Stress-induced misfolding and intraneuronal aggregation of the highly conserved nucleic acid binding protein TDP-43 (transactive response DNA binding protein 43 kDa) and its fragments have been implicated in amyotrophic lateral sclerosis and several other neurodegenerative diseases. However, the physicochemical mechanism of its misfolding from the functional folded state is poorly understood. TDP-43 is a four-domain protein and performs the essential nucleic acid binding function with the help of its two tandem RNA recognition motif domains naturally tethered by a linker (called here the tethered RRM domain of TDP-43 or TDP-43 tRRM ). Here, we show that the monomeric native form of TDP-43 tRRM remains in a pH-dependent and reversible thermodynamic equilibrium with a protonated, nanosized, 40-meric form (the A form). Under the stress-like low-pH condition, the A form becomes predominantly populated. In the A form, protein molecules have restricted dynamics of surface side-chain residues but native-like secondary structure. This self-assembled form possesses a loosely packed core in which the intrinsically disordered and aggregation-prone regions are in the proximity. The A form is metastable and swiftly aggregates into a highly stable amyloid-like protofibrillar form (β form) mediated by the disorder-to-order transition of intrinsically disordered regions upon small environmental perturbations. Interestingly, the A form and the β form are not formed when TDP-43 tRRM is bound to DNA, indicating that the nucleic acid binding regions of the protein participate in their formation. Our results reveal how the energy landscapes of folding and aggregation of TDP-43 tRRM are coupled by a metastable molten-globule like oligomeric form and modulated by stress-like conditions.

TDP-43 protein travels between the cytosol and the nucleus to perform its nucleic acid binding functions through its two tandem RNA recognition motif domains (TDP-43tRRM). When exposed to various environmental stresses, it forms abnormal aggregates in the cytosol of neurons, which are the hallmarks of amyotrophic lateral sclerosis and other TDP-43 proteinopathies. However, the nature of early structural changes upon stress sensing and the consequent steps during the course of aggregation are not well understood. In this study, we show that under low-pH conditions, mimicking starvation stress, TDP-43tRRM undergoes a conformational opening reaction linked to the protonation of buried ionizable residues and grows into a metastable oligomeric assembly (called the “low-pH form” or the “L form”). In the L form, the protein molecules have disrupted tertiary structure, solvent-exposed hydrophobic patches, and mobile side chains but the native-like secondary structure remains intact. The L form structure is held by weak interactions and has a steep dependence on ionic strength. In the presence of as little as 15 mM KCl, it fully misfolds and further oligomerizes to form a β-sheet rich “β form” in at least two distinct steps. The β form has an ordered, stable structure that resembles worm-like amyloid fibrils. The unstructured regions of the protein gain structure during L ⇌ β conversion. Our results suggest that TDP-43tRRM could function as a stress sensor and support a recent model in which stress sensing during neurodegeneration occurs by assembly of proteins into metastable assemblies that are precursors to the solid aggregates.

The mechanism of protein aggregation can be broadly viewed as a shift from the native-state stabilizing intramolecular to the aggregated-phase sustaining intermolecular interactions. Understanding the role of electrostatic forces on the extent of modulation of this switch has recently evolved as a topic of monumental significance as protein aggregation has lately been connected to charge modifications of an aging proteome. To decipher the distinctive role of electrostatic forces on the extremely complicated phase separation landscape, we opted for a combined in vitro–in silico approach to ascertain the structure–dynamics–stability–aggregability relationship of the functional tandem RRM domains of the ALS-related protein TDP-43 (TDP-43tRRM), under a bivariate solution condition in terms of pH and salt concentration. Under acidic pH conditions, the native TDP-43tRRM protein creates an aggregation-prone entropically favorable partially unfolded conformational landscape due to enthalpic destabilization caused by the protonation of the buried ionizable residues and consequent overwhelming fluctuations of selective segments of the sequence leading to anti-correlated movements of the two domains of the protein. The evolved fluffy ensemble with a comparatively exposed backbone then easily interacts with incoming protein molecules in the presence of salt via typical amyloid-aggregate-like intermolecular backbone hydrogen bonds with a considerable contribution originating from the dispersion forces. Subsequent exposure to excess salt at low pH conditions expedites the aggregation process via an electrostatic screening mechanism where salt shows preferential binding to the positively charged side chain. The applied target observable-specific approach complementarity unveils the hidden information landscape of an otherwise complex process with unquestionable conviction.