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Fibril structures of diabetes-related amylin variants reveal a basis for surface-templated assembly

An Author Correction to this article was published on 09 October 2020

This article has been updated

Abstract

Aggregation of the peptide hormone amylin into amyloid deposits is a pathological hallmark of type-2 diabetes (T2D). While no causal link between T2D and amyloid has been established, the S20G mutation in amylin is associated with early-onset T2D. Here we report cryo-EM structures of amyloid fibrils of wild-type human amylin and its S20G variant. The wild-type fibril structure, solved to 3.6-Å resolution, contains two protofilaments, each built from S-shaped subunits. S20G fibrils, by contrast, contain two major polymorphs. Their structures, solved at 3.9-Å and 4.0-Å resolution, respectively, share a common two-protofilament core that is distinct from the wild-type structure. Remarkably, one polymorph contains a third subunit with another, distinct, cross-β conformation. The presence of two different backbone conformations within the same fibril may explain the increased aggregation propensity of S20G, and illustrates a potential structural basis for surface-templated fibril assembly.

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Fig. 1: Morphology of amylin fibrils.
Fig. 2: Structure of protofilaments and near-atomic resolution model for the wild-type amylin fibril.
Fig. 3: Conformation of 2PF fibrils and details of its molecular structure.
Fig. 4: Conformation of S20G 3PF fibrils and details of their molecular structure.
Fig. 5: Schematic views of backbone fold and interprotofilament interactions in the structures of wild-type, S20G 2PF and S20G 3PF amylin fibrils.

Data availability

The cryo-EM maps and atomic models have been deposited in the EMDB and wwPDB, respectively, with the following accession codes: wild-type amylin (EMD-11380, PDB 6ZRF), S20G 2PF (EMD-11382, PDB 6ZRQ) and S20G 3PF (EMD-11383, PDB 6ZRR).

Change history

  • 09 October 2020

    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

R.G., Y.X., R.F., N.A.R. and S.E.R thank Wellcome for generous support (no. 204963). M.G.I. is supported by the MRC (no. MR/P018491/1). All EM was performed at the Astbury Biostructure Laboratory, which was funded by the University of Leeds and the Wellcome Trust (no. 108466/Z/15/Z), and we thank R. Thompson, E. Hesketh, D. Maskell and C. Scarff for assistance with cryo-EM data collection. We thank V. Zorzini for assistance in model building. Peptide synthesis was performed using instrumentation funded by EPSRC (no. EP/N013573/1). The AFM experiments were performed with instrumentation funded by Wellcome (no. 101497/Z/13/Z). All data processing was performed using ARC4 at the University of Leeds. Finally, we thank colleagues in the Radford and Ranson laboratories for many helpful discussions while preparing this manuscript.

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Authors and Affiliations

Authors

Contributions

R.G., M.G.I., Y.X., R.F., S.E.R. and N.A.R. designed the experiments. Y.X. synthesized and characterized peptides. R.G. and Y.X. optimized fibril growth conditions and prepared fibrils. R.G. and G.R.H. performed AFM experiments. R.G. prepared EM samples and collected data. R.G. and M.G.I. performed image processing and reconstruction. R.G. performed model building and refinement. All authors analyzed some or all of the data, and wrote or edited the manuscript.

Corresponding authors

Correspondence to Sheena E. Radford or Neil A. Ranson.

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The authors declare no competing interests.

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Peer review information 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.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Negative-stain TEM and AFM characterization of wild-type and S20G amylin fibrils.

a, Wild-type (WT) amylin fibrils as observed by negative-stain TEM. The proportion of all fibrils in the dominant polymorph determined here (~80%) are indicated by the pie chart in row (a). b, As in (a), but digitally magnified. c, S20G mutant amylin fibrils as observed by negative-stain TEM. Again, the proportion of all fibrils in the dominant two polymorphs determined here (2PF (60%) and 3PF (19%)) are indicated by the pie chart in row (c). d, Same as (c) but digitally magnified. e, In solution AFM analysis of wild type amylin fibrils. f, In solution AFM analysis of S20G mutant amylin fibrils. Scale bars and their accompanying dimensions are indicated on each panel.

Extended Data Fig. 2 2D-classification and class average details of wild-type amylin data-set.

a, 2D classification of the initial cryoEM data-set. Shown are selected classes ranked on class distribution from higher to lower (left to right and then top to bottom). b, Representative 2D class showing a cross-over point along the long fibril axis. c, Detail of the representative 2D class shown in (b) marked in red. d, 2D-projection of the final electron potential map. e, Detail of the 2D-projection of the final electron potential map marked in red shown in D.

Extended Data Fig. 3 Inter-protofilament interactions that stabilised the wild-type amylin fibril and secondary structure assignments.

a, Monomer “i” in red, and apposing monomer “j” in brown have 10º tilt angle respect to the main fibril axis, which generates an effective 20º tilt between monomers in opposing protofilaments. b, Top view of the interaction depicted in (a), showing monomer “i” in red, and the stack of opposing monomers “j-1”, “j”, “j+1” and “j+2” in brown shades, with which monomer “i” interacts. The N-terminal and C-terminal ends of monomers in both protofilaments are indicated by letters (N and C) in the respective colours. c, Side-view of the interaction shown in (b), showing the opposing monomers as molecular surface in shades of brown, where the red surface indicates the contacts with monomer “i”, which is shown as red ribbon. d, Secondary structure assignments for wild-type and S20G amylin subunits in their amyloid fibril state as determined in this study and compared to previously published structures33,35,36,43. Dashed lines indicate sequence segments for which structure is not determined in the experiment. Continuous lines indicate loops. Arrows indicate β-strands.

Extended Data Fig. 4 2D-classification and class average details of the S20G amylin data-set.

a, 2D classification of the initial cryo-EM data-set. Shown are selected classes ranked on class distribution (from left to right and then top to bottom). b, Representative 2D class showing a cross-over point along the long fibril axis. c, Detail of representative 2D class shown in (b) highlighted in red. d, 2D-projection of the final 3D-reconstructed map of 2PF fibrils. e, Detail of the 2D-projection of the final 3D-reconstructed map highlighted in red shown in (d). f, 2D-projection of the final 3D-reconstructed map of 3PF fibrils. g, Detail of the 2D-projection of the final 3D-reconstructed map highlighted in red shown in f.

Extended Data Fig. 5 Inter-protofilament interactions in the 2PF and 3PF polymorphs of S20G amylin.

a, Monomer “i” in red, and apposing monomer “j-1” in brown have 1º tilt angle respect to the main fibril axis, which generates an effective 2º tilt between monomers in opposing protofilaments. b, Top view of the interaction depicted in (a), showing monomer “i” in red, and the stack of opposing monomers “j-1” and “j” in brown shades, with which monomer “i” interacts. The N-terminal (N) and C-terminal (C) ends of monomer “i” are indicated by red letters. c, Side-view of the interaction shown in (b), showing the opposing monomers “j” and “j-1” as molecular surface in shades of brown, where the red surface indicates the contacts with monomer “i”, which is shown as a red ribbon. d, Side view as in (b), where the backbone of each protofilament is shown as ribbons. The even inter-strand distance of 2.4-Å is indicated by alternating black and yellow lines. e, Monomers “jB” and “jA” are out of register and form the “2PF-like” core of 3PF polymorph. Monomer “i”, in red, binds on the side of monomer “jB” and inserts its C-terminal end in the groove between “jA” and jB”. The N-terminal end of monomer “i” is in register with monomer “jB”, while the C-terminal end of monomer “i” is in register with monomer “jA”. f, Top view of the interaction depicted in (e), showing monomer “i” in red, and the stack of opposing monomers “jA-1”, “jA” and “jA+1” in brown shades that form protofilament A. Protofilament B also contains 3 monomers, but their labels have not been included. The N-terminal (N) and C-terminal (C) ends of monomer “i” are indicated by red letters. g, Side-view of the interaction shown in (f), where monomers of protofilaments jA and jB are indicated. The red surface in protofilaments A and B represents the surfaces of interaction with monomer “i” of protofilament C. h, Side view as in (f), where monomer C has been removed for clarity, and where the backbone of each protofilament is shown as ribbons. The uneven inter-strand distance of 1.3-Å and 3.4-Å is indicated by alternating black and yellow lines.

Extended Data Fig. 6 Monomer superposition from amyloid fibrils form by wild-type and S20G amylin, and dependency of fibril stability on number of molecular layers for amylin fibrils.

a, Global superposition of the Cα trace of the wild-type fibril monomer (blue) on the Cα trace of S20G 2PF fibril monomer (red). b, The same structures superposed in the segment 31NVGSNT36 (residues numbered in red on figure), where the side chains of this segment (plus the terminal Tyrosine residue) are shown as sticks. c, Global superposition of the Cα trace of the S20G 2PF monomer (red) on the Cα trace of S20G 3PF symmetric monomers A or B (green). d, Superposition of the Cα trace of the S20G 3PF monomer C (green) on the Cα trace of wild-type monomers A or B (blue) in the region of residues 15–29 (indicated by numbers of the respective colours for each structure). In all the above panels the N-terminal and C-terminal ends of each structure are indicated with letters (N and C) in the respective colour of each monomer. e, The ΔGdiss for wild-type (blue circles) or S20G mutant polymorph 2PF (orange circles) or 3PF (red circles) as estimated by PDBePISA is plotted in function of the number of molecular layers on each model. The data is well represented by linear fit (shown as segmented lines).

Extended Data Fig. 7 Comparison of recently published structures of wild-type and sumoylated amylin fibrils.

Structural superposition of a molecular layer of a, S-shaped monomers of wild-type amylin from a right-handed fibril (blue, PDB code 6Y1A39) onto S-shaped monomers of wild-type amylin determined in our study (left-handed fibril, red), b, N-terminally sumoylated amylin monomers (green, PDB code 6VW242) superposed on the S-shaped monomers determined in our study (red).

Extended Data Fig. 8 Previously proposed structure and model for wild-type amylin, and superposition of X-ray structures from fragments onto cryo-EM structure of wild-type amylin.

a, Structural model for the striated ribbon polymorph studied by Luca and co-workers43 where two amylin monomers are represented as ribbons coloured from N-terminal yellow to C-terminal blue. b, Stick representation of one of the atomic models proposed by Luca and co-workers43. c, Diagram of the “β-serpentine” fold proposed by Kajava and co-workers35 for wildtype amylin. d, Ball-and-stick model of the “β-serpentine” shown in (c). e, Cα trace of fragments of amylin determined by X-ray crystallography (aa 14-20, PDB: 3FTH46, green; aa 14-19, PDB: 3FR146, yellow; aa 21-27, PDB: 3DGJ27, red; aa 28-33, PDB: 3DG127, orange; aa 31-37, PDB: 3FTL46, cyan; aa 31-37, PDB: 3FTK46, purple) superposed to the Cα trace of the S-shaped monomer of wild-type amylin fibrils (blue). The N-terminal and C-terminal ends of the S-shaped monomer of wild-type amylin are indicated by letters N and C in blue colour, respectively. f, As in (e) but also showing the side chains of those amino acids that superpose.

Extended Data Fig. 9 The electron potential map around His18 in the wild-type amylin fibril structure is difficult to interpret.

The density could accommodate two distinct His rotamers (shown in panels a and b). It is possible that both rotomers are present in the structure, or that the dataset contains two polymorphs that differ only in this position. His18 has been proposed to be involved in binding metal ions such as zinc and calcium48. Interestingly, it has been proposed that Lys1 is also part of the coordination system of Zn2+, together with His18, which would require the N-terminal sequence of amylin, including Lys1, to fold back towards His1848. This hypothesis would be consistent with the position of the N-terminal LDR in the wild-type fibril structure presented here (Fig. 1c).

Extended Data Fig. 10 The S-shaped fold of wild-type amylin is similar to the fold of Aβ42 fibril fold.

a, Sequence alignment of human amylin and Aβ42. The highest sequence similarity region (56%) is highlighted by a black box. The aggregation prone region with highest similarity between amylin (residues 22-27) and Aβ42 (residues 27-32) is highlighted by a red box. Conservation symbols and sequence colour are according to Clustal. b, c, d and e, Cα trace of wild-type amylin (blue) superposed onto the Cα trace of Aβ42 fibrils solved by cryo-EM (panel b, cyan, PDB: 5OQV29), and solved by ssNMR (panel c, green, PDB: 2NAO30; panel d, pink, PDB: 5KK328; panel e, orange, PDB: 2MXU31). The side chains of S20 in amylin, and E22 in Aβ, are shown as sticks in panel (b). The N-terminal and C-terminal ends of each structure are indicated by N and C letters, respectively.

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Gallardo, R., Iadanza, M.G., Xu, Y. et al. Fibril structures of diabetes-related amylin variants reveal a basis for surface-templated assembly. Nat Struct Mol Biol 27, 1048–1056 (2020). https://doi.org/10.1038/s41594-020-0496-3

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