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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

In silico peptide design to fill the Ser239mutant cavity. (A) Root mean-squared deviation (RMSD) from the backbone as a representation of structural stability (29). The dynamic conformations of the three structural models (hGBE1 WT, hGBE1-Y329S and peptide-bound hGBE1-Y329S) at 0.5-ns intervals along the simulation were superimposed with the initial backbone of the WT structure, in order to obtain RMSD values. (B) The molecular mechanics force field calculated binding free energy contributions of individual amino acids in the tetra-peptide LTKE. The Leu N-terminus contributes more than half of total binding free energy. (C) Homology model of hGBE1-Y329S in complex with the LTKE peptide (purple sticks) at the Ser329mutant cavity. (D) Close-up view of the LTKE peptide, where the side-chain of the N-terminal leucine (Leui) residue fills the cavity. (E) The LTKE peptide can bind hGBE1 via a number of hydrophobic and polar interactions (predicted hydrogen bonds in dotted lines).
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DDV280F5: In silico peptide design to fill the Ser239mutant cavity. (A) Root mean-squared deviation (RMSD) from the backbone as a representation of structural stability (29). The dynamic conformations of the three structural models (hGBE1 WT, hGBE1-Y329S and peptide-bound hGBE1-Y329S) at 0.5-ns intervals along the simulation were superimposed with the initial backbone of the WT structure, in order to obtain RMSD values. (B) The molecular mechanics force field calculated binding free energy contributions of individual amino acids in the tetra-peptide LTKE. The Leu N-terminus contributes more than half of total binding free energy. (C) Homology model of hGBE1-Y329S in complex with the LTKE peptide (purple sticks) at the Ser329mutant cavity. (D) Close-up view of the LTKE peptide, where the side-chain of the N-terminal leucine (Leui) residue fills the cavity. (E) The LTKE peptide can bind hGBE1 via a number of hydrophobic and polar interactions (predicted hydrogen bonds in dotted lines).

Mentions: Screening around the solvent exposed Ser329mutant region in our hGBE1-Y329S structural model, the ab initio peptide design algorithm gave as best hit a Leu-Thr-Lys-Glu (LTKE) peptide among the six top scores (Supplementary Material, Table S3), in terms of favourable binding affinities and solubility. Molecular dynamics simulation of wild-type hGBE1, hGBE1-Y329S and LTKE peptide-bound hGBE1-Y329S models (Fig. 5A; Supplementary Material, Methods) corroborated our prediction that LTKE stabilizes the mutated enzyme. Modelling of the LTKE peptide onto our hGBE1-Y329S model suggests that the N-terminal Leu (position i) is the primary contributor to peptide-binding energy (Fig. 5B), with a calculated dissociation constant (Kd) of 1.6 µm (Supplementary Material, Table S3). Replacement of Leu at position i with Ala (ATKE peptide) or with acetyl-Leu (Ac-LTKE peptide) was predicted to severely reduce peptide-binding energy (Supplementary Material, Fig. S7; Supplementary Material, Methods), strongly suggesting a specific mode of action for the LTKE peptide. In our LTKE-bound hGBE1-Y329S model, the Leu side-chain can penetrate the cavity formed by the p.Y329S mutation (Fig. 5C and D), recovering some of the hydrophobic interactions (e.g. with Phe327, Met362) offered by the wild-type tyrosyl aromatic ring, albeit with a different hydrogen bond pattern (Fig. 5E). The charged peptidyl N-terminus also hydrogen-bonds with Ser329mutant and forms a salt bridge with Asp386. The peptidyl Thr at position ii hydrogen bonds to Asp386, whereas the side chains of Lys at position iii and Glu at position iv further provide long-range electrostatic interactions with hGBE1.Figure 5.


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)

In silico peptide design to fill the Ser239mutant cavity. (A) Root mean-squared deviation (RMSD) from the backbone as a representation of structural stability (29). The dynamic conformations of the three structural models (hGBE1 WT, hGBE1-Y329S and peptide-bound hGBE1-Y329S) at 0.5-ns intervals along the simulation were superimposed with the initial backbone of the WT structure, in order to obtain RMSD values. (B) The molecular mechanics force field calculated binding free energy contributions of individual amino acids in the tetra-peptide LTKE. The Leu N-terminus contributes more than half of total binding free energy. (C) Homology model of hGBE1-Y329S in complex with the LTKE peptide (purple sticks) at the Ser329mutant cavity. (D) Close-up view of the LTKE peptide, where the side-chain of the N-terminal leucine (Leui) residue fills the cavity. (E) The LTKE peptide can bind hGBE1 via a number of hydrophobic and polar interactions (predicted hydrogen bonds in dotted lines).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

DDV280F5: In silico peptide design to fill the Ser239mutant cavity. (A) Root mean-squared deviation (RMSD) from the backbone as a representation of structural stability (29). The dynamic conformations of the three structural models (hGBE1 WT, hGBE1-Y329S and peptide-bound hGBE1-Y329S) at 0.5-ns intervals along the simulation were superimposed with the initial backbone of the WT structure, in order to obtain RMSD values. (B) The molecular mechanics force field calculated binding free energy contributions of individual amino acids in the tetra-peptide LTKE. The Leu N-terminus contributes more than half of total binding free energy. (C) Homology model of hGBE1-Y329S in complex with the LTKE peptide (purple sticks) at the Ser329mutant cavity. (D) Close-up view of the LTKE peptide, where the side-chain of the N-terminal leucine (Leui) residue fills the cavity. (E) The LTKE peptide can bind hGBE1 via a number of hydrophobic and polar interactions (predicted hydrogen bonds in dotted lines).
Mentions: Screening around the solvent exposed Ser329mutant region in our hGBE1-Y329S structural model, the ab initio peptide design algorithm gave as best hit a Leu-Thr-Lys-Glu (LTKE) peptide among the six top scores (Supplementary Material, Table S3), in terms of favourable binding affinities and solubility. Molecular dynamics simulation of wild-type hGBE1, hGBE1-Y329S and LTKE peptide-bound hGBE1-Y329S models (Fig. 5A; Supplementary Material, Methods) corroborated our prediction that LTKE stabilizes the mutated enzyme. Modelling of the LTKE peptide onto our hGBE1-Y329S model suggests that the N-terminal Leu (position i) is the primary contributor to peptide-binding energy (Fig. 5B), with a calculated dissociation constant (Kd) of 1.6 µm (Supplementary Material, Table S3). Replacement of Leu at position i with Ala (ATKE peptide) or with acetyl-Leu (Ac-LTKE peptide) was predicted to severely reduce peptide-binding energy (Supplementary Material, Fig. S7; Supplementary Material, Methods), strongly suggesting a specific mode of action for the LTKE peptide. In our LTKE-bound hGBE1-Y329S model, the Leu side-chain can penetrate the cavity formed by the p.Y329S mutation (Fig. 5C and D), recovering some of the hydrophobic interactions (e.g. with Phe327, Met362) offered by the wild-type tyrosyl aromatic ring, albeit with a different hydrogen bond pattern (Fig. 5E). The charged peptidyl N-terminus also hydrogen-bonds with Ser329mutant and forms a salt bridge with Asp386. The peptidyl Thr at position ii hydrogen bonds to Asp386, whereas the side chains of Lys at position iii and Glu at position iv further provide long-range electrostatic interactions with hGBE1.Figure 5.

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