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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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Structure and topology of wild-type and mutant zebrafish Ig-like domains belonging to the α-DG C-terminal region.The secondary structure elements (panel A) are named according to Harpaz and Chothia [46]. The β-strands are colored according to the sheet to which they belong and the N and C termini are indicated. The topology diagram of the domains is shown in panel B; β-strands are shown as circles and the small helix as a triangle.
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pone-0103866-g002: Structure and topology of wild-type and mutant zebrafish Ig-like domains belonging to the α-DG C-terminal region.The secondary structure elements (panel A) are named according to Harpaz and Chothia [46]. The β-strands are colored according to the sheet to which they belong and the N and C termini are indicated. The topology diagram of the domains is shown in panel B; β-strands are shown as circles and the small helix as a triangle.

Mentions: In both systems, only β-strands and coils were found in the 475–574 region of α-DG, whereas coils, α helices and strands were found in the extreme C-terminus. Figure S1 shows the predicted secondary structures of the two systems. Not surprisingly, the I-TASSER threading procedure identified immunoglobulin-like domains as the best templates for wild-type, specifically, 1U2C (α-DG N-terminal region [19]), 2WCP (mouse chaderin-23 [43]), 2YST (human protocadherin 7, to be published) and 3Q2V (mouse E-cadherin ectodomain [44]). The same templates, with the exception of 2YST were identified for the mutant V567D. The overall sequence identity shared between the α-DG C-terminal regions and each of the templates is approximately 24%. Although this value is quite low, it is similar to other cases in which modelling has been applied [45]. The quality of the generated models was assessed in I-TASSER based on two major criteria, the C- and the TM-scores. The C-score is calculated based on the significance of the threading alignments and the convergence of the I-TASSER simulations. C-scores typically range from −5 to 2, with higher scores reflecting a model of better quality. The TM-score is a measure of structural similarity between the predicted model and the native or experimentally determined structure, with a value >0.5 indicating a model of correct topology. Assessments for the zebrafish α-DG C-terminal regions are reported in Table 1 indicating reasonable models and accurate topology. In all cases, search of the PDB, quantified by TM-score, indicated 1U2C as the structure with the highest structural similarity (Table 1). As expected, the zebrafish α-DG model, as well as that of the V567D mutant, is similar in structure to the murine α-DG [17] with a root mean square deviation (RMSD) of the Cα atoms of 1.92Å (wild-type) and 1.68Å (V567D). According to I-TASSER then, the region encompassing residues 475–574 of the α-DG C-terminus adopts the typical I-frame immunoglobulin superfamily fold and is stabilized by extensive hydrophobic core interactions between the two β-sheets [46] (Fig. 2). Consistently with the 1U2C structure and our previous results, a small helix was detected between β-strands B and C (residues 495–498). The rest of the region (residues 575–626) displayed two coil-strand-coil regions separated by a helix.


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)

Structure and topology of wild-type and mutant zebrafish Ig-like domains belonging to the α-DG C-terminal region.The secondary structure elements (panel A) are named according to Harpaz and Chothia [46]. The β-strands are colored according to the sheet to which they belong and the N and C termini are indicated. The topology diagram of the domains is shown in panel B; β-strands are shown as circles and the small helix as a triangle.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103866-g002: Structure and topology of wild-type and mutant zebrafish Ig-like domains belonging to the α-DG C-terminal region.The secondary structure elements (panel A) are named according to Harpaz and Chothia [46]. The β-strands are colored according to the sheet to which they belong and the N and C termini are indicated. The topology diagram of the domains is shown in panel B; β-strands are shown as circles and the small helix as a triangle.
Mentions: In both systems, only β-strands and coils were found in the 475–574 region of α-DG, whereas coils, α helices and strands were found in the extreme C-terminus. Figure S1 shows the predicted secondary structures of the two systems. Not surprisingly, the I-TASSER threading procedure identified immunoglobulin-like domains as the best templates for wild-type, specifically, 1U2C (α-DG N-terminal region [19]), 2WCP (mouse chaderin-23 [43]), 2YST (human protocadherin 7, to be published) and 3Q2V (mouse E-cadherin ectodomain [44]). The same templates, with the exception of 2YST were identified for the mutant V567D. The overall sequence identity shared between the α-DG C-terminal regions and each of the templates is approximately 24%. Although this value is quite low, it is similar to other cases in which modelling has been applied [45]. The quality of the generated models was assessed in I-TASSER based on two major criteria, the C- and the TM-scores. The C-score is calculated based on the significance of the threading alignments and the convergence of the I-TASSER simulations. C-scores typically range from −5 to 2, with higher scores reflecting a model of better quality. The TM-score is a measure of structural similarity between the predicted model and the native or experimentally determined structure, with a value >0.5 indicating a model of correct topology. Assessments for the zebrafish α-DG C-terminal regions are reported in Table 1 indicating reasonable models and accurate topology. In all cases, search of the PDB, quantified by TM-score, indicated 1U2C as the structure with the highest structural similarity (Table 1). As expected, the zebrafish α-DG model, as well as that of the V567D mutant, is similar in structure to the murine α-DG [17] with a root mean square deviation (RMSD) of the Cα atoms of 1.92Å (wild-type) and 1.68Å (V567D). According to I-TASSER then, the region encompassing residues 475–574 of the α-DG C-terminus adopts the typical I-frame immunoglobulin superfamily fold and is stabilized by extensive hydrophobic core interactions between the two β-sheets [46] (Fig. 2). Consistently with the 1U2C structure and our previous results, a small helix was detected between β-strands B and C (residues 495–498). The rest of the region (residues 575–626) displayed two coil-strand-coil regions separated by a helix.

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