ABSTRACT
Various pathogenic variants in both mitochondrial tRNAPhe and Phenylalanyl-tRNA synthetase mitochondrial protein coding gene (FARS2) gene encoding for the human mitochondrial PheRS have been identified and associated with neurological and/or muscle-related pathologies. An important Guanine-34 (G34)A anticodon mutation associated with myoclonic epilepsy with ragged red fibers (MERRF) syndrome has been reported in hmit-tRNAPhe . The majority of G34 contacts in available aaRSs-tRNAs complexes specifically use that base as an important tRNA identity element. The network of intermolecular interactions providing its specific recognition also largely conserved. However, their conservation depends also on the invariance of the residues in the anticodon binding domain (ABD) of human mitochondrial Phenylalanyl-tRNA synthetase (hmit-PheRS). A defect in recognition of the anticodon of tRNAPhe may happen not only because of G34A mutation, but also due to mutations in the ABD. Indeed, a pathogenic mutation in FARS2 has been recently reported in a 9-year-old female patient harboring a p.Asp364Gly mutation. Asp364 is hydrogen bonded (HB) to G34 in WT hmit-PheRS. Thus, there are two pathogenic variants disrupting HB between G34 and Asp364: one is associated with G34A mutation, and the other with Asp364Gly mutation. We have measured the rates of tRNAPhe aminoacylation catalyzed by WT hmit-PheRS and mutant enzymes. These data ranked the residues making a HB with G34 according to their contribution to activity and the signal transduction pathway in the hmit-PheRS-tRNAPhe complex. Furthermore, we carried out extensive MD simulations to reveal the interdomain contact topology on the dynamic trajectories of the complex, and gaining insight into the structural and dynamic integrity effects of hmit-PheRS complexed with tRNAPhe . DATABASE: Structural data are available in PDB database under the accession number(s): 3CMQ, 3TUP, 5MGH, 5MGV.
Subject(s)
Genetic Pleiotropy , Mitochondrial Proteins/chemistry , Paraparesis, Spastic/genetics , Phenylalanine-tRNA Ligase/chemistry , RNA, Transfer, Phe/chemistry , Amino Acid Substitution , Anticodon/chemistry , Anticodon/metabolism , Aspartic Acid/chemistry , Child , Consanguinity , DNA, Mitochondrial/genetics , Disease Progression , Female , Guanine/chemistry , Humans , Hydrogen Bonding , MERRF Syndrome/genetics , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Models, Molecular , Molecular Dynamics Simulation , Motion , Mutation, Missense , Phenotype , Phenylalanine-tRNA Ligase/genetics , Phenylalanine-tRNA Ligase/metabolism , Point Mutation , Protein Conformation , Protein DomainsABSTRACT
Mutations in the mitochondrial aminoacyl-tRNA synthetases (mtaaRSs) can cause profound clinical presentations, and have manifested as diseases with very selective tissue specificity. To date most of the mtaaRS mutations could be phenotypically recognized, such that clinicians could identify the affected mtaaRS from the symptoms alone. Among the recently reported pathogenic variants are point mutations in FARS2 gene, encoding the human mitochondrial PheRS. Patient symptoms range from spastic paraplegia to fatal infantile Alpers encephalopathy. How clinical manifestations of these mutations relate to the changes in three-dimensional structures and kinetic characteristics remains unclear, although impaired aminoacylation has been proposed as possible etiology of diseases. Here, we report four crystal structures of HsmtPheRS mutants, and extensive MD simulations for wild-type and nine mutants to reveal the structural changes on dynamic trajectories of HsmtPheRS. Using steady-state kinetic measurements of phenylalanine activation and tRNAPhe aminoacylation, we gained insight into the structural and kinetic effects of mitochondrial disease-related mutations in FARS2 gene.
Subject(s)
Diffuse Cerebral Sclerosis of Schilder/genetics , Mitochondrial Proteins/chemistry , Mutation , Paraplegia/genetics , Phenylalanine-tRNA Ligase/chemistry , RNA, Transfer, Phe/chemistry , Adolescent , Amino Acid Motifs , Aminoacylation , Binding Sites , Child, Preschool , Crystallography, X-Ray , Diffuse Cerebral Sclerosis of Schilder/diagnosis , Diffuse Cerebral Sclerosis of Schilder/metabolism , Diffuse Cerebral Sclerosis of Schilder/pathology , Female , Humans , Kinetics , Male , Mitochondria/genetics , Mitochondria/metabolism , Mitochondria/pathology , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Molecular Dynamics Simulation , Paraplegia/diagnosis , Paraplegia/metabolism , Paraplegia/pathology , Phenylalanine-tRNA Ligase/genetics , Phenylalanine-tRNA Ligase/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , RNA, Transfer, Phe/metabolism , Sequence Alignment , Substrate Specificity , ThermodynamicsABSTRACT
Mitochondria are considered as the primary source of reactive oxygen species (ROS) in nearly all eukaryotic cells during respiration. The harmful effects of these compounds range from direct neurotoxicity to incorporation into proteins producing aberrant molecules with multiple physiological problems. Phenylalanine exposure to ROS produces multiple oxidized isomers: tyrosine, Levodopa, ortho-Tyr, meta-Tyr (m-Tyr), and so on. Cytosolic phenylalanyl-tRNA synthetase (PheRS) exerts control over the translation accuracy, hydrolyzing misacylated products, while monomeric mitochondrial PheRS lacks the editing activity. Recently we showed that "teamwork" of cytosolic and mitochondrial PheRSs cannot prevent incorporation of m-Tyr and l-Dopa into proteins. Here, we present human mitochondrial chimeric PheRS with implanted editing module taken from EcPheRS. The monomeric mitochondrial chimera possesses editing activity, while in bacterial and cytosolic PheRSs this type of activity was detected for the (αß)2 architecture only. The fusion protein catalyzes aminoacylation of tRNA(Phe) with cognate phenylalanine and effectively hydrolyzes the noncognate aminoacyl-tRNAs: Tyr-tRNA(Phe) and m-Tyr-tRNA(Phe) .
