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Fungicidal amphotericin B sponges are assemblies of staggered asymmetric homodimers encasing large void volumes

Abstract

Amphotericin B (AmB) is a powerful but toxic fungicide that operates via enigmatic small molecule–small molecule interactions. This mechanism has challenged the frontiers of structural biology for half a century. We recently showed AmB primarily forms extramembranous aggregates that kill yeast by extracting ergosterol from membranes. Here, we report key structural features of these antifungal ‘sponges’ illuminated by high-resolution magic-angle spinning solid-state NMR, in concert with simulated annealing and molecular dynamics computations. The minimal unit of assembly is an asymmetric head-to-tail homodimer: one molecule adopts an all-trans C1–C13 motif, the other a C6–C7-gauche conformation. These homodimers are staggered in a clathrate-like lattice with large void volumes similar to the size of sterols. These results illuminate the atomistic interactions that underlie fungicidal assemblies of AmB and suggest this natural product may form biologically active clathrates that host sterol guests.

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Fig. 1: An improved AmB sponge preparation technique that reproducibly yields homogeneous SSNMR samples.
Fig. 2: AmB exists in two states.
Fig. 3: DFT calculations of AmB state A and state B conformations.
Fig. 4: SSNMR experiments indicate an asymmetric AmB homodimer structure.
Fig. 5: XPLOR-NIH AmB sponge structure.

Data availability

Data that support the findings of this study are available within the paper and its Supplementary Information. Source data are provided in BMRbig, entry ID bmrbig28. Atomic coordinates are deposited in BMRB, entry ID 21097. Further requests can be directed to the corresponding authors.

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Acknowledgements

This work was supported by the US National Institutes of Health (NIH) R01-GM112845 and R01-GM123455 to C.M.R. and R35-GM118185 to M.D.B. This study made use of the National Magnetic Resonance Facility at Madison, including the technology development program which is supported by NIH grant P41GM136463. C.D.S. was supported by the National Institutes of Health Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases. We would like to dedicate this paper to the memory of our coauthor, A. Khandelwal, who passed away during the course of this investigation.

Author information

Authors and Affiliations

Authors

Contributions

A.L., A.I.G., C.M.R. and M.D.B. designed the research. J.T.H. and G.S.H. prepared and purified [U-13C]AmB. J.T.H. prepared and purified [13C] skip-labeled AmB. A.L. and C.P.S. prepared samples for SSNMR. A.L., G.S.H., C.P.S. and C.M.R. acquired SSNMR data. A.L. and C.P.S. performed UV spectroscopy measurements. A.L, C.P.S. and G.S.H. analyzed SSNMR data. E.N. performed and processed the PM-RESPDOR experiments. A.L., C.P.S. and A.I.G analyzed UV data. A.I.G. designed and together with A.L. and C.P.S. performed PCA analysis. A.K. and J.Z. and A.M.S. performed cell-based assays. A.M.S. synthesized AmB-I. J.Z. and A.M.S. purified AmB-I. T.V.P. performed structural calculations in MOE. A.I.G. and A.M.D.L. performed and analyzed DFT structural calculations. A.L., C.P.S., C.D.S and C.M.R. performed XPLOR-NIH structure calculations. A.M.D.L. and T.V.P. designed and performed clustering analyses. A.L., C.P.S., A.M.D.L., T.V.P, M.D.B and C.M.R. prepared the manuscript.

Corresponding authors

Correspondence to Taras V. Pogorelov, Martin D. Burke or Chad M. Rienstra.

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The authors declare no competing interests.

Additional information

Peer review information Nature Structural & Molecular Biology thanks Hartmut Oschkinat and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Florian Ullrich 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 AmB homogenization validation.

(a) UV-Vis spectra of AmB aggregates before (black) and after (red) homogenization. Grey UV-Vis spectra correspond to representative samples from the middle of the PC1 range (Fig. 1e). (b) LC-MS spectra of AmB before and after homogenization. (c) 1H NMR spectra of AmB before (black line) and after (red line) homogenization.

Extended Data Fig. 2 AmB cluster analysis and Erg docking.

(a) Principal component analysis (PCA) of the 9-dimensional data used for dimer structure clustering analysis for all 11 cluster ensembles. The inset shows the lowest clusters’ energies overlaid on the total energy histogram. (b) Alignment of medoid dimer structures of clusters 1–3 and lattice structures of medoid structures from the lowest energy cluster; states A and B of AmB are marked. (c) Medoid lattices of clusters 1–3 showing representative void volumes in green. Approximate volumes for each void in clusters 1-3 are 390 Å3, 830 Å3, and 530 Å3, respectively. The volume of ergosterol is estimated to be 427 Å3 21. (d) Snapshots of all four void pockets found in cluster 1 lattice - front view (left) and side view (right). The volume of each pocket is approximately 390 Å3. The number density of this lattice is 6.36 molecules per 10,000 Å3. (e) Snapshot of an ergosterol molecule (cpk-blue) docked to the cluster 2 medoid lattice – front view. (f) Close-up snapshot of AmB (white) – ergosterol (blue) docked interaction as seen in (e). Select atom numbers near interaction site are labeled for both AmB and ergosterol molecules.

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Supplementary Figs. 1–7, Discussion and Tables 1–6.

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Lewandowska, A., Soutar, C.P., Greenwood, A.I. et al. Fungicidal amphotericin B sponges are assemblies of staggered asymmetric homodimers encasing large void volumes. Nat Struct Mol Biol 28, 972–981 (2021). https://doi.org/10.1038/s41594-021-00685-4

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