Cervenak, M. | bioRxiv Science

bioRxiv2025

Flexible Motion of T7 bacteriophage Tail Fibers Suggest a Dynamic Viral Infection Mechanism

Viruses are nanoscale infectious agents capable of specifically targeting and reprograming host cells. A unique group of viruses, bacteriophages, have regained popularity in research partly due to the rising number of multidrug-resistant bacterial infections. Phages could potentially replace antibiotics, but only if we understand every detail of their structure and infection cycle. T7 bacteriophages are a group of dsDNA viruses, which infect E. coli bacteria. T7 virions are comprised of an icosahedral protein shell which encapsulates the genomic DNA, and a tail-fiber complex which is primarily used for target recognition and DNA injection. The virus has six" L"-shaped, [~]40 nm long fibers (gp17 protein trimers) attached to the tail-tube, which are thought to be essential for initial host recognition and possibly surface exploration. Using high-speed atomic force microscopy (HS-AFM) and molecular dynamics (MD) simulations combined with small angle X-ray scattering (SAXS) we observed the molecular structure and movements of isolated tail fibers. Firstly, we have identified a hinge region within the fibers, which makes them highly flexible, allowing the bending of their distal region. Furthermore, we have observed the dynamic triple helical coiled coil structure of the proximal region, which would allow fiber rotation. These two points of flexibility allow a more efficient and highly dynamic host recognition and virus anchoring process. The observed flexibility might allow host surface exploration by walking. Such flexibility in the host recognition machinery may not be unique to T7 bacteriophages, getting us one step closer to understanding the intricate details of virus-host interactions.

biophysics↗

bioRxiv2025

Chemical mechanism of allosteric and asymmetric dark reversion in a bacterial phytochrome uncovered by cryo-EM.

Phytochromes are light-sensitive proteins found in plants, fungi, and bacteria. They exist in two functional states, Pr and Pfr, distinguished by Z/E isomers of their bilin chromophore. The chromophore can photoswitch between these states, but also thermally converts in darkness. Despite the importance of the latter reaction, it remains unclear how it is controlled by the phytochrome. Here, we present single-particle cryo-EM measurements on the Pseudomonas aeruginosa bacteriophytochrome (PaBphP) carried out at multiple time points during dark reversion from Pr to Pfr. These experiments resolve the structure of a PrPfr hybrid state. Surprisingly, we find that only protomer B converts back to Pfr in the hybrid, while protomer A remains in Pr. We identify structural asymmetries in the precursor Pr state, which extend from the homodimer interface to a conserved histidine (H277). The hydrogen-bonding network around the chromophore is modulated, explaining how the phytochrome gains control over the activation energy of the isomerization reaction. These findings establish that dark reversion is governed by conformational selection between two substates, whereby one is "dark-reversion ready" and the other one blocks the reaction. Moreover, we explain how the equilibrium of the states is allosterically controlled across the dimer. Together, these findings provide a structural framework for tuning phytochrome signaling lifetimes in optogenetic applications. Significance statementThe dark reversion reaction of phytochromes is crucial to their signalling role in plants, bacteria, and fungi, but it is vastly understudied in terms of its chemical mechanism. It remains unsolved how the reaction can proceed at all, given that the activation energy is prohibitively high for the isomerization to occur in solution. Using time-resolved cryo-EM, we present a chemical and structural framework for understanding how the protein binding pocket regulates the reaction. Our results establish that conformational selection between two substates controls dark reversion, providing a rare example of strongly asymmetric reactivity across a dimeric protein. This opens the way for rational engineering of the lifetimes of the signaling states in phytochromes.

biochemistry↗