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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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I591D mutation partially prevents the post-translational cleavage of murine DG precursor.293-Ebna cells were transfected with the wild-type or the mutated I591D constructs both carrying a myc-tag within the C-terminal domain of α-DG and cloned into the pEGFP vector. A) Immunoprecipitation with an anti-myc-antibody of wild-type and I591D α-DGs. In cells transfected with wild-type DG the slightly broad band detected at 100 kDa (TOT), that is further enriched upon immunoprecipitation (IP), corresponds to the mature α-DG. In cells transfected with the I591D mutant an additional and prominent band is detected at 160 kDa corresponding to the uncleaved DG precursor. B) Western blot of total protein extracts probed with an anti β-DG antibody. The wild-type construct displays a single band at 60 kDa corresponding to the mature β-DG-GFP, while I591D shows an additional band at 160 kDa corresponding to the unprocessed DG precursor (asterisk). The band at 45 kDa represents the endogenous β-DG.
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pone-0103866-g010: I591D mutation partially prevents the post-translational cleavage of murine DG precursor.293-Ebna cells were transfected with the wild-type or the mutated I591D constructs both carrying a myc-tag within the C-terminal domain of α-DG and cloned into the pEGFP vector. A) Immunoprecipitation with an anti-myc-antibody of wild-type and I591D α-DGs. In cells transfected with wild-type DG the slightly broad band detected at 100 kDa (TOT), that is further enriched upon immunoprecipitation (IP), corresponds to the mature α-DG. In cells transfected with the I591D mutant an additional and prominent band is detected at 160 kDa corresponding to the uncleaved DG precursor. B) Western blot of total protein extracts probed with an anti β-DG antibody. The wild-type construct displays a single band at 60 kDa corresponding to the mature β-DG-GFP, while I591D shows an additional band at 160 kDa corresponding to the unprocessed DG precursor (asterisk). The band at 45 kDa represents the endogenous β-DG.

Mentions: Interestingly, the mutation does not prevent or downregulate the expression of DG compared to the wild-type, however the structural rearrangements occurring in I591D partially impair the post-translational cleavage of the mutated DG precursor. In fact, an additional band at about 160 kDa is detected in Western blot using anti β-DG or anti-myc antibodies (Fig. 10 A and B). It was already shown that mutations that affect the stability of the DG precursor, such as the disruption of disulfide bridge within the extracellular domain of β-DG or the perturbation of the interaction between the two subunits, strongly influence its post-translational cleavage and plasma membrane targeting [21], [56], [57].


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)

I591D mutation partially prevents the post-translational cleavage of murine DG precursor.293-Ebna cells were transfected with the wild-type or the mutated I591D constructs both carrying a myc-tag within the C-terminal domain of α-DG and cloned into the pEGFP vector. A) Immunoprecipitation with an anti-myc-antibody of wild-type and I591D α-DGs. In cells transfected with wild-type DG the slightly broad band detected at 100 kDa (TOT), that is further enriched upon immunoprecipitation (IP), corresponds to the mature α-DG. In cells transfected with the I591D mutant an additional and prominent band is detected at 160 kDa corresponding to the uncleaved DG precursor. B) Western blot of total protein extracts probed with an anti β-DG antibody. The wild-type construct displays a single band at 60 kDa corresponding to the mature β-DG-GFP, while I591D shows an additional band at 160 kDa corresponding to the unprocessed DG precursor (asterisk). The band at 45 kDa represents the endogenous β-DG.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103866-g010: I591D mutation partially prevents the post-translational cleavage of murine DG precursor.293-Ebna cells were transfected with the wild-type or the mutated I591D constructs both carrying a myc-tag within the C-terminal domain of α-DG and cloned into the pEGFP vector. A) Immunoprecipitation with an anti-myc-antibody of wild-type and I591D α-DGs. In cells transfected with wild-type DG the slightly broad band detected at 100 kDa (TOT), that is further enriched upon immunoprecipitation (IP), corresponds to the mature α-DG. In cells transfected with the I591D mutant an additional and prominent band is detected at 160 kDa corresponding to the uncleaved DG precursor. B) Western blot of total protein extracts probed with an anti β-DG antibody. The wild-type construct displays a single band at 60 kDa corresponding to the mature β-DG-GFP, while I591D shows an additional band at 160 kDa corresponding to the unprocessed DG precursor (asterisk). The band at 45 kDa represents the endogenous β-DG.
Mentions: Interestingly, the mutation does not prevent or downregulate the expression of DG compared to the wild-type, however the structural rearrangements occurring in I591D partially impair the post-translational cleavage of the mutated DG precursor. In fact, an additional band at about 160 kDa is detected in Western blot using anti β-DG or anti-myc antibodies (Fig. 10 A and B). It was already shown that mutations that affect the stability of the DG precursor, such as the disruption of disulfide bridge within the extracellular domain of β-DG or the perturbation of the interaction between the two subunits, strongly influence its post-translational cleavage and plasma membrane targeting [21], [56], [57].

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