Search

Bacteria use the type VI secretion system (T6SS) to secrete toxins into pro- and eukaryotic cells via machinery consisting of a contractile sheath and a rigid tube. Rearrangement hotspot (Rhs) proteins represent one of the most common T6SS effectors. The Rhs C-terminal toxin domain displays great functional diversity, while the Rhs core is characterized by YD repeats. We elucidate the Rhs core structures of PAAR- and VgrG-linked Rhs proteins from Salmonella bongori and Advenella mimigardefordensis, respectively. The Rhs core forms a large shell of β-sheets with a negatively charged interior and encloses a large volume. The S. bongori Rhs toxin does not lead to ordered density in the Rhs shell, suggesting the toxin is unfolded. Together with bioinformatics analysis showing that Rhs toxins predominantly act intracellularly, this suggests that the Rhs core functions two-fold, as a safety feature for the producer cell and as delivery mechanism for the toxin., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Ion-driven membrane motors, essential across all domains of life, convert a gradient of ions across a membrane into rotational energy, facilitating diverse biological processes including ATP synthesis, substrate transport, and bacterial locomotion. Herein, we highlight recent structural advances in the understanding of two classes of ion-driven membrane motors: rotary ATPases and 5:2 motors. The recent structure of the human F-type ATP synthase is emphasised along with the gained structural insight into clinically relevant mutations. Furthermore, we highlight the diverse roles of 5:2 motors and recent mechanistic understanding gained through the resolution of ions in the structure of a sodium-driven motor, combining insights into potential unifying mechanisms of ion selectivity and rotational torque generation in the context of their function as part of complex biological systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests, intellectual property, or personal relationships that could have influenced the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Enterotoxigenic Escherichia coli (ETEC) is a diverse and poorly characterized E. coli pathotype that causes diarrhea in humans and animals. Phages have been proposed for the veterinary biocontrol of ETEC, but effective solutions require understanding of porcine ETEC diversity that affects phage infection. Here, we sequenced and analyzed the genomes of the PHAGEBio ETEC collection, gathering 79 diverse ETEC strains isolated from European pigs with post-weaning diarrhea (PWD). We identified the virulence factors characterizing the pathotype and several antibiotic resistance genes on plasmids, while phage resistance genes and other virulence factors were mostly chromosome encoded. We experienced that ETEC strains were highly resistant to Enterobacteriaceae phage infection. It was only by enrichment of numerous diverse samples with different media and conditions, using the 41 ETEC strains of our collection as hosts, that we could isolate two lytic phages that could infect a large part of our diverse ETEC collection: vB_EcoP_ETEP21B and vB_EcoS_ETEP102. Based on genome and host range analyses, we discussed the infection strategies of the two phages and identified components of lipopolysaccharides ( LPS) as receptors for the two phages. Our detailed computational structural analysis highlights several loops and pockets in the tail fibers that may allow recognition and binding of ETEC strains, also in the presence of O-antigens. Despite the importance of receptor recognition, the diversity of the ETEC strains remains a significant challenge for isolating ETEC phages and developing sustainable phage-based products to address ETEC-induced PWD.IMPORTANCEEnterotoxigenic Escherichia coli (ETEC)-induced post-weaning diarrhea is a severe disease in piglets that leads to weight loss and potentially death, with high economic and animal welfare costs worldwide. Phage-based approaches have been proposed, but available data are insufficient to ensure efficacy. Genome analysis of an extensive collection of ETEC strains revealed that phage defense mechanisms were mostly chromosome encoded, suggesting a lower chance of spread and selection by phage exposure. The difficulty in isolating lytic phages and the molecular and structural analyses of two ETEC phages point toward a multifactorial resistance of ETEC to phage infection and the importance of extensive phage screenings specifically against clinically relevant strains. The PHAGEBio ETEC collection and these two phages are valuable tools for the scientific community to expand our knowledge on the most studied, but still enigmatic, bacterial species- E. coli ., Competing Interests: The authors declare no conflict of interest.

Genomic manipulation of Yersinia ruckeri, a pathogen of salmonid fish species, is essential for understanding bacterial physiology and virulence. Here, we present a protocol for genomic recombineering in Y. ruckeri, a species reluctant to standard genomic engineering, using CRISPR Cas12a coupled with the λ Red system. We describe steps for identifying protospacer guides, preparing repair template plasmids, and electroporating Yersinia cells with Cpf1 and protospacer plasmids with homologous arms. We then detail procedures for genome editing and plasmid curing., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Bacteriophages in the Agtrevirus genus are known for expressing multiple tail spike proteins (TSPs), but little is known about their genetic diversity and host recognition apart from their ability to infect diverse Enterobacteriaceae species. Here, we aim to determine the genetic differences that may account for the diverse host ranges of Agrevirus phages. We performed comparative genomics of 14 Agtrevirus and identified only a few genetic differences including genes involved in nucleotide metabolism. Most notably was the diversity of the tsp gene cluster, specifically in the receptor-binding domains that were unique among most of the phages. We further characterized agtrevirus AV101 infecting nine diverse Extended Spectrum β-lactamase (ESBL) Escherichia coli and demonstrated that this phage encoded four unique TSPs among Agtrevirus . Purified TSPs formed translucent zones and inhibited AV101 infection of specific hosts, demonstrating that TSP1, TSP2, TSP3, and TSP4 recognize O8, O82, O153, and O159 O-antigens of E. coli , respectively. BLASTp analysis showed that the receptor-binding domain of TSP1, TSP2, TSP3, and TSP4 are similar to TSPs encoded by E. coli prophages and distant related virulent phages. Thus, Agtrevirus may have gained their receptor-binding domains by recombining with prophages or virulent phages. Overall, combining bioinformatic and biological data expands the understanding of TSP host recognition of Agtrevirus and give new insight into the origin and acquisition of receptor-binding domains of Ackermannviridae phages., Competing Interests: The authors declare no competing interests., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

Multidrug resistance-associated protein 4 (MRP4) is an ATP-binding cassette (ABC) transporter expressed at multiple tissue barriers where it actively extrudes a wide variety of drug compounds. Overexpression of MRP4 provides resistance to clinically used antineoplastic agents, making it a highly attractive therapeutic target for countering multidrug resistance. Here, we report cryo-EM structures of multiple physiologically relevant states of lipid bilayer-embedded human MRP4, including complexes between MRP4 and two widely used chemotherapeutic agents and a complex between MRP4 and its native substrate. The structures display clear similarities and distinct differences in the coordination of these chemically diverse substrates and, in combination with functional and mutational analysis, reveal molecular details of the transport mechanism. Our study provides key insights into the unusually broad substrate specificity of MRP4 and constitutes an important contribution toward a general understanding of multidrug transporters., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Bacteria swim using a flagellar motor that is powered by stator units. Vibrio spp. are highly motile bacteria responsible for various human diseases, the polar flagella of which are exclusively driven by sodium-dependent stator units (PomAB). However, how ion selectivity is attained, how ion transport triggers the directional rotation of the stator unit, and how the stator unit is incorporated into the flagellar rotor remained largely unclear. Here, we have determined by cryo-electron microscopy the structure of Vibrio PomAB. The electrostatic potential map uncovers sodium binding sites, which together with functional experiments and molecular dynamics simulations, reveal a mechanism for ion translocation and selectivity. Bulky hydrophobic residues from PomA prime PomA for clockwise rotation. We propose that a dynamic helical motif in PomA regulates the distance between PomA subunit cytoplasmic domains, stator unit activation, and torque transmission. Together, our study provides mechanistic insights for understanding ion selectivity and rotor incorporation of the stator unit of the bacterial flagellum., (© 2023. The Author(s).)

Several new structures of three types of protein complexes, obtained by cryo-electron microscopy (cryo-EM) and published between 2019 and 2021, identify a new family of natural molecular wheels, the "5:2 rotary motors." These span the cytoplasmic membranes of bacteria, and their rotation is driven by ion flow into the cell. They consist of a pentameric wheel encircling a dimeric axle within the cytoplasmic membrane of both Gram-positive and gram-negative bacteria. The axles extend into the periplasm, and the wheels extend into the cytoplasm. Rotation of these wheels has never been observed directly; it is inferred from the symmetry of the complexes and from the roles they play within the larger systems that they are known to power. In particular, the new structure of the stator complex of the Bacterial Flagellar Motor, MotA 5 B 2 , is consistent with a "wheels within wheels" model of the motor. Other 5:2 rotary motors are believed to share the core rotary function and mechanism, driven by ion-motive force at the cytoplasmic membrane. Their structures diverge in their periplasmic and cytoplasmic parts, reflecting the variety of roles that they perform. This review focuses on the structures of 5:2 rotary motors and their proposed mechanisms and functions. We also discuss molecular rotation in general and its relation to the rotational symmetry of molecular complexes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rieu, Krutyholowa, Taylor and Berry.)

The flagellar stator unit is an oligomeric complex of two membrane proteins (MotA 5 B 2 ) that powers bi-directional rotation of the bacterial flagellum. Harnessing the ion motive force across the cytoplasmic membrane, the stator unit operates as a miniature rotary motor itself to provide torque for rotation of the flagellum. Recent cryo-electron microscopic (cryo-EM) structures of the stator unit provided novel insights into its assembly, function, and subunit stoichiometry, revealing the ion flux pathway and the torque generation mechanism. Furthermore, in situ cryo-electron tomography (cryo-ET) studies revealed unprecedented details of the interactions between stator unit and rotor. In this review, we summarize recent advances in our understanding of the structure and function of the flagellar stator unit, torque generation, and directional switching of the motor., Competing Interests: Declaration of interests None declared by authors., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The human peptide transporter hPEPT1 (SLC15A1) is responsible for uptake of dietary di- and tripeptides and a number of drugs from the small intestine by utilizing the proton electrochemical gradient, and hence an important target for peptide-like drug design and drug delivery. hPEPT1 belongs to the ubiquitous major facilitator superfamily that all contain a 12TM core structure, with global conformational changes occurring during the transport cycle. Several bacterial homologues of these transporters have been characterized, providing valuable insight into the transport mechanism of this family. Here we report the overexpression and purification of recombinant hPEPT1 in a detergent-solubilized state. Thermostability profiling of hPEPT1 at different pH values revealed that hPEPT1 is more stable at pH 6 as compared to pH 7 and 8. Micro-scale thermophoresis (MST) confirmed that the purified hPEPT1 was able to bind di- and tripeptides respectively. To assess the in-solution oligomeric state of hPEPT1, negative stain electron microscopy was performed, demonstrating a predominantly monomeric state., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The thyroglobulin (TG) protein is essential to thyroid hormone synthesis, plays a vital role in the regulation of metabolism, development and growth and serves as intraglandular iodine storage. Its architecture is conserved among vertebrates. Synthesis of triiodothyronine (T 3 ) and thyroxine (T 4 ) hormones depends on the conformation, iodination and post-translational modification of TG. Although structural information is available on recombinant and deglycosylated endogenous human thyroglobulin (hTG) from patients with goiters, the structure of native, fully glycosylated hTG remained unknown. Here, we present the cryo-electron microscopy structure of native and fully glycosylated hTG from healthy thyroid glands to 3.2 Å resolution. The structure provides detailed information on hormonogenic and glycosylation sites. We employ liquid chromatography-mass spectrometry (LC-MS) to validate these findings as well as other post-translational modifications and proteolytic cleavage sites. Our results offer insights into thyroid hormonogenesis of native hTG and provide a fundamental understanding of clinically relevant mutations., (© 2022. The Author(s).)

Endolysins are bacteriophage-encoded peptidoglycan hydrolases targeting the cell wall of host bacteria via their cell wall-binding domains (CBDs). The molecular basis for selective recognition of surface carbohydrate ligands by CBDs remains elusive. Here, we describe, in atomic detail, the interaction between the Listeria phage endolysin domain CBD500 and its cell wall teichoic acid (WTA) ligands. We show that 3' O -acetylated GlcNAc residues integrated into the WTA polymer chain are the key epitope recognized by a CBD binding cavity located at the interface of tandem copies of beta-barrel, pseudo-symmetric SH3b-like repeats. This cavity consists of multiple aromatic residues making extensive interactions with two GlcNAc acetyl groups via hydrogen bonds and van der Waals contacts, while permitting the docking of the diastereomorphic ligands. Our multidisciplinary approach tackled an extremely challenging protein-glycopolymer complex and delineated a previously unknown recognition mechanism by which a phage endolysin specifically recognizes and targets WTA, suggesting an adaptable model for regulation of endolysin specificity., Competing Interests: The authors declare no conflicts of interest., (This journal is © The Royal Society of Chemistry.)

Many bacteria use the flagellum for locomotion and chemotaxis. Its bidirectional rotation is driven by a 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 Å-resolution cryoelectron 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 detailed mechanistic model for motor function and switching of rotational direction., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Nucleotide excision repair (NER) is an essential pathway to remove bulky lesions affecting one strand of DNA. Defects in components of this repair system are at the ground of genetic diseases such as xeroderma pigmentosum (XP) and Cockayne syndrome (CS). The XP complementation group G (XPG) endonuclease cleaves the damaged DNA strand on the 3' side of the lesion coordinated with DNA re-synthesis. Here, we determined crystal structures of the XPG nuclease domain in the absence and presence of DNA. The overall fold exhibits similarities to other flap endonucleases but XPG harbors a dynamic helical arch that is uniquely oriented and defines a gateway. DNA binding through a helix-2-turn-helix motif, assisted by one flanking α-helix on each side, shows high plasticity, which is likely relevant for DNA scanning. A positively-charged canyon defined by the hydrophobic wedge and β-pin motifs provides an additional DNA-binding surface. Mutational analysis identifies helical arch residues that play critical roles in XPG function. A model for XPG participation in NER is proposed. Our structures and biochemical data represent a valuable tool to understand the atomic ground of XP and CS, and constitute a starting point for potential therapeutic applications., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Cryo-electron microscopy (cryo-EM) is a powerful tool for investigating the structure of macromolecules under near-native conditions. Especially in the context of membrane proteins, this technique has allowed researchers to obtain structural information at a previously unattainable level of detail. Specimen preparation remains the bottleneck of most cryo-EM research projects, with membrane proteins representing particularly challenging targets of investigation due to their universal requirement for detergents or other solubilizing agents. Here we describe preparation of negative staining and cryo-EM grids and downstream data collection of membrane proteins in detergent, by far the most common solubilization agent. This protocol outlines a quick and straightforward procedure for screening and determining the structure of a membrane protein of interest under biologically relevant conditions.

Bacteriophages recognize their host cells with the help of tail fiber and tailspike proteins that bind, cleave, or modify certain structures on the cell surface. The spectrum of ligands to which the tail fibers and tailspikes can bind is the primary determinant of the host range. Bacteriophages with multiple tailspike/tail fibers are thought to have a wider host range than their less endowed relatives but the function of these proteins remains poorly understood. Here, we describe the structure, function, and substrate specificity of three tailspike proteins of bacteriophage CBA120-TSP2, TSP3 and TSP4 (orf211 through orf213, respectively). We show that tailspikes TSP2, TSP3 and TSP4 are hydrolases that digest the O157, O77, and O78 Escherichia coli O-antigens, respectively. We demonstrate that recognition of the E. coli O157:H7 host by CBA120 involves binding to and digesting the O157 O-antigen by TSP2. We report the crystal structure of TSP2 in complex with a repeating unit of the O157 O-antigen. We demonstrate that according to the specificity of its tailspikes TSP2, TSP3, and TSP4, CBA120 can infect E. coli O157, O77, and O78, respectively. We also show that CBA120 infects Salmonella enterica serovar Minnesota, and this host range expansion is likely due to the function of TSP1. Finally, we describe the assembly pathway and the architecture of the TSP1-TSP2-TSP3-TSP4 branched complex in CBA120 and its related ViI-like phages., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

ABCG2 is a transporter protein of the ATP-binding-cassette (ABC) family that is expressed in the plasma membrane in cells of various tissues and tissue barriers, including the blood-brain, blood-testis and maternal-fetal barriers 1-4 . Powered by ATP, it translocates endogenous substrates, affects the pharmacokinetics of many drugs and protects against a wide array of xenobiotics, including anti-cancer drugs 5-12 . Previous studies have revealed the architecture of ABCG2 and the structural basis of its inhibition by small molecules and antibodies 13,14 . However, the mechanisms of substrate recognition and ATP-driven transport are unknown. Here we present high-resolution cryo-electron microscopy (cryo-EM) structures of human ABCG2 in a substrate-bound pre-translocation state and an ATP-bound post-translocation state. For both structures, we used a mutant containing a glutamine replacing the catalytic glutamate (ABCG2 EQ ), which resulted in reduced ATPase and transport rates and facilitated conformational trapping for structural studies. In the substrate-bound state, a single molecule of estrone-3-sulfate (E 1 S) is bound in a central, hydrophobic and cytoplasm-facing cavity about halfway across the membrane. Only one molecule of E 1 S can bind in the observed binding mode. In the ATP-bound state, the substrate-binding cavity has collapsed while an external cavity has opened to the extracellular side of the membrane. The ATP-induced conformational changes include rigid-body shifts of the transmembrane domains, pivoting of the nucleotide-binding domains (NBDs), and a change in the relative orientation of the NBD subdomains. Mutagenesis and in vitro characterization of transport and ATPase activities demonstrate the roles of specific residues in substrate recognition, including a leucine residue that forms a 'plug' between the two cavities. Our results show how ABCG2 harnesses the energy of ATP binding to extrude E 1 S and other substrates, and suggest that the size and binding affinity of compounds are important for distinguishing substrates from inhibitors.

ABCG2 is an ATP-binding cassette (ABC) transporter that protects tissues against xenobiotics, affects the pharmacokinetics of drugs and contributes to multidrug resistance. Although many inhibitors and modulators of ABCG2 have been developed, understanding their structure-activity relationship requires high-resolution structural insight. Here, we present cryo-EM structures of human ABCG2 bound to synthetic derivatives of the fumitremorgin C-related inhibitor Ko143 or the multidrug resistance modulator tariquidar. Both compounds are bound to the central, inward-facing cavity of ABCG2, blocking access for substrates and preventing conformational changes required for ATP hydrolysis. The high resolutions allowed for de novo building of the entire transporter and also revealed tightly bound phospholipids and cholesterol interacting with the lipid-exposed surface of the transmembrane domains (TMDs). Extensive chemical modifications of the Ko143 scaffold combined with in vitro functional analyses revealed the details of ABCG2 interactions with this compound family and provide a basis for the design of novel inhibitors and modulators.

Contractile tail bacteriophages, or myobacteriophages, use a sophisticated biomolecular structure to inject their genome into the bacterial host cell. This structure consists of a contractile sheath enveloping a rigid tube that is sharpened by a spike-shaped protein complex at its tip. The spike complex forms the centerpiece of a baseplate complex that terminates the sheath and the tube. The baseplate anchors the tail to the target cell membrane with the help of fibrous proteins emanating from it and triggers contraction of the sheath. The contracting sheath drives the tube with its spiky tip through the target cell membrane. Subsequently, the bacteriophage genome is injected through the tube. The structural transformation of the bacteriophage T4 baseplate upon binding to the host cell has been recently described in near-atomic detail. In this review we discuss structural elements and features of this mechanism that are likely to be conserved in all contractile injection systems (systems evolutionary and structurally related to contractile bacteriophage tails). These include the type VI secretion system (T6SS), which is used by bacteria to transfer effectors into other bacteria and into eukaryotic cells, and tailocins, a large family of contractile bacteriophage tail-like compounds that includes the P. aeruginosa R-type pyocins., (© 2018 John Wiley & Sons Ltd.)

The bacteriophage T4 contractile tail (containing a tube and sheath) was the first biological assembly reconstructed in three dimensions by electron microscopy at a resolution of ∼35 Å in 1968. A single-particle reconstruction of the T4 baseplate was able to generate a 4.1 Å resolution map for the first two rings of the tube using the overall baseplate for alignment. We have now reconstructed the T4 tail tube at a resolution of 3.4 Å, more than a 1,000-fold increase in information content for the tube from 1968. We have used legacy software (Spider) to show that we can do better than the typical 2/3 Nyquist frequency. A reasonable map can be generated with only 1.5 electrons/Å 2 using the higher dose images for alignment, but increasing the dose results in a better map, consistent with other reports that electron dose does not represent the main limitation on resolution in cryo-electron microscopy., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

ABCG2 is a constitutively expressed ATP-binding cassette (ABC) transporter that protects many tissues against xenobiotic molecules. Its activity affects the pharmacokinetics of commonly used drugs and limits the delivery of therapeutics into tumour cells, thus contributing to multidrug resistance. Here we present the structure of human ABCG2 determined by cryo-electron microscopy, providing the first high-resolution insight into a human multidrug transporter. We visualize ABCG2 in complex with two antigen-binding fragments of the human-specific, inhibitory antibody 5D3 that recognizes extracellular loops of the transporter. We observe two cholesterol molecules bound in the multidrug-binding pocket that is located in a central, hydrophobic, inward-facing translocation pathway between the transmembrane domains. Combined with functional in vitro analyses, our results suggest a multidrug recognition and transport mechanism of ABCG2, rationalize disease-causing single nucleotide polymorphisms and the allosteric inhibition by the 5D3 antibody, and provide the structural basis of cholesterol recognition by other G-subfamily ABC transporters.