The N‐Terminal Domain of ALS‐Linked TDP‐43 Assembles without Misfolding

Tsoi, Phoebe S.; Choi, Kyoung‐Jae; Leonard, Paul G.; Sizovs, Antons; Moosa, Mahdi Muhammad; MacKenzie, Kevin R.; Ferreon, Josephine C.; Ferreon, Allan Chris M.

doi:10.1002/anie.201706769

Cited by 54 publications

(48 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Several groups have reported that TDP‐43 NTD mediates assembly into dimers or other higher‐order structures (Shiina et al , ; Chang et al , ; Wang et al , ), though the details of the assembly have only recently begun to be visualized with atomistic resolution (Afroz et al , ; Jiang et al , ; Mompean et al , ; Tsoi et al , ). Recombinant TDP‐43 NTD (in our case: residues 1–80, with only three additional N‐terminal residues, GHM, resulting from the cloning strategy and cleavage with TEV protease) is highly soluble when expressed and purified from bacteria.…”

Section: Resultsmentioning

confidence: 99%

A single N‐terminal phosphomimic disrupts TDP‐43 polymerization, phase separation, and RNA splicing

et al. 2018

View full text Add to dashboard Cite

TDP‐43 is an RNA‐binding protein active in splicing that concentrates into membraneless ribonucleoprotein granules and forms aggregates in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Although best known for its predominantly disordered C‐terminal domain which mediates ALS inclusions, TDP‐43 has a globular N‐terminal domain (NTD). Here, we show that TDP‐43 NTD assembles into head‐to‐tail linear chains and that phosphomimetic substitution at S48 disrupts TDP‐43 polymeric assembly, discourages liquid–liquid phase separation (LLPS) in vitro, fluidizes liquid–liquid phase separated nuclear TDP‐43 reporter constructs in cells, and disrupts RNA splicing activity. Finally, we present the solution NMR structure of a head‐to‐tail NTD dimer comprised of two engineered variants that allow saturation of the native polymerization interface while disrupting higher‐order polymerization. These data provide structural detail for the established mechanistic role of the well‐folded TDP‐43 NTD in splicing and link this function to LLPS. In addition, the fusion‐tag solubilized, recombinant form of TDP‐43 full‐length protein developed here will enable future phase separation and in vitro biochemical assays on TDP‐43 function and interactions that have been hampered in the past by TDP‐43 aggregation.

show abstract

Section: Resultsmentioning

confidence: 99%

A single N‐terminal phosphomimic disrupts TDP‐43 polymerization, phase separation, and RNA splicing

et al. 2018

View full text Add to dashboard Cite

show abstract

“…Single-molecule measurements coupled to bulk biophysical techniques indicated that TDP-43 converts into amyloid-like fibrils following two distinct pathways. In one pathway, the CTDs of different molecules assemble directly [ 34 ]. In a second, parallel, pathway, fibrillation is induced by the NTD following a two-step process in which first the NTD domains dock, then the CTDs lock the molecules into an amyloid-like aggregate [ 34 ].…”

Section: Discussionmentioning

confidence: 99%

“…In one pathway, the CTDs of different molecules assemble directly [ 34 ]. In a second, parallel, pathway, fibrillation is induced by the NTD following a two-step process in which first the NTD domains dock, then the CTDs lock the molecules into an amyloid-like aggregate [ 34 ]. This is consistent with a previous finding that the first 10 residues of the TDP-43 sequence are crucial for both the folding and misfolding of the protein [ 31 ].…”

Section: Discussionmentioning

confidence: 99%

“…Secondly, it dimerizes and oligomerizes in vitro when dissected from the remainder of the protein and is proposed to be responsible for the dimerization and oligomerization of the entire full-length protein [ 10 , 11 , 12 , 13 ]. The NTD-driven oligomerization state of TDP-43 modulates the function and propensity to aggregate the entire protein into pathological inclusions [ 11 , 12 , 13 , 30 , 31 , 34 ].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Insight into the Folding and Dimerization Mechanisms of the N-Terminal Domain from Human TDP-43

Vega

Guri

Chiti

et al. 2020

IJMS

View full text Add to dashboard Cite

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.

show abstract

“…Protein valency increases with the presence of oligomerization domains. For example, TDP-43, a highly expressed nuclear RBP, contains an N-terminal domain that forms oligomers (117,(121)(122)(123)(124). Recently, Wang et al (117) established that polymerization of the N-terminal domain promotes LLPS of TDP-43 in vitro and that a single phosphomimetic mutant in the N-terminal domain can reduce the propensity of TDP-43 to phase separate.…”

Section: Oligomerization Domainsmentioning

confidence: 99%

The molecular language of membraneless organelles

Gomes

Shorter

2019

Journal of Biological Chemistry

625

600

View full text Add to dashboard Cite

Eukaryotic cells organize their intracellular components into organelles that can be membrane-bound or membraneless. A large number of membraneless organelles, including nucleoli, Cajal bodies, P-bodies, and stress granules, exist as liquid droplets within the cell and arise from the condensation of cellular material in a process termed liquid-liquid phase separation (LLPS). Beyond a mere organizational tool, concentrating cellular components into membraneless organelles tunes biochemical reactions and improves cellular fitness during stress. In this review, we provide an overview of the molecular underpinnings of the formation and regulation of these membraneless organelles. This molecular understanding explains emergent properties of these membraneless organelles and shines new light on neurodegenerative diseases, which may originate from disturbances in LLPS and membraneless organelles.

show abstract

The N‐Terminal Domain of ALS‐Linked TDP‐43 Assembles without Misfolding

Cited by 54 publications

References 27 publications

A single N‐terminal phosphomimic disrupts TDP‐43 polymerization, phase separation, and RNA splicing

A single N‐terminal phosphomimic disrupts TDP‐43 polymerization, phase separation, and RNA splicing

Insight into the Folding and Dimerization Mechanisms of the N-Terminal Domain from Human TDP-43

The molecular language of membraneless organelles

Contact Info

Product

Resources

About