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Atomic structures of TDP-43 LCD segments and insights into reversible or pathogenic aggregation

Nature Structural & Molecular Biology volume 25, pages 463–471 (2018)Cite this article

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An Author Correction to this article was published on 17 September 2019

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Abstract

The normally soluble TAR DNA-binding protein 43 (TDP-43) is found aggregated both in reversible stress granules and in irreversible pathogenic amyloid. In TDP-43, the low-complexity domain (LCD) is believed to be involved in both types of aggregation. To uncover the structural origins of these two modes of β-sheet-rich aggregation, we have determined ten structures of segments of the LCD of human TDP-43. Six of these segments form steric zippers characteristic of the spines of pathogenic amyloid fibrils; four others form LARKS, the labile amyloid-like interactions characteristic of protein hydrogels and proteins found in membraneless organelles, including stress granules. Supporting a hypothetical pathway from reversible to irreversible amyloid aggregation, we found that familial ALS variants of TDP-43 convert LARKS to irreversible aggregates. Our structures suggest how TDP-43 adopts both reversible and irreversible β-sheet aggregates and the role of mutation in the possible transition of reversible to irreversible pathogenic aggregation.

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Fig. 1: Segments from the LCD of TDP-43 form steric zippers.
Fig. 2: Validation of steric-zipper-forming segments.
Fig. 3: The 312NFGAFS317 segments form a kinked β-sheet that is strengthened by familial variants A315T and A315E.
Fig. 4: Speculative model showing the alternative pathways of formation of stress granules with pathogenic amyloid.

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  • 17 September 2019

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank S. Sangwan and P. Seidler for discussion, D. Shi and T. Gonen at Janelia for microscope support and M. Collazo at UCLA-DOE Macromolecular Crystallization Core Technology Center for crystallization support. This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which is funded by the National Institute of General Medical Sciences from the National Institutes of Health (P41 GM103403). The Eiger 16 M detector on 24-ID-E beam line is funded by a NIH-ORIP HEI grant (S10OD021527). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. This research was supported in part by grants from the National Institutes of Health NIH NIA AG029430, NIH NIA AG054022, Howard Hughes Medical Institute and the Janelia Research Campus visitor program. This material is based upon work supported by the National Science Foundation under Grant No. NSF 1616265. We acknowledge the use of instruments at the Electron Imaging Center for Nanomachines supported by UCLA and by instrumentation grants from NIH (1S10RR23057 and 1U24GM116792) and NSF (DBI-1338135). D.R.B. was supported by the National Science Foundation Graduate Research Fellowship Program.

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  1. Hamilton Trinh

    Present address: Wayne State University School of Medicine, Detroit, MI, USA

  2. These authors contributed equally: Elizabeth L. Guenther, Qin Cao.

Authors and Affiliations

  1. Howard Hughes Medical Institute, University of California, Los Angeles, Los Angeles, CA, USA

    Elizabeth L. Guenther, Qin Cao, Hamilton Trinh, Jiahui Lu, Michael R. Sawaya, Duilio Cascio, David R. Boyer, Jose A. Rodriguez, Michael P. Hughes & David S. Eisenberg

  2. UCLA-DOE Institute, University of California, Los Angeles, Los Angeles, CA, USA

    Elizabeth L. Guenther, Qin Cao, Hamilton Trinh, Jiahui Lu, Michael R. Sawaya, Duilio Cascio, David R. Boyer, Jose A. Rodriguez, Michael P. Hughes & David S. Eisenberg

  3. Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA, USA

    Elizabeth L. Guenther, Qin Cao, Hamilton Trinh, Jiahui Lu, Michael R. Sawaya, Duilio Cascio, David R. Boyer, Jose A. Rodriguez, Michael P. Hughes & David S. Eisenberg

  4. Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, CA, USA

    Elizabeth L. Guenther, Qin Cao, Hamilton Trinh, Jiahui Lu, Michael R. Sawaya, Duilio Cascio, David R. Boyer, Jose A. Rodriguez, Michael P. Hughes & David S. Eisenberg

  5. Chemistry and Biochemistry Department, University of California, Los Angeles, Los Angeles, CA, USA

    Elizabeth L. Guenther, Qin Cao, Hamilton Trinh, Jiahui Lu, Michael R. Sawaya, Duilio Cascio, David R. Boyer, Jose A. Rodriguez, Michael P. Hughes & David S. Eisenberg

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Contributions

E.L.G., Q.C. and D.S.E. designed the project and wrote the manuscript with input from all other authors, especially M.R.S. E.L.G. and H.T. conducted fibril growth assays and prepared peptide crystals. E.L.G., Q.C. and J.L. cloned and purified TDP-43 constructs and performed the protein aggregation assay and fibril diffraction. M.P.H. predicted putative LARKS. E.L.G., H.T. and M.R.S. processed and solved 300GNNQGSN306. E.L.G. and M.R.S. processed and solved 312NFGAFS317, 312NFGTFS317, 370GNNSYS375 and 396GFNGGFG402. E.L.G., Q.C., J.L. and M.R.S. processed data and solved the 321AMMAAA326 and 328AALQSS333. J.A.R. collected MicroED data on 312NFGEFS317 and 333SWGMMGMLASQ343, M.R.S., D.C. and E.L.G. processed data and solved the structure of 312NFGEFS317 and 333SWGMMGMLASQ343. D.R.B. collected MicroED data for 312NFGpTFS317. Q.C., M.R.S., D.C. and D.R.B. processed data and solved the structure of 312NFGpTFS317. E.L.G., Q.C., M.R.S., M.P.H. and D.S.E. analyzed structures and designed the model of the LCD in SG formation and pathological aggregation.

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Correspondence to David S. Eisenberg.

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D.S.E. is an advisor and equity shareholder in ADDRx, Inc.

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Integrated Supplementary Information

Supplementary Figure 1 321AMMAAA326 and 333SWGMMGMLASQ343 form antiparallel steric-zipper structures.

Views perpendicular to the fibril axis (vertical) are shown for three steric-zipper structures of Fig. 1. 321AMMAAA326 and 333SWGMMGMLASQ343 are two zippers that adopt anti-parallel packing, as shown by the alternating orientation of arrows representing the direction of strands. 328AALOSS333 is shown as an example of the zippers which adopt parallel packing, as shown by the same orientation of arrows in each sheet.

Supplementary Figure 2 Validation of TDP-CTF and TDP-LCD constructs.

The SUMO tagged TDP-CTF and TDP-LCD were analyzed by SDS-PAGE and stained with coomassie blue and anti-TDP-43 antibody. For TDP-CTF, notice that 1 hour after (1h) ULP1 cleavage, it generates SUMO protein and tag-free TDP-CTF, and both SUMO-tagged and tag-free TDP-CTF can be recognized by anti-TDP-43 antibody. SUMO tagged TDP-LCD also can be recognized by anti-TDP-43 antibody, and generates SUMO protein and tag-free TDP-LCD after cleavage. The molecular weight of SUMO and tag-free TDP-LCD are so close to each other that the two bands are indistinguishable on SDS-PAGE when stained by coomassie blue. However, the existence of tag-free TDP-LCD can be detected by anti-TDP-43 antibody, forming a band (although very dim) at its expected position. Tag-free TDP-LCD can also be detected by SDS-PAGE of the TDP-LCD pellet, showing a band at its expected position, and SUMO protein remains soluble as shown in Fig. 2a.

Supplementary Figure 3 Positive control of denaturing conditions shown in Fig. 3b and structural details of 312NFGEFS317 and 312NFGpTFS317.

(A) Negative stain EM images of Aβ1-42 fibrils, typical amyloid fibrils, before and after treatment with 2% SDS and 70 °C for 15 mins. Notice that Aβ1-42 fibrils are stable and maintain their morphology after treatment in denaturing conditions. Scale bar = 200 nm. (B) Omit map of phosphate in 312NFGpTFS317 structure viewed from two angles. Green mesh shows the electron density of the Fo-Fc map with sigma > 4.5. Notice that the electron density of the same map with sigma <-4.5 is also computed as red mesh, however there is no observable negative density. TPO: phosphorylated threonine. (C) Hydrogen bond networks that stabilizes the packing of 312NFGEFS317 and 312NFGpTFS317. In each structure, three Glu/pThr residues from three adjacent strands of same sheet are shown, as well as the residues hydrogen-bonded to them. The hydrogen bonds formed by all Glu and the central pThr are shown as yellow dash lines, with distances between 2.3 and 3.2 Å.

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Guenther, E.L., Cao, Q., Trinh, H. et al. Atomic structures of TDP-43 LCD segments and insights into reversible or pathogenic aggregation. Nat Struct Mol Biol 25, 463–471 (2018). https://doi.org/10.1038/s41594-018-0064-2

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