domain with a helical extension.
Mentions: hGBE1 is an elongated molecule (longest dimension >85 Å) composed of four structural regions (Fig. 1A and B): the N-terminal helical segment (aa 43–75), a carbohydrate-binding module 48 (CBM48; aa 76–183), a central catalytic core (aa 184–600) and the C-terminal amylase-like barrel domain (aa 601–702). A structural overlay of hGBE1 with reported branching enzyme structures from O. sativa SBE1 (17) (PDB: 3AMK, Cα-RMSD: 1.4 Å, sequence identity: 54%) and M. tuberculosis GBE (19) (3K1D, 2.1 Å, 28%) (Fig. 1C) highlights the conserved catalytic core housing the active site within a canonical (βα)6 barrel (16). Nevertheless, the different branching enzymes show greater structural variability in the N-terminal region preceding the catalytic core, as well as in two surface-exposed loops of the TIM barrel (Fig. 1C). For example, in O. sativa SBE1 and human GBE1 structures, the helical segment precedes the CBM48 module, whereas in M. tuberculosis GBE, the helical segment is replaced by an additional β-sandwich module (N1 in Fig. 1C and D). The closer homology of hGBE1 with O. sativa SBE1, whose substrate is starch, than with the bacterial paralog M. tuberculosis GBE, suggests a similar evolutionary conservation in the branching enzyme mechanism for glycogen and starch, both involving a growing linear α1,4-linked glucan chain as substrate.Figure 1.

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