Conformational insights into the unfolding pathway of RNA recognition motif (RRM) domain of TAF15 associated with amyotrophic lateral sclerosis

The RNA recognition motif (RRM) domain of TAF15 protein plays an essential role in RNA binding and regulation of diverse cellular processes. However, the molecular mechanism underlying its structural stability and folding behaviour associated with disease state remains poorly understood. Herein, we performed an extensive molecular dynamics (MD) simulations study to investigate the conformational dynamics and unfolding pathway of the TAF15-RRM domain in 8 M urea. The elevated temperatures (300-500 K) were employed to accelerate the unfolding and capture different conformational states. The results from 3 μs cumulative MD simulations revealed that unfolding proceeds through stable, denaturant/temperature-accelerated partially unfolded intermediate ensembles. The native conformation of TAF15-RRM largely confined to marginal perturbation in urea at 300-350 K. The major conformational changes occurred in urea at 400-450 K, revealing distinct conformational ensembles corresponding to native (N), intermediate (I), and unfolded (U) populations. Whereas the native structure of protein was lost abruptly within ∼0-50 ns, at 500 K. At 400 K, the unfolding dynamics restricted to I-state with the loss of ∼55% native contacts. All three (N, I and U) stages are captured more distinctly in urea at 450 K. Furthermore, the secondary structure analyses demonstrated a higher susceptibility of β-sheets toward urea-induced unfolding, accompanied by the transient structures of non-native extended β-conformations and helical intermediates. FELs analyses suggest that the I-state populations are heterogeneous in nature which are largely stabilized by the partial retention and rearrangement of native H-bonds. These results thus highlight conformational signatures of unfolding intermediates that, by analogy with aggregation-prone intermediates characterized in other proteins, may be relevant to TAF15 self-assembly.