Channel width modulates the permeability of DNA origami based nuclear pore mimics.

Nucleoporins (nups) in the central channel of nuclear pore complexes (NPCs) form a selective barrier that suppresses the diffusion of most macromolecules while enabling rapid transport of nuclear transport receptors (NTRs) with bound cargos. The complex molecular interactions between nups and NTRs have been thought to underlie the gatekeeping function of the NPC. Recent studies have shown considerable variation in NPC diameter but how altering NPC diameter might impact the selective barrier properties remains unclear. Here, we build DNA nanopores with programmable diameters and nup arrangement to mimic NPCs of different diameters. We use hepatitis B virus (HBV) capsids as a model for large-size cargos. We find that Nup62 proteins form a dynamic cross-channel meshwork impermeable to HBV capsids when grafted on the interior of 60-nm wide nanopores but not in 79-nm pores, where Nup62 cluster locally. Furthermore, importing substantially changes the dynamics of Nup62 assemblies and facilitates the passage of HBV capsids through NPC mimics containing Nup62 and Nup153. Our study shows the transport channel width is critical to the permeability of nup barriers and underscores the role of NTRs in dynamically remodeling nup assemblies and mediating the nuclear entry of viruses.

Structural insights into DarT toxin neutralization by cognate DarG antitoxin: ssDNA mimicry by DarG C-terminal domain keeps the DarT toxin inhibited.

In the DarTG toxin-antitoxin system, the DarT toxin ADP-ribosylates single-stranded DNA (ssDNA), which stalls DNA replication and plays a crucial role in controlling bacterial growth and bacteriophage infection. This toxic activity is reversed by the N-terminal macrodomain of the cognate antitoxin DarG. DarG also binds DarT, but the role of these interactions in DarT neutralization is unknown. Here, we report that the C-terminal domain of DarG (DarG toxin-binding domain [DarGTBD]) interacts with DarT to form a 1:1 stoichiometric heterodimeric complex. We determined the 2.2 Å resolution crystal structure of the Mycobacterium tuberculosis DarT-DarGTBD complex. The comparative structural analysis reveals that DarGTBD interacts with DarT at the DarT/ssDNA interaction interface, thus sterically occluding substrate ssDNA binding and consequently inactivating toxin by direct protein-protein interactions. Our data support a unique two-layered DarT toxin neutralization mechanism of DarG, which is important in keeping the toxin molecules in check under normal growth conditions.

Investigating the Process of Sheath Maturation in Antifeeding Prophage: a Phage Tail-Like Protein Translocation Structure.

The antifeeding prophage (Afp) produced by the bacterium Serratia entomophila is the archetypical external contractile injection system (eCIS). Afp and its orthologues are characterized by three sheath proteins, while contractile bacteriophages and pyocins encode only one. Using targeted mutagenesis, transmission electron microscopy (TEM), and pulldown studies, we interrogated the roles of the three sheath proteins (Afp2, Afp3, and Afp4) in Afp assembly, in particular the interaction between the two sequence-related helical-sheath-forming proteins Afp2 and Afp3 and their cross talk with the tail termination sheath capping protein (TrP) Afp16 in the sheath maturation process. The expressed assemblies for the afp2-deficient mutant were mostly a mixture of isolated tail fibers, detached baseplates without tail fibers, and sheathless inner tube baseplate complexes (TBCs) with a length similar to that of mature Afp, which were surrounded in many cases by fibrillar polymerized material. In the afp3-deficient mutant, variable-length TBCs with similar but shorter fibrillar polymerized material, largely bereft of tail fibers, were observed, while only detached baseplate assemblies were seen for the afp4-deficient mutant. Furthermore, we found that (i) only trans complementation of afp2 with its mutated counterpart restored mature Afp particles with full biological activity, (ii) purified Afp3 pulled down Afp2 by forming a sodium dodecyl sulfate (SDS)-resistant complex but not vice versa, (iii) Afp16 had a higher affinity for binding Afp2 or Afp3 than Afp4, and (iv) Afp4 is required for the association of the polymerized sheath on the baseplate via Afp2. A proposed model for sheath maturation and assembly in Afp is presented. IMPORTANCE Members of the contractile bacteriophage-related but evolutionarily divergent eCIS contain not one but three sheath proteins, two of which, namely, Afp2 and Afp3 in the Afp, arranged as alternate hexameric stacks constitute the helical sheath. We revealed that Afp2 and Afp3, even though they are highly similar, possess markedly distinct, crucial roles in Afp assembly. We find that Afp3, by virtue of its interaction with the tail-terminating protein Afp16, regulates tube and sheath length, while Afp2 is critical for proper sheath polymerization and the assembly of the baseplate. The resulting model for the Afp assembly will further guide the manipulation of Afp and its related eCISs as nanodelivery vehicles for pest control and phage therapy.