Abstract
SummaryMislocalization, cleavage, and aggregation of the human protein TDP-43 is found in many neurodegenerative diseases. As is the case with many other proteins that are completely or partially structurally disordered, production of full-length recombinant TDP-43 in the quantities necessary for structural characterization has proved difficult. We show that the full-length TDP-43 protein and two truncated N-terminal constructs 1-270 and 1-263 can be heterologously expressed in E. coli. Full-length TDP-43 could be prevented from aggregation during purification using a detergent. Crystals grown from an N-terminal construct (1-270) revealed only the N-terminal domain (residues 1-80) with molecules arranged as parallel spirals with neighboring molecules arranged in head-to-tail fashion. To obtain detergent-free, full-length TDP-43 we mutated all six tryptophan residues to alanine. This provided sufficient soluble protein to collect small-angle X-ray scattering data. Refining relative positions of individual domains and intrinsically disordered regions against this data yielded a model of full-length TDP-43.
Highlights
The deposition of intracellular transactivation response element (TAR) DNA-binding protein 43 (TDP-43) inclusions is the hallmark of TDP-43 pathology
We show that the full-length TDP-43 protein and two truncated N-terminal constructs 1-270 and 1-263 can be heterologously expressed in E. coli
Crystals grown from an N-terminal construct (1-270) revealed only the N-terminal domain with molecules arranged as parallel spirals with neighboring molecules arranged in head-to-tail fashion
Summary
The deposition of intracellular TDP-43 inclusions is the hallmark of TDP-43 pathology. Observed in neural tissues from individuals with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) (Neumann et al, 2006), TDP-43 pathology is associated with many neurodegenerative diseases These include, but are by no means limited to, Alzheimer disease (Amador-Ortiz et al, 2007), Parkinson disease (Nakashima-Yasuda et al, 2007), hippocampal sclerosis (Amador-Ortiz et al, 2007), and chronic traumatic encephalopathy (McKee et al, 2010). At the N terminus, a ubiquitin or dix-like domain (Mompean et al, 2016; Qin et al, 2014) provides a polymerization surface that enables formation of dimer and higher-order oligomers predominantly found in the cell nucleus (Afroz et al, 2017)
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