Limits...
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

p.Y329S mutation results in destabilized hGBE1 protein. (A) Tyr329 is highly conserved across various GBE orthologs (Uniprot ID for each sequence is shown). (B) SDS–PAGE of affinity-purified hGBE1 WT and p.Y329S, showing much reduced level of soluble mutant protein. (C) Structural analysis of Tyr329 and its neighbourhood reveals a number of hydrophobic interactions that are removed by its substitution with serine. (D) Tyr329 (magenta, left panel) is accessible to the protein exterior, and its mutation to Ser329 (magenta, right panel) creates a cavity (circled).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4581599&req=5

DDV280F4: p.Y329S mutation results in destabilized hGBE1 protein. (A) Tyr329 is highly conserved across various GBE orthologs (Uniprot ID for each sequence is shown). (B) SDS–PAGE of affinity-purified hGBE1 WT and p.Y329S, showing much reduced level of soluble mutant protein. (C) Structural analysis of Tyr329 and its neighbourhood reveals a number of hydrophobic interactions that are removed by its substitution with serine. (D) Tyr329 (magenta, left panel) is accessible to the protein exterior, and its mutation to Ser329 (magenta, right panel) creates a cavity (circled).

Mentions: The c.986A>C mutation results in the p.Y329S amino acid substitution, the most common APBD-associated mutation (27). This residue is highly conserved across different GBE orthologs supporting its associated pathogenicity (Fig. 4A). We observed drastically reduced recombinant expression and protein solubility from an hGBE1 construct harbouring the p.Y329S substitution, compared with wild type (Fig. 4B). We therefore sought a molecular explanation by inspecting our hGBE1 structure. Tyr329 is a surface-exposed residue in the catalytic domain and confers stability to the local environment by interacting with the hydrophobic residues Phe327, Val334, Leu338, Met362 and Ala389. Additionally, the tyrosyl hydroxyl group hydrogen bonds with the His289 backbone oxygen (Fig. 4C, left). Mutation of Tyr329 to the smaller amino acid serine (Ser329mutant) likely removes these interactions (Fig. 4C, right) and creates a solvent accessible cavity within this hydrophobic core (Fig. 4D), both of which could lead to destabilized protein. Together, our expression and structural analyses demonstrate that the p.Y329S mutation commonly associated with APBD results in protein destabilization.Figure 4.


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)

p.Y329S mutation results in destabilized hGBE1 protein. (A) Tyr329 is highly conserved across various GBE orthologs (Uniprot ID for each sequence is shown). (B) SDS–PAGE of affinity-purified hGBE1 WT and p.Y329S, showing much reduced level of soluble mutant protein. (C) Structural analysis of Tyr329 and its neighbourhood reveals a number of hydrophobic interactions that are removed by its substitution with serine. (D) Tyr329 (magenta, left panel) is accessible to the protein exterior, and its mutation to Ser329 (magenta, right panel) creates a cavity (circled).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

DDV280F4: p.Y329S mutation results in destabilized hGBE1 protein. (A) Tyr329 is highly conserved across various GBE orthologs (Uniprot ID for each sequence is shown). (B) SDS–PAGE of affinity-purified hGBE1 WT and p.Y329S, showing much reduced level of soluble mutant protein. (C) Structural analysis of Tyr329 and its neighbourhood reveals a number of hydrophobic interactions that are removed by its substitution with serine. (D) Tyr329 (magenta, left panel) is accessible to the protein exterior, and its mutation to Ser329 (magenta, right panel) creates a cavity (circled).
Mentions: The c.986A>C mutation results in the p.Y329S amino acid substitution, the most common APBD-associated mutation (27). This residue is highly conserved across different GBE orthologs supporting its associated pathogenicity (Fig. 4A). We observed drastically reduced recombinant expression and protein solubility from an hGBE1 construct harbouring the p.Y329S substitution, compared with wild type (Fig. 4B). We therefore sought a molecular explanation by inspecting our hGBE1 structure. Tyr329 is a surface-exposed residue in the catalytic domain and confers stability to the local environment by interacting with the hydrophobic residues Phe327, Val334, Leu338, Met362 and Ala389. Additionally, the tyrosyl hydroxyl group hydrogen bonds with the His289 backbone oxygen (Fig. 4C, left). Mutation of Tyr329 to the smaller amino acid serine (Ser329mutant) likely removes these interactions (Fig. 4C, right) and creates a solvent accessible cavity within this hydrophobic core (Fig. 4D), both of which could lead to destabilized protein. Together, our expression and structural analyses demonstrate that the p.Y329S mutation commonly associated with APBD results in protein destabilization.Figure 4.

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