Daniel Nemecek

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

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.

Assembly and Architecture of Biogenesis of Lysosome-related Organelles Complex-1 (BLOC-1)

Background: The BLOC-1 complex is critical for biogenesis of lysosome-related organelles. Results: BLOC-1 is elongated, bends by up to 45º, and contains two heterotrimeric subcomplexes. Conclusion: BLOC-1 is flexible hetero-octameric complex built up from two elongated heterotrimeric subcomplexes. Significance: The length and flexibility of BLOC-1 may contribute to its interactions with membranes and SNAREs in tubular endosome biogenesis. BLOC-1 (biogenesis of lysosome-related organelles complex-1) is critical for melanosome biogenesis and has also been implicated in neurological function and disease. We show that BLOC-1 is an elongated complex that contains one copy each of the eight subunits pallidin, Cappuccino, dysbindin, Snapin, Muted, BLOS1, BLOS2, and BLOS3. The complex appears as a linear chain of eight globular domains, ∼300 Å long and ∼30 Å in diameter. The individual domains are flexibly connected such that the linear chain undergoes bending by as much as 45°. Two stable subcomplexes were defined, pallidin-Cappuccino-BLOS1 and dysbindin-Snapin-BLOS2. Both subcomplexes are 1:1:1 heterotrimers that form extended structures as indicated by their hydrodynamic properties. The two subcomplexes appear to constitute flexible units within the larger BLOC-1 chain, an arrangement conducive to simultaneous interactions with multiple BLOC-1 partners in the course of tubular endosome biogenesis and sorting.

Subunit Folds and Maturation Pathway of a dsRNA Virus Capsid

The cystovirus ϕ6 shares several distinct features with other double-stranded RNA (dsRNA) viruses, including the human pathogen, rotavirus: segmented genomes, nonequivalent packing of 120 subunits in its icosahedral capsid, and capsids as compartments for transcription and replication. ϕ6 assembles as a dodecahedral procapsid that undergoes major conformational changes as it matures into the spherical capsid. We determined the crystal structure of the capsid protein, P1, revealing a flattened trapezoid subunit with an α-helical fold. We also solved the procapsid with cryo-electron microscopy to comparable resolution. Fitting the crystal structure into the procapsid disclosed substantial conformational differences between the two P1 conformers. Maturation via two intermediate states involves remodeling on a similar scale, besides huge rigid-body rotations. The capsid structure and its stepwise maturation that is coupled to sequential packaging of three RNA segments sets the cystoviruses apart from other dsRNA viruses as a dynamic molecular machine.

Packaging accessory protein P7 and polymerase P2 have mutually occluding binding sites inside the bacteriophage 6 procapsid.

ABSTRACT Bacteriophage ϕ6 is a double-stranded RNA (dsRNA) virus whose genome is packaged sequentially as three single-stranded RNA (ssRNA) segments into an icosahedral procapsid which serves as a compartment for genome replication and transcription. The procapsid shell consists of 60 copies each of P1A and P1B, two nonequivalent conformers of the P1 protein. Hexamers of the packaging ATPase P4 are mounted over the 5-fold vertices, and monomers of the RNA-dependent RNA polymerase (P2) attach to the inner surface, near the 3-fold axes. A fourth protein, P7, is needed for packaging and also promotes assembly. We used cryo-electron microscopy to localize P7 by difference mapping of procapsids with different protein compositions. We found that P7 resides on the interior surface of the P1 shell and appears to be monomeric. Its binding sites are arranged around the 3-fold axes, straddling the interface between two P1A subunits. Thus, P7 may promote assembly by stabilizing an initiation complex. Only about 20% of the 60 P7 binding sites were occupied in our preparations. P7 density overlaps P2 density similarly mapped, implying mutual occlusion. The known structure of the ϕ12 homolog fits snugly into the P7 density. Both termini—which have been implicated in RNA binding—are oriented toward the adjacent 5-fold vertex, the entry pathway of ssRNA segments. Thus, P7 may promote packaging either by interacting directly with incoming RNA or by modulating the structure of the translocation pore.

Stepwise expansion of the bacteriophage ϕ6 procapsid: possible packaging intermediates.

The initial assembly product of bacteriophage ϕ6, the procapsid, undergoes major structural transformation during the sequential packaging of its three segments of single-stranded RNA. The procapsid, a compact icosahedrally symmetric particle with deeply recessed vertices, expands to the spherical mature capsid, increasing the volume available to accommodate the genome by 2.5-fold. It has been proposed that expansion and packaging are linked, with each stage in expansion presenting a binding site for a particular RNA segment. To investigate procapsid transformability, we induced expansion by acidification, heating, and elevated salt concentration. Cryo-electron microscopy reconstructions after all three treatments yielded the same partially expanded particle. Analysis by cryo-electron tomography showed that all vertices of a given capsid were either in a compact or an expanded state, indicating a highly cooperative transition. To benchmark the mature capsid, we analyzed filled (in vivo packaged) capsids. When these particles were induced to release their RNA, they reverted to the same intermediate state as expanded procapsids (intermediate 1) or to a second, further expanded state (intermediate 2). This partial reversibility of expansion suggests that the mature spherical capsid conformation is obtained only when sufficient outward pressure is exerted by packaged RNA. The observation of two intermediates is consistent with the proposed three-step packaging process. The model is further supported by the observation that a mutant capable of packaging the second RNA segment without previously packaging the first segment has enhanced susceptibility for switching spontaneously from the procapsid to the first intermediate state.

Phage capsid-like structure of Myxococcus xanthus encapsulin, a protein shell that stores iron

Iron is both an essential cofactor of many enzymes and a producer of highly reactive hydroxyl radicals that can cause cellular damage. To regulate the supply of intracellular iron, cells have developed protein-based organelles, like ferritins, that act as iron storage containers. Myxococcus xanthus, a soil-dwelling gram-negative myxobacterium, produces another type of protein-based organelle that has been related to iron metabolism, the encapsulin nanocompartment [1]. It has a spherical shell composed of a 32 kDa protein called EncA or encapsulin, and three minor proteins in the 11 kDa to 17 kDa range [1]. To gain insight on the molecular architecture of this complex, we have studied encapsulin nanocompartments purified from M. xanthus and recombinant EncA shells produced in E. coli using single-particle cryo-electron microscopy (cryo-EM). This analysis was supplemented with conventional TEM with and without negative staining and scanning transmission electron microscopy (STEM).

Expansion of the Bacteriophage φ6 Procapsid Revealed by Electron Cryo-Microscopy

Bacteriophage φ6 is a spherical enveloped dsRNA virus (diameter 860 Å) that infects the bacterium Pseudomonas syringae and shares many properties with viruses of the Reoviridae family [1]. The first assembly product is the icosahedral procapsid composed of the proteins P1, P2, P4 and P7, and with deeply recessed 5-fold vertices. During packaging of the 3-segment genome, the P1 shell expands to a near-spherical shape. The P1 subunits must undergo significant conformational changes to achieve the expansion. The P2 protein (the RNA-dependent RNA polymerase) was identified inside the unexpanded procapsid shell on the 3-fold axis [2], and should remain in close proximity to the 5-fold vertex on expansion for minus RNA strand synthesis. To shed more light on the mechanics of procapsid expansion, we imaged empty, expanded, and RNA-packaged procapsids.