Abstract
TDP-43 and hnRNPA1 contain tandemly-tethered RNA-recognition-motif (RRM) domains, which not only functionally bind an array of nucleic acids, but also participate in aggregation/fibrillation, a pathological hallmark of various human diseases including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), alzheimer's disease (AD) and Multisystem proteinopathy (MSP). Here, by DSF, NMR and MD simulations we systematically characterized stability, ATP-binding and conformational dynamics of TDP-43 and hnRNPA1 RRM domains in both tethered and isolated forms. The results reveal three key findings: (1) upon tethering TDP-43 RRM domains become dramatically coupled and destabilized with Tm reduced to only 49 °C. (2) ATP specifically binds TDP-43 and hnRNPA1 RRM domains, in which ATP occupies the similar pockets within the conserved nucleic-acid-binding surfaces, with the affinity slightly higher to the tethered than isolated forms. (3) MD simulations indicate that the tethered RRM domains of TDP-43 and hnRNPA1 have higher conformational dynamics than the isolated forms. Two RRM domains become coupled as shown by NMR characterization and analysis of inter-domain correlation motions. The study explains the long-standing puzzle that the tethered TDP-43 RRM1–RRM2 is particularly prone to aggregation/fibrillation, and underscores the general role of ATP in inhibiting aggregation/fibrillation of RRM-containing proteins. The results also rationalize the observation that the risk of aggregation-causing diseases increases with aging.
Highlights
Aggregation/fibrillation of TDP-43 in the cytoplasm of neurons is a pathological hallmark of ~ 97% amyotrophic lateral sclerosis (ALS) and ~ 45% frontotemporal dementia-TDP (FTLD-TDP), that lack any efficacious medicine so far[1,2,3]
With experimental methods including differential scanning fluorimetry (DSF) and NMR as well as molecular dynamics (MD) simulations, we aimed to address this problem by characterizing the thermal stability, ATP-binding and conformational dynamics of TDP-43 and hnRNPA1 RNA-recognition motif (RRM) domains in both tethered and isolated forms
Comparison of NMR structures determined in the tethered and isolated forms (2CQG for RRM1 and 1WF0 for RRM2) revealed that RRM1 has Cα atom root-mean-square deviations (RMSD) value of 1.63 Å but RRM2 only 0.82 Å, indicating that the overall structures of both RRM1 and RRM2 domains of TDP-43 are well-folded and adopt the same RRM fold in both forms
Summary
Aggregation/fibrillation of TDP-43 in the cytoplasm of neurons is a pathological hallmark of ~ 97% amyotrophic lateral sclerosis (ALS) and ~ 45% frontotemporal dementia-TDP (FTLD-TDP), that lack any efficacious medicine so far[1,2,3]. ~ 70 human RRM-containing proteins including FUS, TDP-43 and hnRNPA1 have the intrinsically disordered prion-like domains of low-complexity sequences with amino acid compositions similar to those of the prion domains in yeast responsible for driving the formation of infectious c onformers[2,3,4] These proteins functionally mediate direct interactions with various nucleic acids to control both RNA processing and gene expression, but their aggregation/fibrillation is pathologically characteristic of an increasing spectrum of human diseases including ALS, frontotemporal dementia (FTD), Alzheimer’s disease (AD), chronic traumatic encephalopathy, muscle regeneration/degeneration and multisystem proteinopathy (MSP)[1,2,3,4,5,6,7,8,9,10]. We found that under exactly the same conditions, the tethered RRM1–RRM2 of TDP-43 has only one transition with Tm of only 49 °C, lower than Tm of the isolated RRM1
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