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. 2014 Dec 30;111(52):18619-24.
doi: 10.1073/pnas.1413994112. Epub 2014 Dec 12.

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

Haina Qin  1 , Liang-Zhong Lim  1 , Yuanyuan Wei  2 , Jianxing Song  3
Affiliations

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

Haina Qin et al. Proc Natl Acad Sci U S A. .

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 long-range 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.

Keywords: FTLD-TDP; NMR spectroscopy; TDP-43; amyotrophic lateral sclerosis; ubiquitin-like fold.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Characterization of two coexisting conformations adopted by the TDP-43 N-domain. (A) Domain organization of the 414-residue TDP-43 protein, which is composed of the N-domain determined in the present study, nuclear localization signal (L), two RNA recognition motifs (RRM1 and RRM2) hosting a nuclear export signal (E), and C-terminal glycine-rich domain. (B) Far-UV CD spectra at protein concentrations of 15 µM of the N-domain (1–102) in Milli-Q water at pH 4.0 (blue line) and in 1 mM phosphate buffer at pH 7.5 (blue dotted line); N-domain (1–80) in Milli-Q water at pH 4.0 (red line) and in 1 mM phosphate buffer at pH 7.5 (red dotted line); and N-domain (10–102) in Milli-Q water at pH 4.0 (green line) and in 1 mM phosphate buffer at pH 7.5 (green dotted line). (C) Superimposition of the 2D NMR 1H-15N HSQC spectra of the N-domain (1–102) in Milli-Q water at pH 4.0 at a at protein concentration of 40 µM (blue) and 1 mM (red). Cyan arrows are used to indicate the peaks from the well-folded form whose relative intensities are much higher at 40 µM (blue) than those at 1 mM (red). Purple arrows and oval are used to indicate the peaks from the unfolded form whose relative intensities are much lower at 40 µM (blue) than those at 1 mM (red). (D) Residue specific (ΔCα–ΔCβ) chemical shifts of the N-domain (1–102) in the folded (blue) and unfolded (red) forms.
Fig. 2.
Fig. 2.
NMR structure and dynamics of the TDP-43 N-domain. (A) {1H}-15N heteronuclear steady-state NOE (hNOE) of the N-domain (1–102) in the folded (blue) and unfolded (red) forms. (B) The lowest energy structure of the TDP-43 N-domain calculated by CS-Rosetta program with chemical shifts and unambiguous long-range NOEs. Residues having hNOE >0.7 are colored in red. (C) Overlay of five lowest energy structure of the TDP-43 N-domain. Blue is used for coloring β-strands, red for helix, and light yellow for loop. (D) Overlay of structures of the TDP-43 N-domain (purple) and ubiquitin (yellow; PDB ID code 3EHV). (E) Sequence alignment of TDP-43 N-domain and ubiquitin. Identical residues are colored in red and conserved in blue.
Fig. 3.
Fig. 3.
Binding of ssDNA to N-domain shifts the conformational equilibrium. (A) Far-UV CD spectra of the N-domain (1–102) at a protein concentration of 15 µM in the absence (blue) and in the presence of the single-stranded (TG)6 DNA at molar ratio of 1:0.5 (cyan), 1:1 (green), and 1:1.5 (red) (N-domain:DNA). (B) Superimposition of NMR 1H-15N HSQC spectra of the N-domain (1–102) at a protein concentration of 40 µM in the absence (blue) and in the presence of the (TG)6 DNA at molar ratio of 1:1 (red). Purple arrows are used to indicate HSQC peaks from the unfolded form which disappeared in the HSQC spectra in the presence of the (TG)6 DNA at molar ratio of 1:1 (red). (C) The structure of the TDP-43 N-domain in ribbon with residues having HSQC-peak intensity ratios < average value (0.65) colored in green. (D) The electrostatic potential surface of the TDP-43 N-domain structure with the identical orientation as in C and (E) with 180° rotation.
Fig. 4.
Fig. 4.
Proposed model for the functional dichotomy of TDP-43 mediated by N-domain.
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