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Atomic-level evidence for packing and positional amyloid polymorphism by segment from TDP-43 RRM2

Nature Structural & Molecular Biology volume 25, pages 311–319 (2018)Cite this article

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Abstract

Proteins in the fibrous amyloid state are a major hallmark of neurodegenerative disease. Understanding the multiple conformations, or polymorphs, of amyloid proteins at the molecular level is a challenge of amyloid research. Here, we detail the wide range of polymorphs formed by a segment of human TAR DNA-binding protein 43 (TDP-43) as a model for the polymorphic capabilities of pathological amyloid aggregation. Using X-ray diffraction, microelectron diffraction (MicroED) and single-particle cryo-EM, we show that the 247DLIIKGISVHI257 segment from the second RNA-recognition motif (RRM2) forms an array of amyloid polymorphs. These associations include seven distinct interfaces displaying five different symmetry classes of steric zippers. Additionally, we find that this segment can adopt three different backbone conformations that contribute to its polymorphic capabilities. The polymorphic nature of this segment illustrates at the molecular level how amyloid proteins can form diverse fibril structures.

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Fig. 1: Segments from the RRM2 domain of TDP-43 exhibit characteristic amyloid properties.
Fig. 2: Atomic structures of 248LIIKGI253 and 247DLIIKGISVHI257, determined by diffraction methods, illustrate different steric zipper interfaces.
Fig. 3: Single-particle cryo-EM structure of the 247DLIIKGISVHI257 fibril illustrates a 27-filament, polymorphic three-start helix.
Fig. 4: Seven unique sheet-to-sheet interfaces, seen in the three structures presented.
Fig. 5: The conformational flexibility of the 247DLIIKGISVHI257 backbone provides insight into amyloid fibril formation and polymorphism.

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Acknowledgements

We thank Q. Cao, L. Saelices, S. Sangwan, and P. Seidler for discussion, D. Shi at Janelia for microscope support, and M. Collazo at UCLA-DOE Macromolecular Crystallization Core Technology Center for crystallization support. We thank the staff at the Argonne Photon Source (APS) NECAT Beamline 24-ID-E, which is funded by National Institutes of Health (P41 GM103403). This research was supported in part by grants from the National Science Foundation (NSF MCB 1616265 to D.S.E.) and the National Institutes of Health (GM071940 to Z.H.Z.), Howard Hughes Medical Institute and the Janelia Research Campus visitor program. D.R.B. was supported by the National Science Foundation Graduate Research Fellowship. 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).

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Author notes

  1. Hamilton Trinh

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

  2. Tamir Gonen

    Present address: Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, CA, USA

Authors and Affiliations

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

    Elizabeth L. Guenther, Hamilton Trinh, Michael R. Sawaya, Duilio Cascio, David R. Boyer & David S. Eisenberg

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

    Elizabeth L. Guenther, Michael R. Sawaya, Duilio Cascio, David R. Boyer & David S. Eisenberg

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

    Elizabeth L. Guenther, Michael R. Sawaya, Duilio Cascio, Z. Hong Zhou & David S. Eisenberg

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

    Elizabeth L. Guenther, Hamilton Trinh, Michael R. Sawaya, Duilio Cascio, David R. Boyer & David S. Eisenberg

  5. Electron Imaging Center for Nanomachines, California Nano Systems Institute, University of California, Los Angeles, Los Angeles, CA, USA

    Peng Ge & Z. Hong Zhou

  6. Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA, USA

    Tamir Gonen

  7. Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA

    Z. Hong Zhou

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Contributions

E.L.G. 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 experiments, stability assays and crystallization and structure determination of 248LIIKGI253. E.L.G. and M.R.S. did the fibril diffraction assays. E.L.G. grew crystals of 247DLIIKGISVHI257, while D.R.B. collected MicroED data of the sample. M.R.S., D.C. and E.L.G. processed data and solved the structure of 247DLIIKGISVHI257 crystal. E.L.G. prepared and optimized the 247DLIIKGISVHI257 fibril sample for cryo-EM while P.G. processed and solved the cryo-EM structure. E.L.G., M.R.S. and D.S.E. analyzed structures, conducted computational analysis such as area buried and designed models of fibril nucleation and elongation. T.G. and Z.H.Z. contributed to analysis of microED and cryoEM structures, respectively.

Corresponding author

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 RRM2 segments demonstrate high to moderate stability against treatment with SDS and heat.

Fibril samples were first grown by shaking at 37°C at 20mM concentration in PBS, pH 7.5. Following fibril formation, samples were treated with 2% SDS and heated to 70°C for 15 min. The bar graph shows the mean raw absorbance values as well as individual data points as monitored by the Spectramax. Experiments were done in triplicate and error bars represent standard deviation.

Supplementary Figure 2 Fibril diffraction of the RRM2 fibril segments shows classic amyloid diffraction.

Fibril diffraction was collected at the 5um beam at the Advanced Photon Source. All nine samples exhibit the classic 4.6-4.8Å ring indicative of the stacking of β-strands and the 8-12Å ring indicative of mating sheets. We were unable to collect fibril diffraction patterns for 2 samples (#1: 247DLIIKG252 and #11: 254SVHISN259) Fibril diffraction experiments were completed once at APS. Peptide numbering corresponds with the code provided in Fig. 1.

Supplementary Figure 3 Eight unique fibril morphologies are observed in the 247DLIIKGISVHI257 cryo-EM sample.

(a) Negative stain EM image of the 247DLIIKGISVHI257 cryo-EM sample. 247DLIIKGISVHI257 fibrils were grown by shaking for one week at 1mM in water. Sample was stained with 2% uranyl acetate and visualized on the FEI T12 transmission electron microscope. Fibril samples illustrate multiple morphologies, including twisted helical assemblies. (b) Illustration of the eight different types of fibrils observed by cryo-EM. The eight fibrils demonstrate four different conformations of amyloid fibrils: helices, twists, cylinders, and sheets. (c) The relative abundance of the eight unique fibrils are displayed in a table. The 3-start helix represents the most abundant species, comprising approximately 60% of the sample.

Supplementary Figure 4 Classification of images for the 247DLIIKGISVHI257 cryo-EM sample.

(a) Round 1 classification illustrates the polymorphic assemblies observed in the fibril sample. (b and c) Round 2 and 3 class averages focus on fibrils exhibiting the 3-start helix morphology. Each panel illustrates a unique face of the fibril as it twists along the EM grid. 5-7Å features of the structure become apparent during these rounds of class averaging.

Supplementary Figure 5 2D projection of the final 3D model of the 247DLIIKGISVHI257 cryo-EM fibril structure.

The panels illustrate a side view of the fibril, perpendicular to the fibril axis. Each panel represents an 8° azimuthal rotation of the fibril. The hydrogen bonding network is visualized by the stacking of β-strands.

Supplementary Figure 6 Fourier shell correlation (FSC) curve for the single-particle cryo-EM structure of RRMcore.

FSC cutoff value of 0.5 results in spatial frequency of 3.83Å for the half-maps and 3.63Å for the map-model.

Supplementary Figure 7 The asymmetric unit of the three-start helix is composed of nine strands.

(a) The electron density of the asymmetric unit is displayed. Here, the N-terminus is displayed in red and the C-terminus is displayed in blue. (b,c) The peptide strands are displayed within the electron density. The β-sheets are classified by their relative conformation; kinked = red, straight = blue, curved = green and partial = yellow. Each strand is represented by a unique color within its designated family.

Supplementary Figure 8 The protofilaments of the three-start helix fill space, exclude water, bury hydrophobic surfaces, and exhibit electrostatic interactions.

Here, glycines and apolar residues are white, polar residues are green, positively charged residues are blue, and negatively charged residues are red. The pH of this sample is ~4 and therefore histidine residues are charged. Red and blue circles indicate the presence of electrostatic interactions between the N and C termini as well as between the aspartic acid and histidine side chains. Yellow ellipses illustrate the presence of hydrophobic cores. The three fold screw axis is indicated by the triangle.

Supplementary Figure 9 Eight different symmetry classes of amyloid steric zippers.

Examples of their occurrence among RRMcore segments of TDP-43 are cataloged below their respective classes.

Supplementary Figure 10 The cryo-EM structure demonstrates key features including a 247DLIIKGISVHI257 kinked backbone that flips orientation and a pseudo-two-fold symmetry is observed in the asymmetric unit.

(a) Below, the kinking of the 247DLIIKGISVHI257 backbone allows for the carbonyl of the glycine and isoleucine to both face down. The glycine and isoleucine residues have been marked by one letter abbreviations. (b-c) Strands 1, 2, 5 and 8 form a complex which exhibits two fold symmetry with strands 3, 4, 6 and 9. The oval shape indicates two fold symmetry.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 and Supplementary Tables 1–3

Life Sciences Reporting Summary

Supplementary Video 1

Segment DLIIKGISVHI forms a three-start helix. Here, the 27 β-strands that form the helical fibril can be viewed down the fibril axis. As the fibril axis become parallel to the screen, the twist of the fibril becomes visible

Supplementary Video 2

The asymmetric unit of DLIIKGISVHI is composed of nine strands. These strands adopt kinked, bent and straight morphologies that form five distinct interfaces

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Guenther, E.L., Ge, P., Trinh, H. et al. Atomic-level evidence for packing and positional amyloid polymorphism by segment from TDP-43 RRM2. Nat Struct Mol Biol 25, 311–319 (2018). https://doi.org/10.1038/s41594-018-0045-5

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