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Neurodegenerative disease-associated mutants of a human mitochondrial aminoacyl-tRNA synthetase present individual molecular signatures.

Sauter C, Lorber B, Gaudry A, Karim L, Schwenzer H, Wien F, Roblin P, Florentz C, Sissler M - Sci Rep (2015)

Bottom Line: The effects of these mutations on the structure and function of the enzymes remain to be established.Mutations with mild effects on solubility occur in patients as allelic combinations whereas those with strong effects on solubility or on aminoacylation are necessarily associated with a partially functional allele.The fact that all mutations show individual molecular and cellular signatures and affect amino acids only conserved in mammals, points towards an alternative function besides aminoacylation.

View Article: PubMed Central - PubMed

Affiliation: Architecture et Réactivité de l'ARN, CNRS, Université de Strasbourg, IBMC, 15 rue René Descartes, 67084 STRASBOURG Cedex, France.

ABSTRACT
Mutations in human mitochondrial aminoacyl-tRNA synthetases are associated with a variety of neurodegenerative disorders. The effects of these mutations on the structure and function of the enzymes remain to be established. Here, we investigate six mutants of the aspartyl-tRNA synthetase correlated with leukoencephalopathies. Our integrated strategy, combining an ensemble of biochemical and biophysical approaches, reveals that mutants are diversely affected with respect to their solubility in cellular extracts and stability in solution, but not in architecture. Mutations with mild effects on solubility occur in patients as allelic combinations whereas those with strong effects on solubility or on aminoacylation are necessarily associated with a partially functional allele. The fact that all mutations show individual molecular and cellular signatures and affect amino acids only conserved in mammals, points towards an alternative function besides aminoacylation.

No MeSH data available.


Related in: MedlinePlus

mt-AspRS WT versus mutants.(A) Positions of single point mutations on the 3D model of mt-AspRS including the two C-terminal extensions and a tRNA molecule docked onto the yellow subunit to show its interaction area (adapted from26). (B) Close up views showing the original and the mutated side chains in space filling representation. The color of carbon atoms is the same as in other figures. Oxygen and nitrogen atoms depicted in red and blue, respectively. All substitutions are possible without significant steric hindrance. R263Q and L626Q being close to the enzyme 2-fold axis, WT and mutated residues are shown in the same inset. (C) Five intrinsic properties ranked relative to the WT (red pentagon = 100% of scale). The enzymatic activity of all mutants was comparable to that of the WT, except that of L626Q and R263Q for which moderate 40 and 140-fold decreases were measured, respectively23. Ranges of aggregation temperature determined by DLS, Tm of 2D structure unfolding in SRCD and Tm of dye binding in DSF ranged are 38–41 °C, 45–51 °C and 45–48 °C, respectively. Mitochondrial protein solubilities are taken from top histogram in Fig. 4C. The gray circle delineates the lowest values of each interval. The color code for mutants is indicated. All molecular representations were prepared with PyMOL (Schrödinger, Inc.).
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f6: mt-AspRS WT versus mutants.(A) Positions of single point mutations on the 3D model of mt-AspRS including the two C-terminal extensions and a tRNA molecule docked onto the yellow subunit to show its interaction area (adapted from26). (B) Close up views showing the original and the mutated side chains in space filling representation. The color of carbon atoms is the same as in other figures. Oxygen and nitrogen atoms depicted in red and blue, respectively. All substitutions are possible without significant steric hindrance. R263Q and L626Q being close to the enzyme 2-fold axis, WT and mutated residues are shown in the same inset. (C) Five intrinsic properties ranked relative to the WT (red pentagon = 100% of scale). The enzymatic activity of all mutants was comparable to that of the WT, except that of L626Q and R263Q for which moderate 40 and 140-fold decreases were measured, respectively23. Ranges of aggregation temperature determined by DLS, Tm of 2D structure unfolding in SRCD and Tm of dye binding in DSF ranged are 38–41 °C, 45–51 °C and 45–48 °C, respectively. Mitochondrial protein solubilities are taken from top histogram in Fig. 4C. The gray circle delineates the lowest values of each interval. The color code for mutants is indicated. All molecular representations were prepared with PyMOL (Schrödinger, Inc.).

Mentions: It is widely believed that mutation of strictly conserved residues would be detrimental to the architecture or activity (in the present case, aminoacylation). Therefore we investigated the amino acid conservation of relevant residues in structural sequence alignments made of 180 sequences of bacterial-type AspRSs (60 mitochondrial and 120 bacterial AspRSs). We found that none of them are strictly conserved (Supplementary Table 3), indicating that no gross structural and/or functional perturbations are likely to occur. As seen in Fig. 1A, amino acid substitutions either extend, shorten or delete side chains with either a gain or loss of positive charge or of hydrophobicity. Because of changes in size or the introduction of attractive or repulsive interactions, mutations may compact or expand locally the structure. However, homology models based on the X-ray structure of human WT mt-AspRS suggest that these modifications do not perturb the overall 2D and 3D fold (Fig. 6A,B and Supplementary Table 3). This lack of perturbation is clear for T136S, where mutation replaces a side chain with one of similar volume, and for R58G and L613F where side chains are directed toward the solvent. L613F is the only one occurring in the vicinity of the tRNA binding site but the introduction of a phenyl group at this position is possible without clash with the ligand. In the case of Q184K, R263Q and L626Q at the heart of the dimerization interface, the local structure may cope with the new side chains presenting conformers that do not cause any steric hindrance (Supplementary Table 3).


Neurodegenerative disease-associated mutants of a human mitochondrial aminoacyl-tRNA synthetase present individual molecular signatures.

Sauter C, Lorber B, Gaudry A, Karim L, Schwenzer H, Wien F, Roblin P, Florentz C, Sissler M - Sci Rep (2015)

mt-AspRS WT versus mutants.(A) Positions of single point mutations on the 3D model of mt-AspRS including the two C-terminal extensions and a tRNA molecule docked onto the yellow subunit to show its interaction area (adapted from26). (B) Close up views showing the original and the mutated side chains in space filling representation. The color of carbon atoms is the same as in other figures. Oxygen and nitrogen atoms depicted in red and blue, respectively. All substitutions are possible without significant steric hindrance. R263Q and L626Q being close to the enzyme 2-fold axis, WT and mutated residues are shown in the same inset. (C) Five intrinsic properties ranked relative to the WT (red pentagon = 100% of scale). The enzymatic activity of all mutants was comparable to that of the WT, except that of L626Q and R263Q for which moderate 40 and 140-fold decreases were measured, respectively23. Ranges of aggregation temperature determined by DLS, Tm of 2D structure unfolding in SRCD and Tm of dye binding in DSF ranged are 38–41 °C, 45–51 °C and 45–48 °C, respectively. Mitochondrial protein solubilities are taken from top histogram in Fig. 4C. The gray circle delineates the lowest values of each interval. The color code for mutants is indicated. All molecular representations were prepared with PyMOL (Schrödinger, Inc.).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664897&req=5

f6: mt-AspRS WT versus mutants.(A) Positions of single point mutations on the 3D model of mt-AspRS including the two C-terminal extensions and a tRNA molecule docked onto the yellow subunit to show its interaction area (adapted from26). (B) Close up views showing the original and the mutated side chains in space filling representation. The color of carbon atoms is the same as in other figures. Oxygen and nitrogen atoms depicted in red and blue, respectively. All substitutions are possible without significant steric hindrance. R263Q and L626Q being close to the enzyme 2-fold axis, WT and mutated residues are shown in the same inset. (C) Five intrinsic properties ranked relative to the WT (red pentagon = 100% of scale). The enzymatic activity of all mutants was comparable to that of the WT, except that of L626Q and R263Q for which moderate 40 and 140-fold decreases were measured, respectively23. Ranges of aggregation temperature determined by DLS, Tm of 2D structure unfolding in SRCD and Tm of dye binding in DSF ranged are 38–41 °C, 45–51 °C and 45–48 °C, respectively. Mitochondrial protein solubilities are taken from top histogram in Fig. 4C. The gray circle delineates the lowest values of each interval. The color code for mutants is indicated. All molecular representations were prepared with PyMOL (Schrödinger, Inc.).
Mentions: It is widely believed that mutation of strictly conserved residues would be detrimental to the architecture or activity (in the present case, aminoacylation). Therefore we investigated the amino acid conservation of relevant residues in structural sequence alignments made of 180 sequences of bacterial-type AspRSs (60 mitochondrial and 120 bacterial AspRSs). We found that none of them are strictly conserved (Supplementary Table 3), indicating that no gross structural and/or functional perturbations are likely to occur. As seen in Fig. 1A, amino acid substitutions either extend, shorten or delete side chains with either a gain or loss of positive charge or of hydrophobicity. Because of changes in size or the introduction of attractive or repulsive interactions, mutations may compact or expand locally the structure. However, homology models based on the X-ray structure of human WT mt-AspRS suggest that these modifications do not perturb the overall 2D and 3D fold (Fig. 6A,B and Supplementary Table 3). This lack of perturbation is clear for T136S, where mutation replaces a side chain with one of similar volume, and for R58G and L613F where side chains are directed toward the solvent. L613F is the only one occurring in the vicinity of the tRNA binding site but the introduction of a phenyl group at this position is possible without clash with the ligand. In the case of Q184K, R263Q and L626Q at the heart of the dimerization interface, the local structure may cope with the new side chains presenting conformers that do not cause any steric hindrance (Supplementary Table 3).

Bottom Line: The effects of these mutations on the structure and function of the enzymes remain to be established.Mutations with mild effects on solubility occur in patients as allelic combinations whereas those with strong effects on solubility or on aminoacylation are necessarily associated with a partially functional allele.The fact that all mutations show individual molecular and cellular signatures and affect amino acids only conserved in mammals, points towards an alternative function besides aminoacylation.

View Article: PubMed Central - PubMed

Affiliation: Architecture et Réactivité de l'ARN, CNRS, Université de Strasbourg, IBMC, 15 rue René Descartes, 67084 STRASBOURG Cedex, France.

ABSTRACT
Mutations in human mitochondrial aminoacyl-tRNA synthetases are associated with a variety of neurodegenerative disorders. The effects of these mutations on the structure and function of the enzymes remain to be established. Here, we investigate six mutants of the aspartyl-tRNA synthetase correlated with leukoencephalopathies. Our integrated strategy, combining an ensemble of biochemical and biophysical approaches, reveals that mutants are diversely affected with respect to their solubility in cellular extracts and stability in solution, but not in architecture. Mutations with mild effects on solubility occur in patients as allelic combinations whereas those with strong effects on solubility or on aminoacylation are necessarily associated with a partially functional allele. The fact that all mutations show individual molecular and cellular signatures and affect amino acids only conserved in mammals, points towards an alternative function besides aminoacylation.

No MeSH data available.


Related in: MedlinePlus