Solid-State NMR Reveals the Structural Transformation of the TDP-43 Amyloidogenic Region upon Fibrillation

Abstract

TDP-43 is a primary pathological hallmark protein of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, which may exist in the form of amyloid inclusions in the cells of patients. In addition to serving as a biomarker for these diseases, TDP-43 can also directly trigger neurodegeneration. We previously determined the amyloidogenic core region of TDP-43 (residues 311–360) and showed by solution NMR that this region includes two α-helices [(321–330) and (335–343)] in solution. We suggested that the helix-to-sheet structural transformation initiates TDP-43 aggregation. In the present study, X-ray diffraction shows that TDP-43 (311–360) aggregates adopt a cross-β structure. Thioredoxin (Trx)-fused TDP-43 (311–360) can undergo liquid–liquid phase separation (LLPS) before fibrillation, suggesting that phase separation is an intermediate step before amyloid formation. Solid-state NMR (SSNMR), carried out to elucidate the structural changes of TDP-43 (311–360) at the atomic level, indicates five β-strands of the amyloids formed, with the major two β-strands contributed by the first helical region in the solution structure. The NMR evidence is also in support of the fibril having a parallel in-register conformation, implying a mechanism in which the helix–helix interactions in LLPS are converted into β-strand parallel lateral association upon fibrillation. Our studies have assigned many key interresidue interactions that contribute to the stability of the fibril, including F316 with I318 and Q327 and W334 with A325, A326, A329, and S332. SSNMR with 1H detection reveals a unique close interaction between the indole Nε1–Hε1 of W334 and the side-chain carbonyl of Q343. This interaction could be a very important factor in initiating TDP-43 (311–360) folding/misfolding in LLPS.

Redox-mediated regulation of an evolutionarily conserved cross-β structure formed by the TDP43 low complexity domain

“…In the 500 million years separating fish from humans, not a single amino acid has been changed within this ultraconserved region in any of the 11 species included in this analysis. The overlapping footprinted and ultraconserved regions of the TDP43 LC domain also colocalize with a cross-β structure described recently from cryo-EM and solid-state NMR studies of TDP43 LC domain polymers (23,24). Highly related, dagger-like structures were independently resolved by cryo-EM methods for three different cross-β polymers formed from the TDP43 LC domain (Fig.…”

“…Solution NMR spectroscopic measurements have given evidence of α-helical secondary structure between residues 322 and 344 of the LC domain in a limited population of TDP-43 molecules. Intriguingly, it is this same region that has been shown to: 1) form structurally precise crossβ interactions at physiological pH (7.4-7.5) as deduced by both cryo-EM and solid-state NMR approaches (23,24,28); 2) be protected in a structure-dependent manner from H 2 O 2 -mediated oxidation (Fig. 3A); and 3) be strikingly conserved through evolution (Fig.…”

Cryo-EM structure of amyloid fibrils formed by the entire low complexity domain of TDP-43

“…Furthermore, recent reports indicate that these aggregates can self-propagate by the prion-like mechanism [13][14][15][16] . Consistent with these findings, polypeptides corresponding to TDP-43 LCD or its fragments have been shown to form amyloid fibrils in vitro 3,8,[17][18][19] .…”

Interactions between the Intrinsically Disordered Regions of hnRNP-A2 and TDP-43 Accelerate TDP-43′s Conformational Transition

“…Cryo-EM images of the amyloid core of TDP-43 covering residues 311-360 are in agreement with the loss of the α-helical component observed here [44]. Although the intrinsic α-to-β transition propensity of TDP-43 has been suggested previously by studies of peptides (residues 311-360) [45,46], the present study demonstrates that this transition is accelerated in the presence of its interacting partner hnRNP-A2 ( Figure 5). The monomeric IDR of TDP-43 populates both helix and coil conformations at equilibrium and tends to aggregate in its coil form.…”