A Hydrophobic Core Stabilizes the Residual Structure in the RRM2 Intermediate State of the ALS-linked Protein TDP-43

[Display omitted] •Folding intermediates can mediate misfolding and aggregation in human diseases.•We identified the core structure for a folding intermediate in RRM2 of TDP-43.•The RRM2 intermediate has residual secondary structure in the N-terminal half.•Key stabilizing hydrophobic contacts severely disrupt the fold of RRM2 when mutated.•Results suggest RRM2 intermediate plays a role in normal function and dysfunction. Folding intermediates mediate both protein folding and the misfolding and aggregation observed in human diseases, including amyotrophic lateral sclerosis (ALS), and are prime targets for therapeutic interventions. In this study, we identified the core nucleus of structure for a folding intermediate in the second RNA recognition motif (RRM2) of the ALS-linked RNA-binding protein, TDP-43 (TAR DNA-binding protein-43), using a combination of experimental and computational approaches. Urea equilibrium unfolding studies revealed that the RRM2 intermediate state consists of collapsed residual secondary structure localized to the N-terminal half of RRM2, while the C-terminus is largely disordered. Steered molecular dynamics simulations and mutagenesis studies yielded key stabilizing hydrophobic contacts that, when mutated to alanine, severely disrupt the overall fold of RRM2. In combination, these findings suggest a role for this RRM intermediate in normal TDP-43 function as well as serving as a template for misfolding and aggregation through the low stability and non-native secondary structure.