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

Abstract

Transactive response DNA-binding protein 43 kDa (TDP-43) is an RNA transporting and processing protein whose aberrant aggregates are implicated in neurodegenerative diseases. The C-terminal domain of this protein plays a key role in mediating this process. However, the N-terminal domain (residues 1-77) is needed to effectively recruit TDP-43 monomers into this aggregate. In the present study, we report, for the first time, the essentially complete 1 H, 15N and 13C NMR assignments and the structure of the N-terminal domain determined on the basis of 26 hydrogen-bond, 60 torsion angle and 1058 unambiguous NOE structural restraints. The structure consists of an a-helix and six b-strands. Two b-strands form a b-hairpin not seen in the ubiquitin fold. All Pro residues are in the trans conformer and the two Cys are reduced and distantly separated on the surface of the protein. The domain has a well defined hydrophobic core composed of F35, Y43, W68, Y73 and 17 aliphatic side chains. The fold is topologically similar to the reported structure of axin 1. The protein is stable and no denatured species are observed at pH 4 and 25°C. At 4 kcalÁmol À1 , the conformational stability of the domain, as measured by hydrogen/deuterium exchange, is comparable to ubiquitin (6 kcalÁmol À1 ).The b-strands, a-helix, and three of four turns are generally rigid, although the loop formed by residues 47-53 is mobile, as determined by model-free analysis of the 15 N{ 1 H}NOE, as well as the translational and transversal relaxation rates.

Structural insights into the multi-determinant aggregation of TDP-43 in motor neuron-like cells

“…The idea that TDP-43 inclusions are composed of hybrid molecular species is comforted by the observation that cytoplasmic Q331K TDP-43 inclusions facilitate the recruitment of WT protein, which dramatically accelerates neurodegeneration and disease progression in mice (Mitchell et al, 2015). Moreover, our simulative analyses are in good agreement with the experimental data and with recent biochemical evidence on the structure of the N-terminal domain (Mompean et al, 2016), suggesting that i. the most accessible N-terminal cysteine residues may be involved in a first step of oligomers formation, with the other cysteines contributing in a second step, and ii. that the large conformational capability of the C-terminal domains is involved in the formation of large aggregates.…”

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

“…However, we were able to assign the backbone chemical shifts of the domain as a H 6 GB1-TDP-43 NTD fusion (~20 kDa) at pH 6.8 (Supplementary Table 1). The backbone shifts are similar 5 to those reported for the isolated domain at pH 4 ( Supplementary Fig. 10b), indicating a similar fold.…”

“…Studies on the mechanism of TDP-43 action have revealed a complex interplay between the loss of cellular function and gain of toxic dysfunction effects of the TARDBP gene product 1,3,4 . TDP-43 comprises an N-terminal domain (TDP-43 NTD ), two RNA recognition motif single-stranded nucleic acid binding domains, and a disordered C-terminal domain (TDP-43 CTD ) 5 . The glycine rich C-terminal domain, which harbors the vast majority of disease-linked mutations 4 , selfinteracts and undergoes both liquid-liquid phase separation and aggregation [6][7][8] .…”

“…To probe for minor species that might escape detection in ensemble-averaged experiments, and to eliminate any self-association effects, we used single-molecule Förster resonance energy transfer (smFRET) experiments that can directly resolve minor species in a mixture 15 . We labeled TDP-43 NTD with donor and acceptor dyes (Alexa Fluor 488 & Alexa Fluor 594; see Supplementary Methods) at residues 2 and 81, which are close in space in the folded domain (PDB 2n4p 5 ). As a single doubly-labeled protein in the dilute sample (100 pM) traverses through the femtoliter-observation volume, we record fluorescence signals from the donor and acceptor dyes.…”

ALS Mutations Disrupt Phase Separation Mediated by α-Helical Structure in the TDP-43 Low-Complexity C-Terminal Domain

“…TDP-43 is a multidomain protein containing a folded(Mompean et al, 2016) multimer-forming(Chang et al, 2012; Wang et al, 2013) N-terminal domain, tandem RNA-binding domains that bind (UG)-rich sequences(Lukavsky et al, 2013), and a predominantly disordered C-terminal domain (CTD) that is essential for hnRNP interactions and splicing activity(Buratti et al, 2005; D'Ambrogio et al, 2009). ALS-associated TDP-43 inclusions are primarily fragments including the entire CTD(Neumann et al, 2006).…”