Semi-synthesis of TDP-43 reveals the effects of phosphorylation in N-terminal domain on self-association.

TDP-43, a nucleocytoplasmic shuttle protein consisting of 414 residues, forms self-association in the nucleus for physiological gene regulation, while aggregation into amyloid (consisting of aberrant β-sheets) in the cytoplasm causes neurodegenerative diseases such as amyotrophic lateral sclerosis. Post-translational phosphorylation of TDP-43 alters the self-association properties, which affects both the physiological function in the nucleus and the amyloidogenic potential in the cytoplasm, thereby impacting upon disease progression. However, insight into the role of per-residue phosphorylation in the self-association remains limited due to the difficulty in obtaining site-specifically phosphorylated TDP-43. Here, we demonstrate semi-synthesis of full-length TDP-43 that is uniformly phosphorylated at the 48th serine residue (designated as TDP[pS48]). The synthetic scheme consisting of native chemical ligation followed by His-tag affinity chromatography efficiently gave TDP(pS48) with a high purity. Interestingly, unlike non-phosphorylated TDP-43, the phosphorylated TDP-43 was found to have weak self-association property and to form aggregates that were not typical amyloid fibrils. Furthermore, chemical synthesis and three-dimensional structure analysis of the N-terminal domain (NTD, corresponding to TDP) suggested that the phosphate ion at Ser48 weakens the inter-NTD interaction by inducing electrostatic repulsion. It significantly advances understanding of the pathological mechanisms involved in the post-translational modifications of TDP-43 associated with the neurodegenerative diseases.