Identifying interactions between TDP-43’s N-terminal and RNA binding domains

TAR DNA-binding Protein 43 (TDP-43) plays a crucial role in the pathophysiology and progression of Amyotrophic Lateral Sclerosis (ALS), affecting both 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 which 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 along with de novo sequence-to-structure predictions of TDP-43 _1-260 that propose NTD stacking onto the RRM domains. Using Carr-Purcell-Meiboom-Gill (CPMG)-relaxation dispersion 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 (MST) 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.