The Folding and Aggregation Energy Landscapes of Tethered RRM Domains of Human TDP-43 Are Coupled via a Metastable Molten Globule-like Oligomer

Abstract

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.

Early Metastable Assembly during the Stress-Induced Formation of Worm-like Amyloid Fibrils of Nucleic Acid Binding Domains of TDP-43

“…The expression and the purification protocol of TDP-43 tRRM (UniProtKB/ Swiss-Prot entry Q13148) have been described previously. 34 The pure protein was stored in a buffer containing 10 mM potassium phosphate, 150 mM KCl, and 1 mM dithiothreitol (DTT) (pH 7.2). Sodium dodecyl sulfate−polyacrylamide gel electrophoresis revealed that the protein was highly pure.…”

“…The buffers used at different pH values have been reported previously. 34 A molar extinction coefficient of 15470 M −1 cm −1 at 280 nm was used to determine the protein concentration.…”

“…A DynaPro 99 unit (Wyatt) was used for the DLS measurements as reported previously. 34 In brief, all of the buffers used for the experiment were filtered three times with a 0.2 μm filter to remove dust particles. The centrifuge tubes and the tips were rinsed with 0.2 μm filtered Milli-Q water just before use.…”

“…The experimental settings were as reported previously. 34 DynaLS software (Wyatt) was used to obtain the mean autocorrelation function by taking an average of 50 acquisitions and resolving them into Gaussian distributions of hydrodynamic radii (R H ). The mean of all of the acquisitions from the cumulant analysis was used to calculate the total light scattering intensity.…”

Structural dissection of amyloid aggregates of TDP‐43 and its C‐terminal fragments TDP‐35 and TDP‐16

“…The NTD is folded [38][39][40][41] and essential to fulfill native functional processes and necessary for pathological aggregation [36,[42][43][44]. RRM1 and RRM2 domains are also folded and bind UG-rich segments of RNA [45][46][47], and it has been proposed that RRM2 may contribute to the pathological misfolding [48,49]. The pathological form of TDP-43 in ALS and FTLD is hyperphosphorylated, ubiquitinated, and proteolytically cleaved into C-terminal fragments (CTFs) [16,50].…”