Phenol-soluble modulins PSMα3 and PSMβ2 form nanotubes that are cross-α amyloids

Abstract

Phenol-soluble modulins (PSMs) are peptide-based virulence factors that play significant roles in the pathogenesis of staphylococcal strains in community-associated and hospital-associated infections. In addition to cytotoxicity, PSMs display the propensity to self-assemble into fibrillar species, which may be mediated through the formation of amphipathic conformations. Here, we analyze the self-assembly behavior of two PSMs, PSMα3 and PSMβ2, which are derived from peptides expressed by methicillin-resistant Staphylococcus aureus (MRSA), a significant human pathogen. In both cases, we observed the formation of a mixture of self-assembled species including twisted filaments, helical ribbons, and nanotubes, which can reversibly interconvert in vitro. Cryo–electron microscopy structural analysis of three PSM nanotubes, two derived from PSMα3 and one from PSMβ2, revealed that the assemblies displayed remarkably similar structures based on lateral association of cross-α amyloid protofilaments. The amphipathic helical conformations of PSMα3 and PSMβ2 enforced a bilayer arrangement within the protofilaments that defined the structures of the respective PSMα3 and PSMβ2 nanotubes. We demonstrate that, similar to amyloids based on cross-β protofilaments, cross-α amyloids derived from these PSMs display polymorphism, not only in terms of the global morphology (e.g., twisted filament, helical ribbon, and nanotube) but also with respect to the number of protofilaments within a given peptide assembly. These results suggest that the folding landscape of PSM derivatives may be more complex than originally anticipated and that the assemblies are able to sample a wide range of supramolecular structural space.

Keywords: bacterial pathogenesis; cross-α amyloid; cryo-EM; peptide nanotube.

Fig. 1.

Macromolecular structure of PSMs PSMα3 and PSMβ2. (A) Sequences of S. aureus peptides PSMα3 and PSMβ2. Amphipathic helices are highlighted in cyan. (B and C) Helical wheel diagrams corresponding to the amphipathic helices of PSMα3 (B) and PSMβ2 (C). (Amino acid color code: yellow, hydrophobic; blue, basic; red, acidic; orange, polar). (D) Solution NMR structure of PSMα3 (PDB ID: 5KGY). (E) Solution NMR structure of PSMβ2 (PDB ID: 5KGZ). (F) Top (Upper) and side (Lower) views of a bilayer sheet in cross-α array in the crystal structure of PSMα3 (PDB ID: 5I55). The coloring in the ribbon diagrams is derived from the Kyte-Doolittle hydrophobicity scale in which cyan corresponds to polar residues, white corresponds to neutral residues, and yellow corresponds to hydrophobic residues.

Fig. 2.

Structural analysis of PSMα3 assemblies. (A) Negative-stain TEM image of twisted filaments of PSMα3 (380 μM) assembled in TAPS buffer (10 mM, pH 8.0). (Scale bar, 50 nm.) (B) Positive-stain TEM image of helical ribbons (white arrow) and nanotubes of PSMα3 (300 μM) assembled from under acidic conditions (pH 2) after 1-h incubation. (Scale bar, 100 nm.) (C) Darkfield STEM image of unstained PSMα3 (300 μM) at pH 2, in which twisted filaments are observed to emerge from the ends of the nanotubes (white arrow). (Scale bar, 50 nm.) (D) Comparison of the experimental synchrotron SAXS scattering curve for a mature solution of PSMα3 (300 μM) nanotubes at pH 2 to the calculated fit to a hollow cylinder form factor. The black arrow indicates the presence of a Bragg peak at a q value of 0.61 Å⁻¹ (d = 10.3 Å), which corresponds to the helical stacking distance within the solvated nanotubes. A.U., arbitrary units.

Fig. 3.

Cryo-EM structure of the PSMα3 nanotubes. (A) Cryo-electron micrograph of PSMα3 nanotubes of different diameters, the two most common diameters being near 370 Å (blue double arrow) and 410 Å (red double arrow). (B) Model of the smaller diameter tube fit into its density map (light gray). The top image shows a view through the lumen of the tube, while the bottom image shows a side view. The protofilaments are distinguished by alternating gold and green strands. (C) Model of the larger diameter tube fit into its density map (light gray). Top and bottom images are the same views as in B. The protofilaments are indicated as alternating pink and blue strands. (D) Model of a single PSMα3 peptide fit into its corresponding density map. (E) Helical net of the smaller PSMα3 dimeter tubes. The convention used is that the surface of the tube is unrolled and is being viewed from the outside surface of the nanotube. The positions of the blue dots correspond to the helical arrangement of the asymmetric units. These smaller diameter tubes are generated from 13 protofilaments with a left-handed twist (light blue lines) in the absence of rotational point group symmetry (C₁ symmetry). Each asymmetric unit is related to an adjacent one in the same protofilament by a rise of 8.2 Å and rotation of −2.1°. (F) Helical net of the larger diameter PSMα3 tubes. The asymmetric units are indicated by the red dots. This larger diameter tube has C₇ rotational point group symmetry with 14 left-handed protofilaments (red lines). Adjacent asymmetric units in each protofilament are related by a rise of 8.3 Å and rotation of −2.2°.

Fig. 4.

Comparison of the organization of cross-α strands of PSMα3 in the crystal structure and nanotubes. (A) Helix packing of the PSMα3 strands in the crystal structure of the WT S. aureus peptide (PDB 5I55). (B) Packing of α-helices within a protofilament cross-section in the smaller diameter PSMα3 nanotube. (C) Packing of α-helices within a protofilament cross-section in the larger diameter PSMα3 nanotube. (D) Side view of the cross-α strand of the PSMα3 crystal structure. (E) Side view of a protofilament strand of the smaller diameter PSMα3 nanotube. The gray dashed line indicates the helical axis of the nanotube. (F) Side view of a protofilament strand of the larger diameter PSMα3 nanotube. The gray dashed line indicates the helical axis of the nanotube. (G) Asymmetric unit (ASU) of the smaller diameter PSMα3 tubes indicating the closest approach of helices across the inner–outer interface of the bilayer. (H) Asymmetric unit of the larger diameter PSMα3 tubes indicating the closest approach of helices across the inner–outer interface of the bilayer.

Fig. 5.

Cryo-EM structure of the PSMβ2 nanotubes. (A) Cryo-electron micrograph of the PSMβ2 nanotubes. The orange double arrow indicates a 310-Å diameter. (B) Top view of the PSMβ2 nanotube model fit into its density map (light gray). (C) Side view of the PSMβ2 nanotube model fit into the density map. Protofilaments are colored in alternating orange and purple. (D) Single PSMβ2 peptide model fit into its corresponding density map. (E) Asymmetric unit of the PSMβ2 nanotubes. The asymmetric unit consists of four peptides with eight helices. (F) Helical net of the PSMβ2 nanotube. The asymmetric units are depicted as blue dots, and the yellow lines indicate the presence of the 12 left-handed protofilaments in the structure.

Fig. 6.

Comparison of PSMα3 and PSMβ2 nanotube protofilaments. (A) PSMα3 subunits on the inner and outer walls are arranged antiparallel to each other. (B) PSMβ2 subunits on the inner and outer walls are arranged parallel to each other. (C) The tilt of the PSMα3 cross-α protofilaments with respect to the filament’s helical axis is −45°. The orientations of inner and outer wall subunits are the same. (D) The tilt of the PSMβ2 protofilaments with respect to the filament axis is −22°. The outer wall subunits are oriented in a plane perpendicular to the filament axis, while the inner wall subunits are tilted relative to the filament axis.