Biology subjects

Roa-Eguiara, A.

Publications and source records attributed to Roa-Eguiara, A..

7 recordsLinked to original sources

bioRxiv2026

Piercing mechanism in the minimal contractile tail of bacteriophage P2.

Bacteriophage P2 is a long-standing model for contractile injection systems, yet high-resolution structures of the intact virion and the mechanism of baseplate activation have remained unresolved. Here, we combine high-resolution cryo-electron microscopy and in situ cryo-electron tomography to characterize P2, using its lytic mutant P2vir, in its pre- and post-contraction states and during host infection. We identify a previously undescribed fold in the head-to-tail adaptor, which mediates the transition from 12-fold portal symmetry to sixfold tail symmetry through interlocking {beta}-strand handshake interactions. Our structures further reveal a compact, threefold-symmetric baseplate established by a distinctive loop-helix module formed by the hub and tube initiator, which bioinformatic and phylogenetic analyses identify as a characteristic architectural feature of the Peduovirus lineage. We also propose a rope-like arrangement that anchors the tail fibre to the baseplate triplex component gpI and obtain evidence that the tape-measure protein adopts a C3 arrangement within the tail lumen, while computational predictions suggest a possible hexameric, pore-like organization after release. Comparison of pre- and post-contraction particles, supported by in situ imaging of infection, suggests that sheath contraction destabilizes the baseplate, promoting its disassembly. The inner tail tube appears to penetrate the host outer membrane but does not appear to reach the inner membrane, leading us to propose that the reorganized tape-measure protein forms a secondary conduit for genome translocation. These findings support a distinct model of contractile tail activation and expand the known architectural diversity of viral injection systems.

microbiology↗

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↗

bioRxiv2025

Structural characterization of an extracellular contractile injection system from Photorhabdus luminescens in extended and contracted states.

Contractile injection systems (CISs) are phage-tail-like nanosyringes that mediate bacterial interactions by puncturing target cell membranes. Within these systems, Photorhabdus Virulence Cassettes (PVCs) can translocate toxins across eukaryotic target cell membranes. The structure of a PVC has been described at atomic level and engineered to deliver diverse protein cargoes into non-natively-targeted organisms. Despite the structural insights into several CISs, information on PVCs from other species and details on the contraction mechanism remain limited. Here, we present the single-particle cryo-electron microscopy structure of PlPVC1, a PVC from the nematode symbiont and insect pathogen Photorhabdus luminescens DJC, in both extended and contracted states. Our structure displays distinct structural features that differ from other CISs, such as a cage surrounding the central spike, a larger sheath adaptor, and a plug exposed to the tube lumen. Moreover, we present the structures of the PlPVC1 fiber as well as the baseplate of the contracted particle, yielding insight into the contraction mechanism. This study provides structural details of the contracted state of the PlPVC1 particle and supports the model in which contraction is triggered. Furthermore, it facilitates the comparison of PlPVC1 with other contractile systems and expands the scope of engineering opportunities for future biomedical and biotechnological applications.

molecular biology↗

bioRxiv2024

Towards a complete phage tail fiber structure atlas.

Bacteriophages use receptor-binding proteins (RBPs) to adhere to bacterial hosts. Understanding the structure of these RBPs can provide insights into their target interactions. Tail fibers, a prominent type of RBP, are typically elongated, flexible, and trimeric proteins, making it challenging to obtain high-resolution experimental data of their full-length structures. Recent advancements in deep learning-based protein structure prediction, such as AlphaFold2-multimer (AF2M) and ESMfold, allow for the generation of high-confidence predicted models of complete tail fibers. In this paper, we introduce RBPseg, a method that combines monomeric ESMfold predictions with a novel sigmoid distance pair (sDp) protein segmentation technique. This method segments the tail fiber sequences into smaller fractions, preserving domain boundaries. These segments are then predicted in parallel using AF2M and assembled into a full fiber model. We demonstrate that RBPseg significantly improves AF2M v2.3.2 in terms of model confidence, running time, and memory usage. To validate our approach, we used single-particle cryo-electron microscopy to analyze five tail fibers from three phages of the BASEL collection. Additionally, we conducted a structural classification of 67 fibers and their domains, which identified 16 well-defined tail fiber classes and 89 domains. Our findings suggest the existence of modular fibers as well as fibers with different sequences and shared structure, indicating possible sequence convergence, divergence, and domain swapping. We further demonstrate that these structural classes account for at least 24% of the known tail fiber universe.

microbiology↗

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↗

bioRxiv2020

Structure and function of stator units of the bacterial flagellar motor

Many bacteria use the flagellum for locomotion and chemotaxis. Its bi-directional rotation is driven by the membrane-embedded motor, which uses energy from the transmembrane ion gradient to generate torque at the interface between stator units and rotor. The structural organization of the stator unit (MotAB), its conformational changes upon ion transport and how these changes power rotation of the flagellum, remain unknown. Here we present ~3 [A]-resolution cryo-electron microscopy reconstructions of the stator unit in different functional states. We show that the stator unit consists of a dimer of MotB surrounded by a pentamer of MotA. Combining structural data with mutagenesis and functional studies, we identify key residues involved in torque generation and present a mechanistic model for motor function and switching of rotational direction. One Sentence SummaryStructural basis of torque generation in the bidirectional bacterial flagellar motor

biophysics↗