Biology subjects

Rutbeek, N. R.

Publications and source records attributed to Rutbeek, N. R..

6 recordsLinked to original sources

bioRxiv2026

Molecular basis of the Druantia anti-phage defense system

Eukaryotes and prokaryotes have evolved diverse antiviral immune systems, many containing helicase modules central to defense. Druantia are widespread bacterial anti-phage defense systems, each built around a large helicase domain-containing protein, DruE, paired with variable subunits. Here, we investigate the molecular basis of a minimal two-protein module Druantia system, DruH-E. We demonstrate that DruH-E is sufficient to confer robust anti-phage defense. DruE exists in equilibrium between a monomer and an asymmetric dimer, with dimerization required for in vivo immunity. Cryo-EM structures define DruE asymmetric dimer assembly and its dsDNA unwinding mechanism, revealing a topologically closed architecture that is specifically activated by dsDNA substrates with a 3' overhang. We further identify DruH as an ssDNA-binding protein regulated by a metabolic switch, where its activity is inhibited by ATP at physiological concentrations through direct competition with ssDNA. Supported by mass spectrometry and single-cell microscopy data, we establish key determinants of the Druantia defense system and reveal how it mediates a direct antiviral immune mechanism.

microbiology↗

bioRxiv2026

Genome delivery of a contractile tailed phage and its superinfection exclusion mechanism

Successful viral infection requires efficient adsorption to the target cell, followed by membrane penetration for genome translocation into the host cytoplasm. Bacteriophage T4 initiates infection by binding to Escherichia coli receptors, triggering sequential conformational changes that culminate in genome delivery through viral channels spanning the host cell envelope. Here, we resolved structures of bacteriophage T4 at discrete stages. This revealed how long tail fiber extension affects the baseplate to initiate tail contraction, and the channel formation by the tape measure protein for genome translocation. We further demonstrate that the virus-encoded superinfection exclusion protein Imm binds to the tape measure protein to prevent secondary infections. Our findings offer insights into the coordinated process of genome delivery and phage-encoded superinfection exclusion proteins that prevent genome translocation.

microbiology↗

bioRxiv2024

The phage protein paratox is a multifunctional metabolic regulator of Streptococcus

Streptococcus pyogenes, or Group A Streptococcus (GAS), is a commensal bacteria and human pathogen. Central to GAS pathogenesis is the presence of prophage encoded virulence genes. The conserved phage gene for the protein paratox (Prx) is genetically linked to virulence genes, but the reason for this linkage is unknown. Prx inhibits GAS quorum sensing and natural competence by binding the transcription factor ComR. However, inhibiting ComR does not explain the virulence gene linkage. To address this, we took a mass-spectrometry approach to search for other Prx interaction partners. The data demonstrates that Prx binds numerous DNA binding proteins and transcriptional regulators. We show binding of Prx in vitro with the GAS protein EndoS1 (SpyM3_0890) and the phage protein JM3 (SpyM3_1246). An EndoS1:Prx complex X-ray crystal structure reveals that EndoS1 and ComR possess a conserved Prx binding helix. Computational modelling predicts that the Prx binding helix is present in several, but not all, binding partners. Namely, JM3 lacks the Prx binding helix. As Prx is conformationally dynamic, this suggests partner-dependent binding modes. Overall, Prx acts as a metabolic regulator of GAS to maintain the phage genome. As such, Prx maybe a direct contributor to the pathogenic conversion of GAS.

biochemistry↗

bioRxiv2024

The Streptococcus phage protein paratox is an intrinsically disordered protein.

The bacteriophage protein paratox blocks quorum sensing in its streptococcal host by directly binding the signal receptor and transcription factor ComR. This reduces ability of Streptococcus to uptake environmental DNA and protects phage DNA from damage by recombination. Past work characterizing the paratox:ComR molecular interaction revealed that paratox adopts a well-ordered globular fold when bound to ComR. However, solution-state biophysical measurements suggested that paratox may be conformationally dynamic. To address this discrepancy, we investigated the stability and dynamic properties of paratox in solution using circular dichroism, nuclear magnetic resonance, and several fluorescence-based protein folding assays. Our work shows that under dilute buffer conditions paratox is intrinsically disordered. We also show that the addition of kosmotropic salts or protein stabilizing osmolytes induces paratox folding. However, only the addition of ComR was able to induce paratox to adopt its previously characterized globular fold. Furthermore, as we can induce different paratox folding-states we characterize Prx folding thermodynamics and folding kinetics using stopped flow measurements. Based upon the kinetic results, paratox is a highly dynamic protein in dilute solution, folding and refolding within the 10 ms timescale. Overall, our results demonstrate that the streptococcal phage protein paratox is an intrinsically disordered protein in a two-state equilibrium with a solute-stabilized folded form. Furthermore, the solute-stabilized paratox fold is likely the predominant form of paratox in a solute-crowded bacterial cell. Finally, our work suggests that Prx binds and inhibits ComR, and thus quorum sensing in Streptococcus, by a combination of conformational selection and induced-fit binding mechanisms.

biochemistry↗

bioRxiv2023

Structure and mechanism of Zorya anti-phage defense system

Zorya is a recently identified and widely distributed bacterial immune system, which protects against phage invasion. It consists of a predicted membrane-embedded complex (ZorAB) and soluble components that differ among Zorya subtypes, notably ZorC and ZorD, in type I Zorya systems. Here, we reveal the molecular basis of the Zorya defense system using cryo-electron microscopy, mutagenesis, fluorescence microscopy, proteomics, and functional studies. We demonstrate that ZorAB shares the stoichiometry of other 5:2 inner membrane ion-driven rotary motors. Additionally, ZorA5B2 features a dimeric ZorB peptidoglycan binding domain and a pentameric -helical coiled-coil tail made of ZorA that projects approximately 700 [A] into the cytoplasm. We further characterize the structure and function of the soluble Zorya components, ZorC and ZorD, and find that they harbour DNA binding and nuclease activity, respectively. Comprehensive functional and mutational analyses demonstrates that all Zorya components work in concert to protect bacterial cells against invading phages. We present evidence that ZorAB operates as an ion-driven motor that becomes activated and anchors to the cell wall upon sensing of cell envelope perturbations during phage invasion. Subsequently, ZorAB transfers the phage invasion signal through the ZorA cytoplasmic tail to the soluble effectors, which function to prevent phage propagation. In summary, our study elucidates the foundational mechanisms of Zorya function and reveals a novel triggering signal for the rapid activation of an anti-phage defense system.

molecular biology↗

bioRxiv2021

Molecular mechanism of quorum sensing inhibition in Streptococcus by the phage protein paratox

Streptococcus pyogenes, or Group A Streptococcus, is a Gram-positive bacterium that can be both a human commensal and pathogen. Central to this dichotomy are temperate bacteriophages that incorporate into the bacterial genome as a prophage. These genetic elements encode both the phage proteins as well as toxins harmful to the human host. One such conserved phage protein paratox (Prx) is always found encoded adjacent to the toxin genes and this linkage is preserved during transduction. Within Streptococcus pyogenes, Prx functions to inhibit the quorum-sensing ComRS receptor-signal pair that is the master regulator of natural competence, or the ability to uptake endogenous DNA. Specifically, Prx directly binds and inhibits the receptor ComR by unknown mechanism. To understand how Prx inhibits ComR at the molecular level we pursued an X-ray crystal structure of Prx bound to ComR. The structural data supported by solution X-ray scattering data demonstrate that Prx induces a conformational change in ComR to directly access the DNA binding domain. Furthermore, electromobility shift assays and competition binding assays reveal that Prx effectively uncouples the inter-domain conformational change that is required for activation of ComR by the signaling molecule XIP. Although to our knowledge the molecular mechanism of quorum-sensing inhibition by Prx is unique, it is analogous to the mechanism employed by the phage protein Aqs1 in Pseudomonas aeruginosa. Together, this demonstrates an example of convergent evolution between Gram-positive and Gram-negative phages to inhibit quorum-sensing, and highlights the versatility of small phage proteins.

biochemistry↗