Pathological Mutations D169G and P112H Electrostatically\nAggravate the Amyloidogenicity of the Functional Domain of TDP-43

Aggregation\nof TDP-43 is linked to the pathogenesis of many neurodegenerative\ndiseases, including amyotrophic lateral sclerosis (ALS). Notably,\nelectrostatic point mutations such as D169G and P112H, located within\nthe highly conserved functional tandem RNA recognition motif (RRM)\ndomains of the TDP-43 protein (TDP-43<sup>tRRM</sup>), have been identified\nin diseased patients as well. In this study, we address how the electrostatic\nmutations alter both the native state stability and aggregation propensity\nof TDP-43<sup>tRRM</sup>. The mutants D169G and P112H show increased\nchemical stability compared to the TDP-43<sup>tRRM</sup> at physiological\npH. However, at low pH, both the mutants undergo a conformational\nchange to form amyloid-like fibrils, though with variable ratesthe\nP112H mutant being substantially faster than the other two sequences\n(TDP-43<sup>tRRM</sup> and D169G mutant) showing comparable rates.\nMoreover, among the three sequences, only the P112H mutant undergoes\na strong ionic strength-dependent aggregability trend. These observations\nsignify the substantial contribution of the excess charge of the P112H\nmutant to its unique aggregation process. Complementary simulated\nobservables with atomistic resolution assign the experimentally observed\nsequence-, pH-, and ionic strength-dependent aggregability pattern\nto the degree of thermal lability of the mutation site-containing\nRRM1 domain and its extent of dynamical anticorrelation with the RRM2\ndomain whose combination eventually dictate the extent of generation\nof aggregation-prone partially unfolded conformational ensembles.\nOur choice of a specific charge-modulated pathogenic mutation-based\nexperiment-simulation-combination approach unravels the otherwise\nhidden residue-wise contribution to the individual steps of this extremely\ncomplicated multistep aggregation process.