Membrane insertion of α-xenorhabdolysin in near-atomic detail

ABSTRACTα-Xenorhabdolysins (Xax) are α-pore-forming toxins (α-PFT) from pathogenic bacteria that form 1-1.3 MDa large pore complexes to perforate the host cell membrane. PFTs are used by a variety of bacterial pathogens as an offensive or defensive mechanism to attack host cells. Due to the lack of structural information, the molecular mechanism of action of Xax toxins is poorly understood. Here, we report the cryo-EM structure of the XaxAB pore complex from Xenorhabdus nematophila at an average resolution of 4.0 Å and the crystal structures of the soluble monomers of XaxA and XaxB at 2.5 Å and 3.4 Å, respectively. The structures reveal that XaxA and XaxB are built similarly and appear as heterodimers in the 12-15 subunits containing pore. The structure of the XaxAB pore represents therefore the first structure of a bi-component α-PFT. Major conformational changes in XaxB, including the swinging out of an amphipathic helix are responsible for membrane insertion. XaxA acts as an activator and stabilizer for XaxB that forms the actual transmembrane pore. Based on our results, we propose a novel structural model for the mechanism of action of Xax toxins.

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Membrane insertion of α-xenorhabdolysin in near-atomic detail

eLife ◽

10.7554/elife.38017 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 7

Author(s):

Evelyn Schubert ◽

Ingrid R Vetter ◽

Daniel Prumbaum ◽

Pawel A Penczek ◽

Stefan Raunser

Keyword(s):

Conformational Changes ◽

Structural Model ◽

Structural Information ◽

Host Cells ◽

Membrane Insertion ◽

Amphipathic Helix ◽

Xenorhabdus Nematophila ◽

Host Cell Membrane ◽

Molecular Mechanism Of Action ◽

Pore Complexes

α-Xenorhabdolysins (Xax) are α-pore-forming toxins (α-PFT) that form 1–1.3 MDa large pore complexes to perforate the host cell membrane. PFTs are used by a variety of bacterial pathogens to attack host cells. Due to the lack of structural information, the molecular mechanism of action of Xax toxins is poorly understood. Here, we report the cryo-EM structure of the XaxAB pore complex from Xenorhabdus nematophila and the crystal structures of the soluble monomers of XaxA and XaxB. The structures reveal that XaxA and XaxB are built similarly and appear as heterodimers in the 12–15 subunits containing pore, classifying XaxAB as bi-component α-PFT. Major conformational changes in XaxB, including the swinging out of an amphipathic helix are responsible for membrane insertion. XaxA acts as an activator and stabilizer for XaxB that forms the actual transmembrane pore. Based on our results, we propose a novel structural model for the mechanism of Xax intoxication.

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Cloning, Expression, Purification, and Oligomeric Characterization of the AopB-C-terminus Domain in T3SS Major Translocator Protein of Aeromonas hydrophila

VNU Journal of Science Natural Sciences and Technology ◽

10.25073/2588-1140/vnunst.5293 ◽

2021 ◽

Vol 37 (3) ◽

Author(s):

Nguyen Van Sang ◽

Nguyen Thi Uyen

Keyword(s):

Aeromonas Hydrophila ◽

Structural Information ◽

Secretion System ◽

Host Cells ◽

Translocator Protein ◽

Dimensional Structure ◽

Soluble Form ◽

Salmonella Spp ◽

Host Cell Membrane ◽

C Terminus

Type three secretion system (T3SS) is found exclusively in gram-negative pathogens such as Yersinia spp., Escherichia coli, Salmonella spp., Shigella spp., Pseudomonas spp., Vibrio parahaemolyticus, and Aeromonas hydrophila. The translocon pore of T3SS comprises major and minor translocator proteins that assemble to provide passage of effectors through the host cell membrane. Major translocator protein AopB from Aeromonas hydrophila plays an important role in translocon pore formation. Despite tremendous efforts, structural information regarding the C-terminus domain of major translocator AopB remains elusive. In this study, the DNA fragment encoding for the C-terminus domain of the AopB major translocator from Aeromonas hydrophila AH-1 was cloned in to pET-M expression vector and expressed in BL21 (DE3) host cells. The recombinant AopB-C-terminus domain was successfully purified using immobilized nickel affinity chromatography as a soluble form. Crosslinking analysis among AopB-C-terminus molecules in solution showed that this domain exists as a mixture of tetramer, trimer, dimer and monomer forms. The three-dimensional structure model of AopB-C-terminus oligomerization was built by SWISS-MODEL and PyMol. The oligomeric model of AopB-C-terminus can be used for structural studies of the AopB-C-terminus domain which can contribute to the elucidation of the structure of the type III secretion system.

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Structural Basis of the Pore-Forming Toxin/Membrane Interaction

Toxins ◽

10.3390/toxins13020128 ◽

2021 ◽

Vol 13 (2) ◽

pp. 128

Author(s):

Yajuan Li ◽

Yuelong Li ◽

Hylemariam Mihiretie Mengist ◽

Cuixiao Shi ◽

Caiying Zhang ◽

...

Keyword(s):

Cell Membrane ◽

Pathogenic Bacteria ◽

Therapeutic Strategies ◽

Host Cells ◽

Cell Membrane Permeability ◽

Structural Basis ◽

Resistant Bacteria ◽

Host Cell Membrane ◽

Target Cell Membrane ◽

Novel Therapeutic Strategies

With the rapid growth of antibiotic-resistant bacteria, it is urgent to develop alternative therapeutic strategies. Pore-forming toxins (PFTs) belong to the largest family of virulence factors of many pathogenic bacteria and constitute the most characterized classes of pore-forming proteins (PFPs). Recent studies revealed the structural basis of several PFTs, both as soluble monomers, and transmembrane oligomers. Upon interacting with host cells, the soluble monomer of bacterial PFTs assembles into transmembrane oligomeric complexes that insert into membranes and affect target cell-membrane permeability, leading to diverse cellular responses and outcomes. Herein we have reviewed the structural basis of pore formation and interaction of PFTs with the host cell membrane, which could add valuable contributions in comprehensive understanding of PFTs and searching for novel therapeutic strategies targeting PFTs and interaction with host receptors in the fight of bacterial antibiotic-resistance.

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Microscale communication between bacterial pathogens and the host epithelium

Genes and Immunity ◽

10.1038/s41435-021-00149-1 ◽

2021 ◽

Author(s):

Ann-Kathrin Mix ◽

Griseldis Goob ◽

Erik Sontowski ◽

Christof R. Hauck

Keyword(s):

Host Cell ◽

Pathogenic Bacteria ◽

Bacterial Pathogens ◽

Infection Process ◽

Bacterial Attachment ◽

Host Cells ◽

Mammalian Host ◽

Transcriptional Responses ◽

Host Cell Membrane ◽

Host Epithelium

AbstractPathogenic bacteria have evolved a variety of highly selective adhesins allowing these microbes to engage specific surface determinants of their eukaryotic host cells. Receptor clustering induced by the multivalent microorganisms will not only anchor the bacteria to the tissue, but will inevitably trigger host cell signaling. It has become clear, that these bacteria-initiated signaling events can be seen as a form of localized communication with host epithelial cells. Such a microscale communication can have immediate consequences in the form of changes in host cell membrane morphology or cytoskeletal organization, but can also lead to transcriptional responses and medium- and long-term alterations in cellular physiology. In this review, we will discuss several examples of this form of microscale communication between bacterial pathogens and mammalian host cells and try to delineate their downstream ramifications in the infection process. Furthermore, we will highlight recent findings that specialized pathogenic bacteria utilize the adhesin-based interaction to diffuse the short-range messenger molecule nitric oxide into the host tissue. While anti-adhesive strategies to disrupt the initial bacterial attachment have not yet translated into medical applications, the ability to interfere with the microscale communication emanating on the host side provides an unconventional approach for preventing infectious diseases.

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Structural transition and antibody binding of Ebola GP and Zika E proteins from pre-fusion to fusion-initiation states

10.1101/285130 ◽

2018 ◽

Author(s):

A Lappala-Vernon ◽

W Nishima ◽

J C Miner ◽

P W Fenimore ◽

W Fischer ◽

...

Keyword(s):

Conformational Changes ◽

Viral Entry ◽

Fusion Proteins ◽

Neutralizing Antibodies ◽

Structural Information ◽

Host Cells ◽

Host Immune System ◽

E Proteins ◽

Disease Etiology ◽

Viral Genomes

AbstractMembrane fusion proteins are responsible for viral entry into host cells– a crucial first step in viral infection. These proteins undergo large conformational changes from pre-fusion to fusion initiation structures, and, despite differences in viral genomes and disease etiology, many fusion proteins are arranged as trimers. Structural information for both pre-fusion and fusion initiation states is critical for understanding virus neutralization by the host immune system. In the case of Ebola glycoprotein (GP) and Zika envelope protein (Zika E), pre-fusion state structures have been identified experimentally, but only partial structures of fusion initiation states have been described. While the fusion initiation structure is in an energetically unfavorable state that is difficult to solve experimentally, the existing structural information combined with computational approaches enabled the modeling of fusion initiation state structures of both proteins. These structural models provide an improved understanding of four different neutralizing antibodies in the prevention of viral host entry.

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Structural basis of SARS-CoV-2 spike protein induced by ACE2

10.1101/2020.05.24.113175 ◽

2020 ◽

Author(s):

Tomer Meirson ◽

David Bomze ◽

Gal Markel

Keyword(s):

Conformational Changes ◽

Viral Entry ◽

Structural Information ◽

Active Form ◽

Intramolecular Interactions ◽

Host Cells ◽

Structural Basis ◽

S Protein ◽

Binding Interface ◽

Recent Emergence

AbstractMotivationThe recent emergence of the novel SARS-coronavirus 2 (SARS-CoV-2) and its international spread pose a global health emergency. The viral spike (S) glycoprotein binds the receptor (angiotensin-converting enzyme 2) ACE2 and promotes SARS-CoV-2 entry into host cells. The trimeric S protein binds the receptor using the distal receptor-binding domain (RBD) causing conformational changes in S protein that allow priming by host cell proteases. Unravelling the dynamic structural features used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal novel therapeutic targets. Using structures determined by X-ray crystallography and cryo-EM, we performed structural analysis and atomic comparisons of the different conformational states adopted by the SARS-CoV-2-RBD.ResultsHere, we determined the key structural components induced by the receptor and characterized their intramolecular interactions. We show that κ-helix (also known as polyproline II) is a predominant structure in the binding interface and in facilitating the conversion to the active form of the S protein. We demonstrate a series of conversions between switch-like κ-helix and β-strand, and conformational variations in a set of short α-helices which affect the proximal hinge region. This conformational changes lead to an alternating pattern in conserved disulfide bond configurations positioned at the hinge, indicating a possible disulfide exchange, an important allosteric switch implicated in viral entry of various viruses, including HIV and murine coronavirus. The structural information presented herein enables us to inspect and understand the important dynamic features of SARS-CoV-2-RBD and propose a novel potential therapeutic strategy to block viral entry. Overall, this study provides guidance for the design and optimization of structure-based intervention strategies that target SARS-CoV-2.

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Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1

Biochemical Journal ◽

10.1042/bcj20190289 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3227-3240 ◽

Cited By ~ 1

Author(s):

Shanshan Wang ◽

Yanxiang Zhao ◽

Long Yi ◽

Minghe Shen ◽

Chao Wang ◽

...

Keyword(s):

Crystal Structures ◽

Conformational Changes ◽

Magnaporthe Oryzae ◽

Structural Information ◽

Complex Structure ◽

Critical Role ◽

Catalytic Process ◽

Structural Basis ◽

Salt Bridges ◽

Terminal Domain

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (β3–β4 loop to α0 helix) and movement of a ‘shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a ‘closed' state compared with its ‘open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

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Novel Toxin-antitoxin System Xn-mazEF from Xenorhabdus nematophi-la: Identification, Characterization and Functional Exploration

Current Computer - Aided Drug Design ◽

10.2174/1573409916666200625135850 ◽

2020 ◽

Vol 16 ◽

Author(s):

Jogendra Singh Nim ◽

Mohit Yadav ◽

Lalit Kumar Gautam ◽

Chaitali Ghosh ◽

Shakti Sahi ◽

...

Keyword(s):

Interaction Analysis ◽

Functional Characterization ◽

Host Cells ◽

System Control ◽

Xenorhabdus Nematophila ◽

Functional Features ◽

Dynamics Simulations ◽

Species Specific ◽

Rna Interaction

Background: Xenorhabdus nematophila maintains species-specific mutual interaction with nematodes of Steinernema genus. Type II Toxin Antitoxin (TA) systems, the mazEF TA system controls stress and programmed cell death in bacteria. Objective: This study elucidates the functional characterization of Xn-mazEF, a mazEF homolog in X. nematophila by computational and in vitro approaches. Methods: 3 D- structural models for Xn-MazE toxin and Xn-MazF antitoxin were generated, validated and characterized for protein - RNA interaction analysis. Further biological and cellular functions of Xn-MazF toxin were also predicted. Molecular dynamics simulations of 50ns for Xn-MazF toxin complexed with nucleic acid units (DU, RU, RC, and RU) were performed. The MazF toxin and complete MazEF operon were endogenously expressed and monitored for the killing of Escherichia coli host cells under arabinose induced tightly regulated system. Results: Upon induction, E. coli expressing toxin showed rapid killing within four hours and attained up to 65% growth inhibition, while the expression of the entire operon did not show significant killing. The observation suggests that the Xn-mazEF TA system control transcriptional regulation in X. nematophila and helps to manage stress or cause toxicity leading to programmed death of cells. Conclusion: The study provides insights into structural and functional features of novel toxin, XnMazF and provides an initial inference on control of X. nematophila growth regulated by TA systems.

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Model Analysis and Optimization of BIM Technology in High-rise Shear Wall Residential Structure

MATEC Web of Conferences ◽

10.1051/matecconf/201926702001 ◽

2019 ◽

Vol 267 ◽

pp. 02001

Author(s):

Liangli Xiao ◽

Yan Liu ◽

Zhuang Du ◽

Zhao Yang ◽

Kai Xu

Keyword(s):

Structural Analysis ◽

Collaborative Design ◽

Structural Model ◽

Structural Information ◽

Shear Wall ◽

Structure Design ◽

Secondary Development ◽

Optimized Design ◽

Analysis Model ◽

High Rise

This study combines specific high-rise shear wall residential projects with the Revit to demonstrate BIM application processes. The use of R-Star CAD may help to realize the link barrier of the building information model and the structural analysis software PKPM. Sequentially, the information supplement of the structural analysis model is completed by extracting the structural information with the Revit secondary development. By the collaborative design platform based on BIM technology, the paper examines the collision check of structural model, conducts collision analysis on other professional models and modifies the design scheme for conflict points. After the statistics of material usage, an optimized design is proposed. The findings of this paper could contribute to provide some reference for the specific application of BIM in structural design and realize the application of BIM technology in the process of building structure design.

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Assessing the Molecular Targets and Mode of Action of Furanone C-30 on Pseudomonas aeruginosa Quorum Sensing

Molecules ◽

10.3390/molecules26061620 ◽

2021 ◽

Vol 26 (6) ◽

pp. 1620

Author(s):

Victor Markus ◽

Karina Golberg ◽

Kerem Teralı ◽

Nazmi Ozer ◽

Esti Kramarsky-Winter ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Quorum Sensing ◽

Conformational Changes ◽

Mode Of Action ◽

Pathogenic Bacteria ◽

Molecular Targets ◽

Resistant Bacteria ◽

Public Healthcare ◽

C 30

Quorum sensing (QS), a sophisticated system of bacterial communication that depends on population density, is employed by many pathogenic bacteria to regulate virulence. In view of the current reality of antibiotic resistance, it is expected that interfering with QS can address bacterial pathogenicity without stimulating the incidence of resistance. Thus, harnessing QS inhibitors has been considered a promising approach to overriding bacterial infections and combating antibiotic resistance that has become a major threat to public healthcare around the globe. Pseudomonas aeruginosa is one of the most frequent multidrug-resistant bacteria that utilize QS to control virulence. Many natural compounds, including furanones, have demonstrated strong inhibitory effects on several pathogens via blocking or attenuating QS. While the natural furanones show no activity against P. aeruginosa, furanone C-30, a brominated derivative of natural furanone compounds, has been reported to be a potent inhibitor of the QS system of the notorious opportunistic pathogen. In the present study, we assess the molecular targets and mode of action of furanone C-30 on P. aeruginosa QS system. Our results suggest that furanone C-30 binds to LasR at the ligand-binding site but fails to establish interactions with the residues crucial for the protein’s productive conformational changes and folding, thus rendering the protein dysfunctional. We also show that furanone C-30 inhibits RhlR, independent of LasR, suggesting a complex mechanism for the agent beyond what is known to date.

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