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Cryo-EM structure and inhibitor design of human IAPP (amylin) fibrils

Nature Structural & Molecular Biology volume 27, pages 653–659 (2020)Cite this article

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Abstract

Human islet amyloid polypeptide (hIAPP) functions as a glucose-regulating hormone but deposits as amyloid fibrils in more than 90% of patients with type II diabetes (T2D). Here we report the cryo-EM structure of recombinant full-length hIAPP fibrils. The fibril is composed of two symmetrically related protofilaments with ordered residues 14–37. Our hIAPP fibril structure (i) supports the previous hypothesis that residues 20–29 constitute the core of the hIAPP amyloid; (ii) suggests a molecular mechanism for the action of the hIAPP hereditary mutation S20G; (iii) explains why the six residue substitutions in rodent IAPP prevent aggregation; and (iv) suggests regions responsible for the observed hIAPP cross-seeding with β-amyloid. Furthermore, we performed structure-based inhibitor design to generate potential hIAPP aggregation inhibitors. Four of the designed peptides delay hIAPP aggregation in vitro, providing a starting point for the development of T2D therapeutics and proof of concept that the capping strategy can be used on full-length cryo-EM fibril structures.

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Fig. 1: Cryo-EM map of hIAPP fibrils and identification of two models.
Fig. 2: Structure analysis of hIAPP fibrils.
Fig. 3: Structure-based inhibitor design of hIAPP 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 accession codes PDB 6VW2 and EMD-21410, respectively. Coordinates for model 2, model 1 (swap) and model 2 (swap) are available as Supplementary Data 1–3. Source data for Fig. 3b,d, Extended Data Fig. 1e,f and Extended Data Fig. 7b are available online.

Code availability

The custom software used for solvation energy calculation is available upon request.

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Acknowledgements

We thank H. Zhou for the use of Electron Imaging Center for Nanomachines (EICN) resources. We acknowledge the use of instruments at the EICN supported by the NIH (1S10RR23057 and IS10OD018111), NSF (DBI-1338135) and CNSI at UCLA. The authors acknowledge NIH AG 054022, NIH AG061847, and DOE DE-FC02-02ER63421 for support. D.R.B. was supported by the National Science Foundation Graduate Research Fellowship Program.

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Authors and Affiliations

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

    Qin Cao, David R. Boyer, Michael R. Sawaya & David S. Eisenberg

  2. California NanoSystem Institute, University of California, Los Angeles, Los Angeles, CA, USA

    Peng Ge

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  1. Qin Cao
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  2. David R. Boyer
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Contributions

Q.C. designed experiments, purified constructs, prepared cryo-EM samples, performed cryo-EM data collection and processing, designed inhibitors, performed biochemical experiments and performed data analysis. D.R.B. and P.G. assisted in cryo-EM data collection and processing. Q.C. and M.R.S. built the inhibitor binding model. M.R.S. performed solvation energy calculation. 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.

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Peer review information Peer reviewer reports are available. Inês Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Cryo-EM data processing.

a, Representative Krios micrograph of hIAPP fibrils. Blue and green arrows indicate two morphologies (twister and ribbon, respectively) identified by 2D classification. Notice they are not distinguishable by eye. b, Representative 2D classes and relative population of twister and ribbon morphologies. c, Representative 2D classes of twister with smaller box size particles showing the 4.8 Å β-sheet spacing, and the computed diffraction pattern from a representative 2D class. d, Central slice (left) and 2D projection (right) from the final reconstruction. Notice the 2D projection of final reconstruction is consistent with 2D classification. e-f, FSC curves between two half-maps (e) and the cryo-EM reconstruction and refined atomic model (f). Data for graphs in e and f are available as source data.

Source data

Extended Data Fig. 2 Potential domain swapping of hIAPP models.

Domain swapped versions of both Model 1 and Model 2 were built to test the possibility of domain swapping. In the swapped models, the residues between the N-terminus and Gly24 from one protofilament were connected to the residues between Ala25 and the C-terminus from the other protofilament of the un-swapped model. The density map with σ=3.0 is shown in blue mesh and that with σ=2.0 is shown in grey mesh. Notice that Gly24 in both swapped Model 1 and Model 2 is clearly out of the density, demonstrating that the domain swapping is not supported by our cryo-EM map.

Extended Data Fig. 3 The fuzzy coat in hIAPP fibril structure may represent the flexible N-terminal of hIAPP and the SUMO-tag.

a, The final reconstruction (left) and 2D classification (right) show a fuzzy coat of ~55 Å surrounding the fibril core. b, Protease cleavage assays indicate the construct we used for fibril structure determination (SUMO-IAPP with 1xG, means one glycine between SUMO-tag and hIAPP) has an un-removable SUMO-tag, whereas the SUMO-tag is removable when we extend the linker to three glycine. c, Plausible N-terminal conformation suggested by the extra densities near Asn14. The density map with σ=3.0 is shown in blue mesh and that with σ=2.0 is shown in grey mesh. The intra-molecular disulfide bond is labeled between Cys2 and Cys7, and the residues occupying the extra densities in our hypothetical model are underlined. d, Crystal structure of SUMO protein (PDB ID 1L2N). e, Hypothetical model of N-terminus of hIAPP and SUMO-tag match the dimensions of the fuzzy coat observed in the hIAPP fibril reconstruction. Notice that in most cases the SUMO-tag is far away from the fibril core therefore should not influence the fibril structure.

Extended Data Fig. 4 Rosetta energy minimization of hIAPP fibril structure and rIAPP homology model.

a, Structure superimposition between (grey) hIAPP fibril structure determined here and (blue) hIAPP fibril structure (upper panels) or rIAPP homology model (lower panels) optimized by Rosetta energy minimization. Calculation was done either allowing only side chain movements (left panels) or allowing both side chain and main chain movements (middle and right panels). Notice that during Rosetta energy minimization, we did not apply non-crystallographic symmetry so that the 5 layers in each model were not forced to be identical. b, Steric clashes of the rIAPP homology model after side chain Rosetta energy minimization were probed with COOT and displayed as red dots. Notice that most of the steric clashes are found near S28P and S29P.

Extended Data Fig. 5 Structural superimposition of Aβ fibril structures and hIAPP fibril structure.

Ten previously reported Aβ fibril structures were superimposed with the hIAPP fibril structure by either directly comparing full-length Aβ fibril structures with the full-length hIAPP structure, or by only comparing residues 24-34 of Aβ fibril structures with residues 19-29 of the hIAPP structure. For the full-length comparison, one Aβ fibril structure (PDB ID 6SHS) shows reasonable alignment with low r.m.s.d., and the structural superimposition is shown on the far left panel, with the Aβ fibril structure shown in grey, the hIAPP structure shown in blue, and the segment that fits best (residues 20-25 of Aβ fibril structure) shown in magenta. For the partial comparison, four Aβ fibril structures show a good fit (middle left), three Aβ fibril structures show a moderate fit (middle right) and four Aβ fibril structures do not fit (far right). In these superimpositions, residues 24-34 of the Aβ fibril structures were colored grey and the highest fitting region (residues 26-31) is colored magenta. Detailed alignment parameters are listed in Supplementary Table 3.

Extended Data Fig. 6 Segments selected for hIAPP fibril inhibitor design.

Three segments of hIAPP, 21NNFGAILSS29 (N9S, left panels), 25AILSSTNVG33 (A9G, middle panels) and 21NNFG24 (N4G, right panels), were selected for design of inhibitors of hIAPP fibrils. For each selected segment, the hIAPP structure with the segment highlighted is shown on the top, with the hIAPP structure shown as lines and the segment shown as sticks. Proposed models of the corresponding inhibitor peptides (before adding N-methylation) binding to the hIAPP structures are shown as top views (middle panels) and side views (bottom panels). Notice there are multiple hydrogen bonds between the designed inhibitors and hIAPP fibrils, providing binding affinities for these inhibitors. For the N4G merged inhibitor, the model indicates the orientation-flipped and chirality-reversed N4G has high structural similarity to the original N4G and recaptures all original inter-layer interactions. Hydrogen bonds with distances between 2.3-3.2 Å are shown as black dashed lines.

Extended Data Fig. 7 Additional inhibitors designed for hIAPP fibrils.

a, Proposed model of designed inhibitors (magenta) bound to hIAPP fibrils (blue and grey for each protofilament). The methyl group of N-methylated inhibitors is shown as a green sphere. The last three residues of N4Gm-A are d-amino acids and are underlined. b, ThT assays measuring inhibitor efficacy shown on the left. A9G-A delays hIAPP fibril formation but not N9S-B and N4Gm-A. For the two inhibitors that were not effective, two effective inhibitors (N9S-A and N4Gm-B, respectively) are tested in the same experiment as controls. Data are shown as mean ± s.d., n = 3 independent experiments. c, Negative stain EM images of hIAPP with N9S-A, A9G-A or A9G-B after 20 hours of incubation. Notice that hIAPP fibril formation is not fully eliminated by these inhibitors. d, Negative stain EM shows hIAPP S20G fibrils present after 3 days of incubation with N4Gm-B, suggesting that fibril formation of hIAPP S20G is not fully eliminated when longer incubation times are examined (compared to 20 hours shown in Fig. 3e). Data for graphs in b are available as source data.

Source data

Extended Data Fig. 8 Connection of N-terminal density.

Slices of 3D maps of the final reconstruction (left) and an earlier reconstruction with lower resolution (right). The positions that represent N-terminus of Model 1 and Model 2 are indicated by arrows. Note the weak density that represents the flexible N-terminus of hIAPP seems to connect to the position that represents the N-terminus of Model 2 in the final reconstruction (left); whereas in the lower resolution reconstruction (right), the weak density seems to connect to the position of N-terminus of Model 1.

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Supplementary Tables 1−4 and Supplementary Note 1.

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Supplementary Data 1

Coordinates of model 2.

Supplementary Data 2

Coordinates of model 1 (swap).

Supplementary Data 3

Coordinates of model 2 (swap).

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Source Data Fig. 3

Statistical source data for Fig.3b,d

Source Data Extended Data Fig. 1

Statistical source data for Extended Data Fig. 1e,f

Source Data Extended Data Fig. 7

Statistical source data for Extended Data Fig. 7b

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Cao, Q., Boyer, D.R., Sawaya, M.R. et al. Cryo-EM structure and inhibitor design of human IAPP (amylin) fibrils. Nat Struct Mol Biol 27, 653–659 (2020). https://doi.org/10.1038/s41594-020-0435-3

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