A virus capsid-like nanocompartment that stores iron and protects bacteria from oxidative stress

Abstract

Living cells compartmentalize materials and enzymatic reactions to increase metabolic efficiency. While eukaryotes use membrane-bound organelles, bacteria and archaea rely primarily on protein-bound nanocompartments. Encapsulins constitute a class of nanocompartments widespread in bacteria and archaea whose functions have hitherto been unclear. Here, we characterize the encapsulin nanocompartment from Myxococcus xanthus, which consists of a shell protein (EncA, 32.5 kDa) and three internal proteins (EncB, 17 kDa; EncC, 13 kDa; EncD, 11 kDa). Using cryo-electron microscopy, we determined that EncA self-assembles into an icosahedral shell 32 nm in diameter (26 nm internal diameter), built from 180 subunits with the fold first observed in bacteriophage HK97 capsid. The internal proteins, of which EncB and EncC have ferritin-like domains, attach to its inner surface. Native nanocompartments have dense iron-rich cores. Functionally, they resemble ferritins, cage-like iron storage proteins, but with a massively greater capacity (~30,000 iron atoms versus ~3,000 in ferritin). Physiological data reveal that few nanocompartments are assembled during vegetative growth, but they increase fivefold upon starvation, protecting cells from oxidative stress through iron sequestration.

Keywords: HK97 fold; cryo‐electron microscopy; encapsulin; ferritin; oxidative stress.

Figure 1. Native encapsulin nanocompartments are composed of four proteins and contain a dense iron-rich core

1. EM of negatively stained nanocompartments. Scale bar, 100 nm.

2. SDS–PAGE of purifed native encapsulin nanocompartments. Mass markers are indicated (in kDa).

3. Domain maps of the four encapsulin proteins. Yellow, HK97-like domain; orange, rubrerythrin/ferritin-like domain; asterisks, ExxH metal coordination motifs; gray, encapsulation signal.

4. EM of unstained/air-dried nanocompartments. Scale bar, 100 nm.

5. STEM and energy-dispersive X-ray spectroscopy (EDX) images of two representative particles. In the STEM image (unstained specimen), positive signals from mass scattering identify two nanocompartments, while the corresponding EDX images map elemental concentrations of Fe and P (strongly above background), and S and Ca (at or close to background). Red crosshairs indicate identical positions on one nanocompartment. Scale bar, 100 nm.

Source data are available online for this figure.

Figure 2. Cryo-EM of encapsulin nanocompartments and EncA shells

A, B Cryo-micrographs of (A) native nanocompartments and (B) recombinantly expressed EncA capsids. C Section through a reconstruction of the EncA capsid. An enlargement of the boxed area showing a longitudinal section through an α-helix is inset (upper right). D Atomic model of the T = 3 EncA capsid. The three quasi-equivalent subunits are colored yellow, green, and red. Symmetry axes are marked. E The 3-subunit asymmetric unit fitted into electron density (transparent). F, G Atomic model of EncA, rainbow-colored with the N-terminus in blue, and the C-terminus in red. Data information: Scale bars: (A, B) 50 nm; (C) 10 nm; (D) 5 nm; (E, C insert) 2 nm.

Figure 3. Cryo-EM of encapsulin nanocompartments with electron-dense cores

1. Cryo-EM image of a native encapsulin nanocompartment without (top) and with (bottom) masking out of the electron-dense core.

2. Sections through the single-particle reconstruction of the native Myxococcus xanthus encapsulin nanocompartment.

3. Isosurface representation of the reconstruction color-coded according to radial distance from the center (given in Å). Left, viewed from the outside; right, cutaway view of the internal structure.

Data information: Scale bars, 10 nm in (A, C); 25 nm in (B).

Figure 4. Cryo-ET of encapsulin nanocompartments with electron-dense cores

1. Tomographic slice showing several native Myxococcus xanthus encapsulin nanocompartments.

2. Tomographic slices through two nanocompartments (top and bottom rows).

3. Gallery of tomographic central sections of eight nanocompartments.

4. Histogram of the diameters of electron-dense granules in the nanocompartment cores.

Data information: Scale bars, 25 nm.

Figure 5. Cryo-EM visualization of internal proteins

A–C Central sections through reconstructions of (A) native nanocompartments lacking a dense core; (B) EncA capsids; (C) the difference map between (A) and (B). In (C), contours delineating the outer and inner surfaces of the EncA capsid are superimposed in half of the section to aid in locating densities of internal proteins. White arrows label densities in direct contact with the inner surface, and black arrows label offset densities. D Isosurface rendering of a cutaway view of the EncA capsid (green-blue) and the positive difference density (orange) attributable to the internal proteins. Scale bar, 10 nm.

Figure 6. STEM of native encapsulin nanocompartments

A, B Dark-field STEM micrograph of unstained encapsulin nanocompartments. The masses (in MDa) of some particles are indicated. The rod-like structures are TMV virions, used as a mass standard. Scale bars, 50 nm in (A); 25 nm in (B). C, D Histograms of the masses measured for (C) the encapsulin nanocompartments and (D) 56-nm-long segments of TMV (mass/length = 131.4 kDa/nm), which contain approximately the same amount of protein as the nanocompartments.

Figure 7. Encapsulin nanocompartments are upregulated during starvation and protect the cells from oxidative stress

A Western blot of EncA from lysate of 1 × 10⁸ vegetative cells in rich CTT medium or starvation TPM medium, and spores after 48 or 96 h of development. B Amounts of purified nanocompartments recovered from different cell types, compared to yield recovered from cells grown in CTT (100%). C EM of unstained longitudinal thin sections of vegetative cells grown in CTT (left panel) or TPM medium (right panel). Arrows mark dense-cored encapsulin nanocompartments. Insets, transverse thin cross-sections. Scale bar, 0.5 μm. D, E Survival of wild-type (blue curve) and encA::pCR2.1 disruption (yellow curve) strains (D) and of wild-type (blue curve) and ΔencA in-frame deletion (orange curve) (E) after exposure to 0.5 mM hydrogen peroxide. Source data are available online for this figure.

Figure 8. Comparison of encapsulin proteins and HK97 gp5* in different maturation states

A EncA from this study (pdb 4PT2; EMDataBank EMD-5917). B Encapsulin from Thermotoga maritima (pdb 3DKT). C Encapsulin from Pyrococcus furiosus (pdb 2E0Z). D, E gp5* from HK97 in the Prohead II (D; pdb 3E8K) and Head II(E; pdb 2FT1) conformations. Models are rainbow-colored, with the N-termini of the solved structures in blue and the C-termini in red. F Top views (upper row) and side views (lower row) of EncA and gp5* hexamers. The EncA hexamer is more similar to Prohead II gp5* than to Head II gp5* despite lacking the skew disclination of the Prohead II gp5* hexamer (cf. the respective frames).

Figure 9. Schematic model of an Myxococcus xanthus encapsulin nanocompartment

1. Shown are cutaway views of the EncA shell (blue) for the particles with (left) and without (right) iron-rich granules. The C-terminal anchor motif common to EncB, EncC, and EncD is depicted as a small gray oval. In EncB and EncC, this motif is connected by an extended flexible linker to the rubrerythrin domain (green ovals). EncD's domain is shown as a brown oval. Since EncB and EncC are predicted to have only two α-helices each, we hypothesize that they dimerize forming a 4-helix bundle in homo- or hetero-dimers with the iron-binding motifs midway along the bundle. However, monomers may also exist, and they may be capable of nucleating iron deposition. The model envisages iron atoms entering the shell through narrow channels, to be incorporated into a nascent granule nucleated on the rubrerythrin domains. Interaction of the iron with the internal proteins probably induces a conformational change that causes the rubrerythrin domains to detach from the EncA shell.

2. The most likely candidates for the entry channels are shown.