Abstract
TAR DNA-binding Protein 43 kilodaltons (TDP-43) plays a crucial role in the pathophysiology and progression of amyotrophic lateral sclerosis, affecting familial and sporadic cases. TDP-43 is an intrinsically disordered multidomain protein that consists of an N-terminal domain (NTD(1-102)), two tandem RNA recognition motifs (RRM1(102-177) and RRM2(191-260)), and an intrinsically disordered glycine-rich C-terminal(261-414) domain. We previously identified a chemical probe that led to allosteric alterations between the RRM and NTD of TDP-43. We attributed these changes to potential interdomain interactions between the NTD and RRM segments. In this work, we compared the 2D [(1)H,(15)N] HSQC-NMR resonances of two constructs, TDP-43(102-260) (RRM domain alone) against TDP-43(1-260) (NTD linked to RRM) and observed clustered shifts in the RNA-binding sites of both RRM domains. To investigate why these shifts appeared in the RRM domains in the absence of RNA, we hypothesized that the NTD domain could be stacking on the RRM domains. Thus, we modeled NTD-RRM interactions using protein-protein docking of TDP-43 subdomains that propose NTD stacking onto the RRM domains. Using Carr-Purcell-Meiboom-Gill NMR spectroscopy, we demonstrated evidence of an interaction between NTD(1-102) and RRMs(102-260). Finally, we investigated the impact of NTD on RNA binding using 2D (15)N-HSQC-NMR and microscale thermophoresis by titration of a short UG-rich RNA sequence and observed significant changes in RNA binding between TDP-43(102-260) and TDP-43(1-260), further suggesting the NTD plays a role in TDP-43 RNA interactions.