Biology subjects

Prokhorov, N. S.

Publications and source records attributed to Prokhorov, N. S..

4 recordsLinked to original sources

bioRxiv2023

Function of the bacteriophage P2 baseplate central spike Apex domain in the infection process

The contractile tail of bacteriophage P2 functions to drive the tail tube across the outer membrane of its host bacterium, a prerequisite event for subsequent translocation of phage genomic DNA into the host cell. The tube is equipped with a spike-shaped protein (product of P2 gene V, gpV or Spike) that contains a membrane-attacking Apex domain carrying a centrally positioned Fe ion. The ion is enclosed in a histidine cage that is formed by three symmetry-related copies of a conserved HxH (histidine, any residue, histidine) sequence motif. Here, we used solution biophysics and X-ray crystallography to characterize the structure and properties of Spike mutants in which the Apex domain was either deleted or its histidine cage was either destroyed or replaced with a hydrophobic core. We found that the Apex domain is not required for the folding of full-length gpV or its middle intertwined {beta}-helical domain. Furthermore, despite its high conservation, the Apex domain is dispensable for infection in laboratory conditions. Collectively, our results show that the diameter of the Spike but not the nature of its Apex domain determines the efficiency of infection, which further strengthens the earlier hypothesis of a drill bit-like function of the Spike in host envelope disruption.

molecular biology↗

bioRxiv2022

Biophysical and Structural Characterization of a Viral Genome Packaging Motor

Like many dsDNA viruses, bacteriophage {lambda} replicates its genome as a concatemer consisting of multiple copies of covalently linked dsDNA genomes. To encapsidate a single genome within a nascent procapsid, {lambda} must: 1) find its own dsDNA amongst the multitude of host nucleic acids; 2) identify the genomic start site; 3) cut the DNA; 4) bring the excised DNA to a procapsid; 5) translocate DNA into the capsid; 6) cut DNA again at a packaging termination site, 7) disengage from the newly filled capsid; and 8) bring the remainder of the genomic concatemer to fill another empty procapsid. These disparate genome processing tasks are carried out by a single virus-encoded enzyme complex called terminase. While it has been shown that {lambda} terminase initially forms a tetrameric complex to cut DNA, it is not clear whether the same configuration translocates DNA. Here, we describe biophysical and initial structural characterization of a {lambda} terminase translocation complex. Analytical ultracentrifugation (AUC) and small angle X-ray scattering (SAXS) indicate that between 4 and 5 protomeric subunits assemble a cone-shaped terminase complex with a maximum dimension of [~]230 and radius of gyration of [~]72 [A]. Two-dimensional classification of cryoEM images of {lambda} terminase are consistent with these dimensions and show that particles assume a preferred orientation in ice. The orientations appear to be end-on, as terminase rings resemble a starfish with approximate pentameric symmetry. While [~]5-fold symmetry is apparent, one of the five "arms" appears partially displaced with weaker more diffuse density in some classes, suggesting flexibility and/or partial occupancy. Charge detection mass spectrometry (CDMS) is consistent with a pentameric complex, with evidence that one motor subunit is weakly bound. Kinetic analysis indicates that the complex hydrolyzes ATP at a rate comparable to the rates of other phage packaging motors. Together with previously published data, these results suggest that {lambda} terminase assembles conformationally and stoichiometrically distinct complexes to carry out different genome processing tasks. We propose a "symmetry resolution" pathway to explain how terminase transitions between these structurally and functionally distinct states.

biophysics↗

bioRxiv2021

Identification of Low Population States in Cryo-EM Using Deep Learning

Cryo-EM has made extraordinary headway towards becoming a semi-automated, high-throughput structure determination technique. In the general workflow, high-to-medium population states are grouped into two- and three-dimensional classes, from which structures can be obtained with near-atomic resolution and subsequently analyzed to interpret function. However, low population states, which are also functionally important, are often discarded. Here, we describe a technique whereby low population states can be efficiently identified with minimal human effort via a deep convolutional neural network classifier. We use this deep learning classifier to describe a transient, low population state of bacteriophage A511 in the midst of infecting its bacterial host. This method can be used to further automate data collection and identify other functionally important low population states.

biophysics↗

bioRxiv2019

Phages associated with horses provide new insights into the dominance of lateral gene transfer in virulent bacteriophages evolution in natural systems

Tailed bacteriophages (Caudovirales order) are omnipresent on our planet. Their impressive ecological and evolutionary success largely relies on the bacteriophage potential to adapt to great variety of the environmental conditions found in the Biosphere. It is believed that the adaptation of bacteriophages, including short time scale adaptation, is achieved almost exclusively via the (micro)evolution processes. In order to analyze the major mechanisms driving adaptation of phage genomes in a natural habitat we used comparative genomics of G7C-like coliphage isolates obtained during 7 years period from the feces of the horses belonging to a local population. The data suggest that even at this relatively short time scale the impact of various recombination events overwhelms the impact of the accumulation of point mutations. The access to the large pool of the genes of a complex microbial and viral community of the animal gut had major effect on the evolutionary trajectories of these phages. Thus the "real world" bacteriophage evolution mechanisms may differ significantly from those observed in the simplified laboratory model systems.

evolutionary biology↗