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

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.