TDP-43 N terminus encodes a novel ubiquitin-like fold and its unfolded form in equilibrium that can be shifted by binding to ssDNA

Abstract

Transactivation response element (TAR) DNA-binding protein 43 (TDP-43) is the principal component of ubiquitinated inclusions characteristic of most forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia-frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP), as well as an increasing spectrum of other neurodegenerative diseases. Previous structural and functional studies on TDP-43 have been mostly focused on its recognized domains. Very recently, however, its extreme N terminus was identified to be a double-edged sword indispensable for both physiology and proteinopathy, but thus far its structure remains unknown due to the severe aggregation. Here as facilitated by our previous discovery that protein aggregation can be significantly minimized by reducing salt concentrations, by circular dichroism and NMR spectroscopy we revealed that the TDP-43 N terminus encodes a well-folded structure in concentration-dependent equilibrium with its unfolded form. Despite previous failure in detecting any sequence homology to ubiquitin, the folded state was determined to adopt a novel ubiquitin-like fold by the CS-Rosetta program with NMR chemical shifts and 78 unambiguous longrange nuclear Overhauser effect (NOE) constraints. Remarkably, this ubiquitin-like fold could bind ssDNA, and the binding shifted the conformational equilibrium toward reducing the unfolded population. To the best of our knowledge, the TDP-43 N terminus represents the first ubiquitin-like fold capable of directly binding nucleic acid. Our results provide a molecular mechanism rationalizing the functional dichotomy of TDP-43 and might also shed light on the formation and dynamics of cellular ribonucleoprotein granules, which have been recently linked to ALS pathogenesis. As a consequence, one therapeutic strategy for TDP-43-causing diseases might be to stabilize its ubiquitin-like fold by ssDNA or designed molecules. 1, 2). Since then, numerous studies have confirmed that TDP43 protein is mechanistically linked to neurodegeneration (3, 4). TDP43 is a 414-residue protein that has been previously recognized to be composed of a nuclear localization signal (NLS), two RNA recognition motifs (RRM1 and RRM2) hosting a nuclear export signal (NES), and C-terminal glycine-rich prion-like domain (Fig. 1A). The NLS and NES regulate the shuttling of TDP-43 between the nucleus and the cytoplasm (5), whereas the RRM1 and RRM2 are responsible for binding to nucleic acids including single-or double-stranded DNA/RNA (5-8). The prion-like domain mediates protein-protein interactions between TDP-43 and other hnRNP members (9), which also hosts most known ALS-associated TDP-43 mutations.TDP43 is an aggregation-prone protein (1-4, 10-14), and its abnormal aggregation has been found in ∼97% ALS and ∼45% frontotemporal dementia (FTD) patients. Additionally, TDP-43 immunoreactive inclusions have also been observed in an increasing spectrum of other neurodegenerative disorders, which include ALS/parkinsonism-dementia complex of Guam, Alzhei...

The TDP‐43 N‐terminal domain structure at high resolution

“…The conformational stability of the NTD in the context of the construct reported by Qin et al [22] is remarkably low, with the majority of the protein being denatured at equilibrium, and it was hypothesized that this low stability is key for the physiological and pathological roles of the protein. Qin et al [22] also claim that DNA binding can stabilize the folded form of a longer construct containing TDP-43 residues 1-102. Despite the significant advance that these results represent, no structure or chemical shift values were deposited and the resolution reported was low.…”

“…This result prompted us to characterize the structure, conformational stability and dynamics of this domain using NMR spectroscopy. When our work was underway, a low resolution structural model of the backbone structure was reported by Qin et al [22] for the NTD construct containing residues 1-80 and a C-terminal His-tag. The model contains five b-strands and an a-helix whose topology matches the ubiquitin fold.…”

“…A disordered peptide corresponding to TDP-43 residues 78-102 can bind DNA oligos Qin et al [22] found the NTD 1-102 to be unstable and also that the folded form could be stabilized by binding of DNA oligos. A speculative model was advanced in which DNA binding might prevent the formation of pathological TDP-43 aggregates by stabilizing the folded form of the NTD.…”

The N-terminal domain of ALS-linked TDP-43 assembles without misfolding

“…2-4). Because this finding contradicts the concentration-dependent unfolding described by Qin et al 13 , we performed smFRET on doubly-labeled 100 pM TDP-43 NTD titrated with up to 120 μM unlabeled TDP-43 NTD (Supplementary Fig. 14).…”

“…Other biophysical work has suggested that the unfolded state of TDP-43 NTD contributes to aggregation during cellular dysfunction 13,14 . sedimentation velocity analytical ultracentrifugation experiment shows self-association in a concentration-dependent fashion (see Supplementary Figure 9 for details).…”