Abstract
TAR DNA-binding protein 43 (TDP-43) is a 414-residue long nuclear protein whose deposition into intraneuronal insoluble inclusions has been associated with the onset of amyotrophic lateral sclerosis (ALS) and other diseases. This protein is physiologically a homodimer, and dimerization occurs through the N-terminal domain (NTD), with a mechanism on which a full consensus has not yet been reached. Furthermore, it has been proposed that this domain is able to affect the formation of higher molecular weight assemblies. Here, we purified this domain and carried out an unprecedented characterization of its folding/dimerization processes in solution. Exploiting a battery of biophysical approaches, ranging from FRET to folding kinetics, we identified a head-to-tail arrangement of the monomers within the dimer. We found that folding of NTD proceeds through the formation of a number of conformational states and two parallel pathways, while a subset of molecules refold slower, due to proline isomerism. The folded state appears to be inherently prone to form high molecular weight assemblies. Taken together, our results indicate that NTD is inherently plastic and prone to populate different conformations and dimeric/multimeric states, a structural feature that may enable this domain to control the assembly state of TDP-43.
Highlights
In 2006, two seminal papers by Neumann et al [1] and Arai et al [2] reported that the nuclear TAR DNA-binding protein 43 (TDP-43) is involved in amyotrophic lateral sclerosis (ALS) and tau-negative, ubiquitin-positive frontotemporal lobar degeneration (FTLD-U)
We studied the folding process of NTD1–77 from its fully unfolded state to its folded native dimer and identified a peculiar mechanism through which the protein domain folds with at least two parallel pathways and partially folded states accumulating in both pathways
I becomes more unstable than CS and this leads to a second, parallel, folding pathway, in which CSt undergoes a direct transition that leads to the formation of the folded state (F), with no need of intermediate states accumulating during the process
Summary
In 2006, two seminal papers by Neumann et al [1] and Arai et al [2] reported that the nuclear TAR DNA-binding protein 43 (TDP-43) is involved in amyotrophic lateral sclerosis (ALS) and tau-negative, ubiquitin-positive frontotemporal lobar degeneration (FTLD-U). We measured fluorescence after a dilution of the unfolded protein to a final urea concentration of 0.1 M, obtaining a spectrum fully superimposable to that of the folded protein (Figure 2b). This shows that the urea-induced denaturation of NTD was a fully reversible process. Since other authors reported the ability of NTD to oligomerize via disulphide bridging in the absence of DTT [12], we repeated the experiment in the presence of 1 mM DTT Under these conditions, NTD eluted as a single peak at 17.70 mL, close to that corresponding to a dimer (17.0 mL).
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