SignificanceThe recent increase in multidrug-resistant pathogenic bacteria is limiting the utility of our current arsenal of clinically important antibiotics. The development of improved antibiotics would therefore benefit from a better understanding of the current resistance mechanisms employed by bacteria. Many Gram-positive bacteria, including pathogenic Staphylococcus aureus and Enterococcus faecalis, utilize ribosome protection proteins to confer resistance to medically relevant antibiotics, such as streptogramins A, lincosamides, and pleuromutilins. We have employed cryo-electron microscopy to reveal the structural basis for how the Bacillus subtilis VmlR protein binds to the ribosome to confer resistance to the streptogramin A antibiotic virginiamycin M, the lincosamide lincomycin, and the pleuromutilin tiamulin.
Ribosomes synthesizing proteins containing consecutive proline residues become stalled and require rescue via the action of uniquely modified translation elongation factors, EF-P in bacteria, or archaeal/eukaryotic a/eIF5A. To date, no structures exist of EF-P or eIF5A in complex with translating ribosomes stalled at polyproline stretches, and thus structural insight into how EF-P/eIF5A rescue these arrested ribosomes has been lacking. Here we present cryo-EM structures of ribosomes stalled on proline stretches, without and with modified EF-P. The structures suggest that the favored conformation of the polyproline-containing nascent chain is incompatible with the peptide exit tunnel of the ribosome and leads to destabilization of the peptidyl-tRNA. Binding of EF-P stabilizes the P-site tRNA, particularly via interactions between its modification and the CCA end, thereby enforcing an alternative conformation of the polyproline-containing nascent chain, which allows a favorable substrate geometry for peptide bond formation.
Potato virus Y (PVY) is among the most economically important plant pathogens. Using cryoelectron microscopy, we determined the near-atomic structure of PVY’s flexuous virions, revealing a previously unknown lumenal interplay between extended carboxyl-terminal regions of the coat protein units and viral RNA. RNA–coat protein interactions are crucial for the helical configuration and stability of the virion, as revealed by the unique near-atomic structure of RNA-free virus-like particles. The structures offer the first evidence for plasticity of the coat protein’s amino- and carboxyl-terminal regions. Together with mutational analysis and in planta experiments, we show their crucial role in PVY infectivity and explain the ability of the coat protein to perform multiple biological tasks. Moreover, the high modularity of PVY virus-like particles suggests their potential as a new molecular scaffold for nanobiotechnological applications.
Bacteriophages from the family Myoviridae use double-layered contractile tails to infect bacteria. Contraction of the tail sheath enables the tail tube to penetrate through the bacterial cell wall and serve as a channel for the transport of the phage genome into the cytoplasm. However, the mechanisms controlling the tail contraction and genome release of phages with “double-layered” baseplates were unknown. We used cryo-electron microscopy to show that the binding of the Twort-like phage phi812 to the Staphylococcus aureus cell wall requires a 210° rotation of the heterohexameric receptor-binding and tripod protein complexes within its baseplate about an axis perpendicular to the sixfold axis of the tail. This rotation reorients the receptor-binding proteins to point away from the phage head, and also results in disruption of the interaction of the tripod proteins with the tail sheath, hence triggering its contraction. However, the tail sheath contraction of Myoviridae phages is not sufficient to induce genome ejection. We show that the end of the phi812 double-stranded DNA genome is bound to one protein subunit from a connector complex that also forms an interface between the phage head and tail. The tail sheath contraction induces conformational changes of the neck and connector that result in disruption of the DNA binding. The genome penetrates into the neck, but is stopped at a bottleneck before the tail tube. A subsequent structural change of the tail tube induced by its interaction with the S. aureus cell is required for the genome’s release.
Background: Nucleoporin Nup159 has multiple recognition motifs for Dyn2, the yeast ortholog of LC8. Results: Nup159 is intrinsically disordered and binds Dyn2 cooperatively at five of the six recognition motifs. Conclusion: Initial binding aligns two Nup chains in a bivalent scaffold; motifs 5 and 6 underlie rigidity. Significance: Multiple recognition sites provide entropy/enthalpy balance to a stable yet entropically unfavorable rigid complex.
Plectin 1c (P1c), an intermediate filament-associated cytolinker protein, antagonizes the microtubule (MT)-stabilizing function of MT-associated proteins. Lack of P1c in keratinocytes leads to the stabilization of MTs and alters basic cellular features and functions, including cell shape, polarized migration, metabolism, and mitotic spindle formation.
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