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Cryo-EM structures of hIAPP fibrils seeded by patient-extracted fibrils reveal new polymorphs and conserved fibril cores

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Abstract

Amyloidosis of human islet amyloid polypeptide (hIAPP) is a pathological hallmark of type II diabetes (T2D), an epidemic afflicting nearly 10% of the world’s population. To visualize disease-relevant hIAPP fibrils, we extracted amyloid fibrils from islet cells of a T2D donor and amplified their quantity by seeding synthetic hIAPP. Cryo-EM studies revealed four fibril polymorphic atomic structures. Their resemblance to four unseeded hIAPP fibrils varies from nearly identical (TW3) to non-existent (TW2). The diverse repertoire of hIAPP polymorphs appears to arise from three distinct protofilament cores entwined in different combinations. The structural distinctiveness of TW1, TW2 and TW4 suggests they may be faithful replications of the pathogenic seeds. If so, the structures determined here provide the most direct view yet of hIAPP amyloid fibrils formed during T2D.

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Fig. 1: Extraction of patient islet cells and seeding of fibril growth.
Fig. 2: Cryo-EM structures of patient extract-seeded hIAPP fibrils.
Fig. 3: Structural comparisons of hIAPP fibrils seeded with patient-extracted fibrils.

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Data availability

Structural data have been deposited into the Worldwide Protein Data Bank (wwPDB) and the Electron Microscopy Data Bank (EMDB) with the following accession codes: PDB 7M61, EMD-23686 (TW1); PDB 7M62, EMD-23687 (TW2); PDB 7M64, EMD-23688 (TW3); PDB 7M65, EMD-23689 (TW4). PDB accession codes for previously reported coordinates used for structural analysis in this study are: 6Y1A, 6ZRR, 6ZRQ, 6ZRF, 6VW2 for hIAPP fibrils and 6OIZ, 2M4J, 2MVX, 5KK3, 5OQV, 2NAO, 2MXU, 2BEG, 2LMN, 2MPZ, 6SHS for amyloid-β fibrils. All data are available in the paper or the Supplementary Information.

Code availability

Energetic calculations were performed using custom written software. The code is available at the MBI website (https://people.mbi.ucla.edu/sawaya/amyloidatlas/accessiblesurfacearea_v07.2d.f).

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Acknowledgements

We thank the Southern California Islet Cell Resources Center for providing human islets for this study. We thank X. Zhao at the HHMI Janelia Cryo-EM Facility for help with microscope operation and data collection. We acknowledge support from NIH AG 054022, NIH AG061847 and DOE DE-FC02-02ER63421. D.R.B. was supported by the National Science Foundation Graduate Research Fellowship Program.

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Author notes

  1. Qin Cao

    Present address: Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, China

  2. Lorena Saelices & Binh A. Nguyen

    Present address: Center for Alzheimer’s and Neurodegenerative Diseases, Department of Biophysics, UT Southwestern Medical Center, Dallas, TX, USA

Authors and Affiliations

  1. Departments of Chemistry and Biochemistry and Biological Chemistry, UCLA-DOE Institute, Molecular Biology Institute, and Howard Hughes Medical Institute, UCLA, Los Angeles, CA, USA

    Qin Cao, David R. Boyer, Michael R. Sawaya, Romany Abskharon, Lorena Saelices, Binh A. Nguyen, Jiahui Lu, Kevin A. Murray & David S. Eisenberg

  2. Department of Translational Research & Cellular Therapeutics, City of Hope, Duarte, CA, USA

    Fouad Kandeel

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Contributions

Q.C. designed experiments and performed data analysis. F.K. prepared islet cells from donors. Q.C. and L.S. performed Congo red staining of islet cells. Q.C., L.S. and B.A.N. performed fibril extraction from islet cells. Q.C. and R.A. performed immunoprecipitation in fibril extraction. R.A. and J.L. performed western blot and MTT assays. K.A.M. helped with western blots. Q.C. prepared hIAPP fibrils and cryo-EM grids. Q.C. and D.R.B. collected cryo-EM data. Q.C. performed cryo-EM data processing and model building, J.L. assisted with particle picking. Q.C. and M.R.S. performed solvation energy calculations. All authors analyzed the results and wrote the manuscript. D.S.E. supervised and guided the project.

Corresponding author

Correspondence to David S. Eisenberg.

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Competing interests

D.S.E. is an advisor and equity shareholder in ADRx, Inc. The remaining authors declare no competing interests.

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Peer review information Nature Structural & Molecular Biology thanks Sjors Scheres and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Supplemental information on extraction of patient islet cells and seeding of fibril growth.

a, Congo Red staining of slices of islet cells from various T2D donors (see Supplementary Table 1). b, Dot blot of fractions from extracted patient islet cells probed by anti-hIAPP (top) and anti-amyloid fibrils OC (bottom) antibodies. c, Negatively stained images of S1 and P fractions in (b). d, Dot blot of fractions from immunoprecipitation of the S1 fraction probed by anti-hIAPP (top), OC (middle) antibodies and no primary antibody (bottom) as a base line. e, Negatively stained images of flow through, elute−2 and elute−3 fractions in (b). The EM image of the elute−1 fraction is shown in Fig. 1b. f, ThT aggregation curves of fresh prepared synthetic hIAPP peptide incubated alone (black) or with elute-1 (red), elute-2 (blue) or elute-3 (green) fraction in (d) as fibril growth seeds. Data are shown as mean ± s.d., n=3 independent experiments. Note the elute-1 fraction from immunoprecipitation shows notable seeding ability as its curve shows shortened lag time and stronger ThT readings compared to hIAPP alone, whereas the elute-2 and elute-3 fractions show no ability in altering the hIAPP aggregation curve. Please see methods for the detailed definitions of fractions in panels b–f.

Extended Data Fig. 2 Cryo-EM data processing.

a, Representative micrographs of 8 identifiable morphologies during data processing (TW1-TW4 and NT1-NT4, scale bar 500 Å). b, Representative 2D classes of NT1-NT4. c, Central slices of final 3D reconstructions of TW1-TW4. d-e, FSC curves between two half-maps (e) and the cryo-EM reconstruction and refined atomic model (f). In half-maps FSC, FSC curves (black) are fitted (red) with the model function 1/(1+exp((x-A)/B)), with A=0.2328 and B=0.01517 for TW1, A=0.2347 and B=0.01234 for TW2, A=0.2255 and B=0.01427 for TW3, and A=0.2252 and B=0.009377 for TW4.

Extended Data Fig. 3 Different views of the cryo-EM maps with five layers shown.

For each morphology, the top view shows clear separation of β-strands, and the tilted views on the middle and bottom show clear separation of the layers of β-sheets along the fibril axis.

Extended Data Fig. 4 Structural comparisons of hIAPP fibrils.

a, Superposition of chain A and B of TW1 (left) and TW4 (right). b, Superposition of TW3, 6Y1A and 6ZRF, note that these three structures are very similar to each other. c, Superposition of chain A of TW1, TW3 and TW4 (top) and of TW1 chain A and 6ZRR chain C (bottom) at CF1 region. d, Superposition of TW1 chain A and TW2 (left), or TW1 chain B and TW2 (right). For superimposition details of panels a, b and d see Supplementary Table 2.

Extended Data Fig. 5 Detailed analysis of hIAPP fibril structures.

a, Above in black: the amino acid sequence of hIAPP; below: the residues visible in different hIAPP fibril structures. b, Plausible conformations of flexible N-termini of TW2 (purple), TW3 (green) and TW4 (red and orange) suggested by weak densities. The superposition of Thr6 to Ala13 region of TW2 and TW4 chain A is shown as an insert. c, The interactions around Tyr37 for TW1 (chain A, blue; chain B, cyan), TW2 (purple), TW3 (green), and TW4 (chain A, red; chain B, orange). d, The detailed view of an unexplained density around Gly33. The red dot represents the center of the unexplained density, and the length of each dash line is: i, 4.0 Å; ii, 2.9 Å; iii, 3.7 Å; iv, 5.2 Å. e, Different conformations of chain A of TW1 (marine) and TW4 (red, left) or chain B of TW1 (cyan) and TW4 (orange, right) outside the CF1 or CF2 region, respectively. TW1 and TW2 are superimposed at the CF1 (chain A) or CF2 (chain B) region. f, The interfaces between protofilaments A and B for TW1 (marine and cyan), TW2 (purple), TW3 (green), TW4 (red and orange), and 6ZRQ (gray), as well as between protofilaments B (top) and C (bottom) for 6ZRR (grey). Ab, area buried. In panels c, e, and f, hydrogen bonds with distances between 2.3 and 3.2 Å are shown as black dashed lines. For superimposition details for panel b see Supplementary Table 2.

Extended Data Fig. 6 Explanation of swapped version of 6VW2.

Original version (left) and swapped version (right) of SUMO-tagged recombinant hIAPP fibril structure. Two symmetrically related chains are colored black and grey. CF2 was shown as surface and colored red.

Extended Data Fig. 7 Distribution of different polymorphs in reported hIAPP cryo-EM datasets.

hIAPP polymorphs are colored by (a) core folds or (b) homotypic vs. heterotypic pairings. From panel a, we found CF2 is more abundant in S20G dataset compared to wildtype ones with the exception of 6VW2 dataset. In S20G dataset, 6ZRQ contributes to 76% of the solvable fibrils and is purely composed of protofilaments with CF2; 6ZRR contributes to the other 24% and contains two protofilaments with CF2 and one protofilament with CF1. In contrast, wild-type fibrils, 6ZRF and 6Y1A are the only solvable species in their datasets and they contain only CF1 in their protofilaments; in the wild-type fibrils in this study, we also observe more protofilaments with CF1 than that with CF2. TW1 and TW4 have equal amount of protofilaments with CF1 and CF2, but TW3 contains only protofilaments with CF1. From panel b, we note when formed in vitro without patient seeds, we observed homo-dimer forms of fibrils in most datasets (Cao et al., Röder et al., and the wile-type of Gallardo et al.) and only in one dataset did we find a small portion of heterotypic species (S20G of Gallardo et al., in which 6ZRR contributes to 24% of the solvable population compared to 76% of 6ZRQ that is homo-dimer form). Whereas in patient-extract-seeded fibrils we see higher populations of heterotypic species (TW1 and TW4 contribute to 40% out of 65% of solvable population).

Extended Data Fig. 8 Solvation energy maps of reported cryo-EM hIAPP fibril structures.

Residues are colored from unfavorable (blue, 2.5 kcal/mol) to favorable stabilization energy (red, −2.5 kcal/mol).

Extended Data Fig. 9 Western blot of S1 fraction of fibril extraction and MTT assays of seeded hIAPP fibrils.

a, Western blot of S1 fraction (originally characterized in Fig. S1b) probed by antibodies that target hIAPP, amyloid-β, tau (K18), and α-synuclein. The only antibody to recognize and label the S1 fraction is anti-hIAPP, suggesting that the S1 fraction consists primarily of hIAPP. Moreover, the molecular weight of the band corresponds to full-length hIAPP. When probed with amyloid-β, tau, and α-synuclein antibodies, bands appear only in positive control lanes (labeled as “+”, see Methods for detail). b, Rin5F cells were treated with different concentrations of patient-fibril-seeded hIAPP fibrils, and significantly less MTT dye reduction was observed compared to cells without adding fibrils (****p<0.0001 using one-way ANOVA test, data are shown as mean ± s.d., n=3 independent experiments).

Extended Data Fig. 10 Main chain tracing of TW2 and TW4.

Low resolution 3D reconstructions of TW2 (a) and TW4 (b) displayed to illustrate the main chain tracing.

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Supplementary Text 1–7, references and Tables 1–3.

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Cao, Q., Boyer, D.R., Sawaya, M.R. et al. Cryo-EM structures of hIAPP fibrils seeded by patient-extracted fibrils reveal new polymorphs and conserved fibril cores. Nat Struct Mol Biol 28, 724–730 (2021). https://doi.org/10.1038/s41594-021-00646-x

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  • DOI: https://doi.org/10.1038/s41594-021-00646-x

  • Springer Nature America, Inc.

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