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Insights from molecular dynamics simulations: structural basis for the V567D mutation-induced instability of zebrafish alpha-dystroglycan and comparison with the murine model.

Pirolli D, Sciandra F, Bozzi M, Giardina B, Brancaccio A, De Rosa MC - PLoS ONE (2014)

Bottom Line: We then ran extensive molecular dynamics (MD) simulations to reveal the structural and dynamic properties of the C-terminal domain and to evaluate the effect of the single mutation on alpha-DG stability.A comparative study has been also carried out on our previously generated model of murine alpha-DG C-terminal domain including the I591D mutation, which is topologically equivalent to the V567D mutation found in zebrafish.Trajectories from MD simulations were analyzed in detail, revealing extensive structural disorder involving multiple beta-strands in the mutated variant of the zebrafish protein whereas local effects have been detected in the murine protein.

View Article: PubMed Central - PubMed

Affiliation: Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Rome, Italy.

ABSTRACT
A missense amino acid mutation of valine to aspartic acid in 567 position of alpha-dystroglycan (DG), identified in dag1-mutated zebrafish, results in a reduced transcription and a complete absence of the protein. Lacking experimental structural data for zebrafish DG domains, the detailed mechanism for the observed mutation-induced destabilization of the DG complex and membrane damage, remained unclear. With the aim to contribute to a better clarification of the structure-function relationships featuring the DG complex, three-dimensional structural models of wild-type and mutant (V567D) C-terminal domain of alpha-DG from zebrafish were constructed by a template-based modelling approach. We then ran extensive molecular dynamics (MD) simulations to reveal the structural and dynamic properties of the C-terminal domain and to evaluate the effect of the single mutation on alpha-DG stability. A comparative study has been also carried out on our previously generated model of murine alpha-DG C-terminal domain including the I591D mutation, which is topologically equivalent to the V567D mutation found in zebrafish. Trajectories from MD simulations were analyzed in detail, revealing extensive structural disorder involving multiple beta-strands in the mutated variant of the zebrafish protein whereas local effects have been detected in the murine protein. A biochemical analysis of the murine alpha-DG mutant I591D confirmed a pronounced instability of the protein. Taken together, the computational and biochemical analysis suggest that the V567D/I591D mutation, belonging to the G beta-strand, plays a key role in inducing a destabilization of the alpha-DG C-terminal Ig-like domain that could possibly affect and propagate to the entire DG complex. The structural features herein identified may be of crucial help to understand the molecular basis of primary dystroglycanopathies.

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Backbone hydrogen bonds along the simulation trajectories for the four models.Shown is the number of backbone hydrogen bonds formed between the A′ and the G strands of zebrafish (panel A) and murine (panel B) α-DG Ig-like domains. The black and gray lines show the trajectories for wild-type and mutant systems, respectively.
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pone-0103866-g006: Backbone hydrogen bonds along the simulation trajectories for the four models.Shown is the number of backbone hydrogen bonds formed between the A′ and the G strands of zebrafish (panel A) and murine (panel B) α-DG Ig-like domains. The black and gray lines show the trajectories for wild-type and mutant systems, respectively.

Mentions: The Ig-like domain is stabilized by hydrophobic core interactions between the two β-sheets and by the hydrogen bonds between the β-strands [39], [49]. Interfering with any of the residues in the sheet by a mutation may lead to a discontinuity in the hydrogen bonding pattern, which is characteristic of the Ig-like domains. This may enhance the conformational flexibility of the mutated residue side chain, which could disrupt the natural bonding of neighbours and might result in loss of secondary structural elements [50], [51]. The external strands A′ and G present geometrical distortions known as β-bulges, as found in some Ig molecules [52], which lead to an imperfect general H-bond network. However, examination of the hydrogen bond patterns involving the β-strands A′–G reveals significant differences among the simulated systems (Fig. 6). Figure 6A shows that the backbone hydrogen bonds formed between the strands A′ and G, where the mutation is located, are stable in zebrafish wild-type but are disrupted in the zebrafish V567D mutant, resulting in a significant separation between the two strands in the β-sheet. By contrast, the corresponding backbone hydrogen bonds in murine DG were not noticeably affected by the I591D mutation (Fig. 6B). The changes in the hydrogen bond pattern observed in zebrafish DG are closely related to the disruption of the native hydrophobic contacts. Val567 residue, located on the G strand, interacts with a number of hydrophobic residues nearby and the strongest interactions are observed with Val481(β-strand A′), Ala483(β-strand A′), Phe489 (β-strand B) and Val491 (β-strand B). Significantly, unlike Val567, the acidic Asp567 residue of mutant DG maintains its side chain exposed to the solvent over the simulation time. Analysis of the MD trajectories shows that the hydrophobic contacts involving the 567 position remain relatively stable in the wild-type with the Val567 residue continuously interacting with residues Val481, Phe489, Ala483 and Val491 whereas they are disrupted upon mutation. This results in a significant disorganization of the A′ strand and in a widening of the cleft between the sheets of the Ig-like domain. This effect is not observed in murine α-DG, in which the hydrophobic contacts established by Ile591 with Val504, Ala506, Phe512 and Val514 are well preserved after the mutation. The Cα-Cα distances between the above-mentioned residues are reported in Figure 7 for both, zebrafish (A, C, E and G panels) and murine (B, D, F, and H panels) protein models, in comparison with their mutated counterpart. Panels A, C, E and G highlight the separation between A′–G (Fig. 7A, C) and B-G (Fig. 7E, G) strands. Notably, the large differences observed between Cα of 489, 491 (strand B) and 567 (strand A′) positions (Fig. 7E, G) indicate the separation between the two sheets of the β-sandwich (Fig. 2). In the case of murine α-DG the I591D replacement produces no effect on the corresponding distances between A′–G (Fig. 7B, D) strands and very little effects on the separation between the sheets (Fig. 7F, H).


Insights from molecular dynamics simulations: structural basis for the V567D mutation-induced instability of zebrafish alpha-dystroglycan and comparison with the murine model.

Pirolli D, Sciandra F, Bozzi M, Giardina B, Brancaccio A, De Rosa MC - PLoS ONE (2014)

Backbone hydrogen bonds along the simulation trajectories for the four models.Shown is the number of backbone hydrogen bonds formed between the A′ and the G strands of zebrafish (panel A) and murine (panel B) α-DG Ig-like domains. The black and gray lines show the trajectories for wild-type and mutant systems, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4117597&req=5

pone-0103866-g006: Backbone hydrogen bonds along the simulation trajectories for the four models.Shown is the number of backbone hydrogen bonds formed between the A′ and the G strands of zebrafish (panel A) and murine (panel B) α-DG Ig-like domains. The black and gray lines show the trajectories for wild-type and mutant systems, respectively.
Mentions: The Ig-like domain is stabilized by hydrophobic core interactions between the two β-sheets and by the hydrogen bonds between the β-strands [39], [49]. Interfering with any of the residues in the sheet by a mutation may lead to a discontinuity in the hydrogen bonding pattern, which is characteristic of the Ig-like domains. This may enhance the conformational flexibility of the mutated residue side chain, which could disrupt the natural bonding of neighbours and might result in loss of secondary structural elements [50], [51]. The external strands A′ and G present geometrical distortions known as β-bulges, as found in some Ig molecules [52], which lead to an imperfect general H-bond network. However, examination of the hydrogen bond patterns involving the β-strands A′–G reveals significant differences among the simulated systems (Fig. 6). Figure 6A shows that the backbone hydrogen bonds formed between the strands A′ and G, where the mutation is located, are stable in zebrafish wild-type but are disrupted in the zebrafish V567D mutant, resulting in a significant separation between the two strands in the β-sheet. By contrast, the corresponding backbone hydrogen bonds in murine DG were not noticeably affected by the I591D mutation (Fig. 6B). The changes in the hydrogen bond pattern observed in zebrafish DG are closely related to the disruption of the native hydrophobic contacts. Val567 residue, located on the G strand, interacts with a number of hydrophobic residues nearby and the strongest interactions are observed with Val481(β-strand A′), Ala483(β-strand A′), Phe489 (β-strand B) and Val491 (β-strand B). Significantly, unlike Val567, the acidic Asp567 residue of mutant DG maintains its side chain exposed to the solvent over the simulation time. Analysis of the MD trajectories shows that the hydrophobic contacts involving the 567 position remain relatively stable in the wild-type with the Val567 residue continuously interacting with residues Val481, Phe489, Ala483 and Val491 whereas they are disrupted upon mutation. This results in a significant disorganization of the A′ strand and in a widening of the cleft between the sheets of the Ig-like domain. This effect is not observed in murine α-DG, in which the hydrophobic contacts established by Ile591 with Val504, Ala506, Phe512 and Val514 are well preserved after the mutation. The Cα-Cα distances between the above-mentioned residues are reported in Figure 7 for both, zebrafish (A, C, E and G panels) and murine (B, D, F, and H panels) protein models, in comparison with their mutated counterpart. Panels A, C, E and G highlight the separation between A′–G (Fig. 7A, C) and B-G (Fig. 7E, G) strands. Notably, the large differences observed between Cα of 489, 491 (strand B) and 567 (strand A′) positions (Fig. 7E, G) indicate the separation between the two sheets of the β-sandwich (Fig. 2). In the case of murine α-DG the I591D replacement produces no effect on the corresponding distances between A′–G (Fig. 7B, D) strands and very little effects on the separation between the sheets (Fig. 7F, H).

Bottom Line: We then ran extensive molecular dynamics (MD) simulations to reveal the structural and dynamic properties of the C-terminal domain and to evaluate the effect of the single mutation on alpha-DG stability.A comparative study has been also carried out on our previously generated model of murine alpha-DG C-terminal domain including the I591D mutation, which is topologically equivalent to the V567D mutation found in zebrafish.Trajectories from MD simulations were analyzed in detail, revealing extensive structural disorder involving multiple beta-strands in the mutated variant of the zebrafish protein whereas local effects have been detected in the murine protein.

View Article: PubMed Central - PubMed

Affiliation: Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Rome, Italy.

ABSTRACT
A missense amino acid mutation of valine to aspartic acid in 567 position of alpha-dystroglycan (DG), identified in dag1-mutated zebrafish, results in a reduced transcription and a complete absence of the protein. Lacking experimental structural data for zebrafish DG domains, the detailed mechanism for the observed mutation-induced destabilization of the DG complex and membrane damage, remained unclear. With the aim to contribute to a better clarification of the structure-function relationships featuring the DG complex, three-dimensional structural models of wild-type and mutant (V567D) C-terminal domain of alpha-DG from zebrafish were constructed by a template-based modelling approach. We then ran extensive molecular dynamics (MD) simulations to reveal the structural and dynamic properties of the C-terminal domain and to evaluate the effect of the single mutation on alpha-DG stability. A comparative study has been also carried out on our previously generated model of murine alpha-DG C-terminal domain including the I591D mutation, which is topologically equivalent to the V567D mutation found in zebrafish. Trajectories from MD simulations were analyzed in detail, revealing extensive structural disorder involving multiple beta-strands in the mutated variant of the zebrafish protein whereas local effects have been detected in the murine protein. A biochemical analysis of the murine alpha-DG mutant I591D confirmed a pronounced instability of the protein. Taken together, the computational and biochemical analysis suggest that the V567D/I591D mutation, belonging to the G beta-strand, plays a key role in inducing a destabilization of the alpha-DG C-terminal Ig-like domain that could possibly affect and propagate to the entire DG complex. The structural features herein identified may be of crucial help to understand the molecular basis of primary dystroglycanopathies.

Show MeSH
Related in: MedlinePlus