Impact of pathogenic mutations on the refolding ability and stability of human mitochondrial Phenylalanyl-tRNA synthetase

Human mitochondrial phenylalanyl-tRNA synthetase (hmtPheRS) requires partial unfolding for mitochondrial import and subsequent refolding to maintain proper functionality. Mutations in the FARS2 gene, encoding hmtPheRS, cause disorders such as early-onset epileptic encephalopathy and spastic paraplegia. This study was intended to investigate the impact of mutations on hmtPheRS refolding ability, stability, and solubility. We have selected two mutations associated with early-onset epileptic encephalopathy (G309S, D325Y) with severe phenotype and three mutations associated with spastic paraplegia (P136H, D142Y, P361L) with less severe phenotypes. Some of those mutations were reported to have diminished aminoacylation activity. However, the molecular connection of pathogenicity remained elusive for these mutants. We observed that hmtPheRS showed exceptional structural flexibility and refolding ability even at lower pH. Mutations associated with severe phenotypes (G309S, D325Y) exhibited impaired refolding ability and stability, whereas other mutant versions of hmtPheRS linked to hereditary spastic paraplegia (P136H, D142Y, P361L) retained some stability and refolding capacity. Mutants exhibited expansion in hydrodynamic diameter, indicating significant perturbation in the internal architecture. Molecular simulation studies suggested the presence of structural deformities in hmtPheRS mutants at mildly acidic pH. This analysis reveals how mutations affect protein stability and function, which may play a role in mitochondrial disorders. It may act as a probable model for predicting pathogenicity-related mutants.