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.

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 ų, 830 ų, and 530 ų, respectively. The volume of ergosterol is estimated to be 427 ų . (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 ų. The number density of this lattice is 6.36 molecules per 10,000 ų. (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.

Fig. 1 |. An improved AmB sponge preparation technique that reproducibly yields homogeneous SSNMR samples.

a, Molecular structure of AmB polyene macrolide. b, Schematic representation of the process for obtaining homogenous AmB assemblies for SSNMR spectroscopy experiments. First, 1 mM AmB aggregate in 10 mM HEPES buffer pH 7.0 is prepared. This sample is then heated at 80 °C for 1 h, followed by bath sonication for 30 min and cooling at room temperature for another 30 min. Next, the sample is pelleted in the ultracentrifuge and spectrophotometrically analyzed before packing it into the SSNMR rotor. c, Biological analysis (MIC and MTC) of the AmB aggregates before (AmB I) and after (AmB II) homogenization. MIC against yeast pathogens (C. albicans, C. krusei, C. tropicalis and A. fumigatus) and MTC values causing 90% toxicity against human primary RPTECs ± percent error standard deviation. MIC and MTC values are given in μM. d, UV–Vis spectra of AmB aggregates before (black) and after (red) homogenization. e, Sample quality validation by PCA of the UV spectrum. PC1/PC2 projection of the heterogeneous (black) and homogenous (red) samples that were obtained after homogenization. f, ¹³C 1D SSNMR spectra (with 50 Hz line broadening) of the [U-¹³C]AmB before (black) and after (red) homogenization. Black arrows represent carbon sites with changes in resolution.

Fig. 2 |. AmB exists in two states.

a, ¹³C–¹³C 2D SPC mixing spectrum of the homogenous AmB sample. b, AmB C1–C13 region showing 1- and 2-bond correlations that allowed for assigning two AmB populations. Chemical shift assignments of state A and state B are presented in blue and green, respectively. c, Graphical representation of the ¹³C chemical shift differences between two states of AmB. The biggest changes are observed on carbons 2, 5, 6 and 7 of the C1–C13 region.

Fig. 3 |. DFT calculations of AmB state A and state B conformations.

Heatmap displaying the r.m.s.d. (from blue as the lowest and yellow as the highest r.m.s.d.) between experimentally measured and calculated chemical shifts for carbon atoms 1–19 and 34–47 for pairs of AmB structures with varied conformations of the C1–C13 regions labeled 1–14. Based on the r.m.s.d. comparisons in the heatmap, the best pair of structures contains structure number 1 as state A and structure number 6 as state B. Carbons 5–8 are highlighted to illustrate the difference in C1–C13 region dihedral geometry between state A and state B.

Fig. 4 |. SSNMR experiments indicate an asymmetric AmB homodimer structure.

a–c, Representative regions of the SSNMR spectra: ¹³C–¹³C 2D 1,000 ms DARR spectrum of the homogenous [¹³C] skip-labeled AmB (a); ¹³C–¹³C–¹³C 3D SPC PAR spectrum of the homogenous [U-¹³C]AmB (b); and ¹³C–¹³C 2D 11 ms PAR spectrum of the homogenous [U-¹³C]AmB showing head-to-tail interactions between AmB molecules as well as interactions between AmB C1–C13 middle region and the tail methyls (c); namely, between C41 and C36, C38, C39; C1′/C13 and C34, C36, C38, C39, C40; C39 and C18 (a); between C1′/C13 and C38, C39, C40; C39 and C1′/C13, C3′, C17, C19; C40 and C1′/C13, C3′, C16, C17, C19 (b); and between C17 and C38, C39, C40; C8 and C39, C40; C9 and C40, C38, C39 (c). d,e, PM-RESPDOR experiments of homogenized [¹³C] skip-labeled AmB. d, experimental ΔS/S₀(t) dipolar curves of ¹³C41 (183 ppm)–¹⁴N under two different lengths of PM-RESPDOR pulses: t_PM = 14T_R and t_PM = 24T_R (black diamonds). Solid blue lines represent Bessel function (equation (1.1)) with optimal distance of 3.91 Å (D = 36.49 Hz). ν₁ = 25kHz, ν_R = 12.5kHz. Dashed blue lines represent the cases when r_CN = 3.41 and 4.41 Å, respectively. e, experimental ΔS/S₀(t) curves (red stars) of ¹³C39 (11 ppm)–¹⁴N, ¹³C6′/¹³C38 (18 ppm)–¹⁴N and ¹³C40 (23 ppm)–¹⁴N, and their comparison with Bessel function (blue solid line) with optimal distances: r = 5.28 Å (D = 14.82 Hz), r = 6.1 Å (D = 9.61 Hz) and r = 5.59 Å (D = 12.49 Hz), respectively. For d and e, distances were found using least squares fitting (Levenberg–Marquardt method). Blue dashed lines represent Bessel functions with ±0.5 Å of deviation in each case, which were estimated according to the experimental errors. experimental parameters for red stars were: v₁ = 25 kHz and t_PM = 14T_R. The first two black diamond points represent the experimental curves with v₁= 0 kHz during S part. The last point (for each methyl) was obtained when the PM-RESDOR pulse was applied before proton excitation. The experimental errors were defined as the noise level intensities of the 1D ¹³C spectra, where the zero signals were expected. f,g, Graphical illustrations of the intermolecular interactions: f for panels a and b, and g for panel c.

Fig. 5 |. XPLoR-NIH AmB sponge structure.

a, Lowest distance restraint energy (6.8 kcal) XPLOR-NIH structure of the AmB sponge (3 × 3 × 3) lattice which consists of asymmetric head-to-tail homodimers. b, Minimal head-to-tail asymmetric unit of the AmB sponge. State A is shown in orange and state B in blue. In state B, the gauche C5–C6–C7–C8 dihedral is outlined in blue. c, Representative head-to-tail interactions between AmB molecules in the lattice derived from ¹³C–¹³C SSNMR experiments (Fig. 4a–c and Supplementary Fig. 4). d, Representative interactions between ¹⁴N and ¹³C derived from PM-RESPDOR experiments (Fig. 4d). e, Representative staggered interactions between AmB molecules in the lattice derived from ¹³C–¹³C SSNMR experiments (Fig. 4c). f, Representative void volume in the AmB lattice.