Biology subjects

Deme, J.

Publications and source records attributed to Deme, J..

13 recordsLinked to original sources

bioRxiv2026

Regulated conformational transitions in seipin define a functional ER–lipid droplet interface

Lipid droplets (LDs) are key organelles in cellular lipid homeostasis that form at the endoplasmic reticulum (ER) through a sequence of membrane rearrangements. While seipin emerged as an essential protein complex for LD biogenesis, how seipin-mediated LD formation proceeds beyond the initial step of neutral lipid nucleation remains unknown. Using a combination of in vitro and in-cell cryogenic electron microscopy (cryo-EM), ultrastructural expansion microscopy, molecular simulations and tailored genetic perturbations, we show that the seipin transmembrane domains undergo large-scale conformational rearrangements that define the architecture of the ER-LD interface and enable LD growth. Cryo-EM of purified Xenopus seipin revealed coexistence of two states: a compact "closed" conformation, consistent with early LD biogenesis, and an "open" conformation in which the transmembrane helices splay out laterally. Molecular dynamics simulations indicate that this open state induces local membrane curvature and promotes triacylglycerol accumulation. We identify conserved flexible linkers between the seipin luminal and transmembrane regions that act as mechanical hinges, enabling this conformational transition. We demonstrate that mutations in these hinge regions hinder seipin opening and affect LD formation in yeast and human cells. Analysis of native ER-LD contacts in human cells using light microscopy and cryo-electron tomography confirms that the seipin complex opens to establish stereotypical ~21-nm necks connecting the ER bilayer and LD monolayer. Moreover, we identify the liver-enriched microprotein SMLR1 as an inhibitor of this seipin conformational transition, providing a regulatory mechanism for seipin-dependent lipid storage in a tissue-specific manner. Together, these data establish seipin opening as a key structural rearrangement at the ER-LD interface that is essential for LD biogenesis and growth.

Cell Biology↗

bioRxiv2025

Distinct immunity protein families mediate compartment-specificneutralisation of a bacterial toxin

Staphylococcus aureus utilises a type VII secretion system (T7SS) to secrete antibacterial toxins that target competitor bacteria. EsxX is a T7SS substrate protein secreted by ST398 strains that has an LXG domain at its N-terminus. Here we show that the EsxX C-terminus is a membrane-depolarising toxin with a glycine zipper motif. EsxX is profoundly toxic to bacteria, displaying toxicity from both cytoplasmic and extracellular compartments. A pair of polytopic membrane proteins, ExiCD, protect cells from intoxication by extracellular EsxX. By contrast, a distinct soluble heterodimer, ExiAB, neutralises cytoplasmic EsxX by sequestration of its glycine zipper motif in a binding groove on ExiB. The exiA-exiB gene pair co-occur in staphylococcal genomes with esxX, invoking a model whereby ExiAB protects EsxX-producing cells from self-toxicity prior to EsxX secretion. By contrast ExiCD is encoded by both EsxX producers and in antitoxin islands of competitor strains that do not encode EsxX, consistent with providing immunity against the secreted form of the toxin. This work defines a new class of antibacterial toxin requiring two distinct types of immunity protein which follow different phylogenetic distributions.

microbiology↗

bioRxiv2023

An interbacterial lipase toxin with an unprecedented reverse domain arrangement defines a new class of type VII secretion system effector

The type VII protein secretion system (T7SS) is found in many Gram-positive bacteria and in pathogenic mycobacteria. All T7SS substrate proteins described to date share a common helical domain architecture at the N-terminus that typically interacts with other helical partner proteins, forming a composite signal sequence for targeting to the T7SS. The C-terminal domains are functionally diverse and in Gram-positive bacteria such as Staphylococcus aureus often specify toxic anti-bacterial activity. Here we describe the first example of a new class of T7 substrate, TslA, that has an unexpected reverse domain organisation. TslA is widely found across Bacillota including Staphylococcus, Enterococcus and Listeria. We show that the S. aureus TslA N-terminal domain is a phospholipase A with anti-staphylococcal activity that is neutralised by the immunity lipoprotein TilA. Two small helical partner proteins, TlaA1 and TlaA2 are essential for T7-dependent secretion of TslA and at least one of these interacts with the TslA C-terminal domain to form a helical stack. Cryo-EM analysis of purified TslA complexes indicate that they share structural similarity with canonical T7 substrates. Our findings suggest that the T7SS has the extraordinary feature of recognising a secretion signal present at either end of a substrate.

microbiology↗

bioRxiv2020

Molecular structure of the intact bacterial flagellar basal body

Bacterial flagella self-assemble a strong, multi-component drive shaft that couples rotation in the inner membrane to the microns-long flagellar filament that powers bacterial swimming in viscous fluids. We here present structures of the intact Salmonella flagellar basal body, solved using cryo-electron microscopy to resolutions between 2.2 and 3.7 [A]. The structures reveal molecular details of how 173 protein molecules of 13 different types assemble into a complex spanning two membranes and a cell wall. The helical drive shaft at one end is intricately interwoven with the inner membrane rotor component, and at the other end passes through a molecular bearing that is anchored in the outer membrane via interactions with the lipopolysaccharide. The in situ structure of a protein complex capping the drive shaft provides molecular insight into the assembly process of this molecular machine.

microbiology↗

bioRxiv2020

Nonameric structures of the cytoplasmic domain of FlhA and SctV in the context of the full-length protein

Type three secretion is the mechanism of protein secretion found in bacterial flagella and injectisomes. At its centre is the export apparatus (EA), a complex of five membrane proteins through which secretion substrates pass the inner membrane. While the complex formed by four of the EA proteins has been well characterised structurally, little is known about the structure of the membrane domain of the largest subunit, FlhA in flagella, SctV in injectisomes. Furthermore, the biologically relevant nonameric assembly of FlhA/SctV has been infrequently observed and differences in conformation of the cytoplasmic portion of FlhA/SctV between open and closed states have been suggested to reflect secretion system specific differences. FlhA has been shown to bind to chaperone-substrate complexes in an open state, but in previous assembled ring structures, SctV is in a closed state. Here, we identify FlhA and SctV homologues that can be recombinantly produced in the oligomeric state and study them using cryo-electron microscopy. The structures of the cytoplasmic domains from both FlhA and SctV are in the open state and we observe a conserved interaction between a short stretch of residues at the N-terminus of the cytoplasmic domain, known as FlhAL/SctVL, with a groove on the adjacent protomers cytoplasmic domain, which stabilises the nonameric ring assembly.

microbiology↗

bioRxiv2020

Molecular basis for B. pertussis interference with complement, coagulation, fibrinolytic and contact activation systems: The cryo-EM structure of the Vag8-C1 inhibitor complex

Complement, contact activation, coagulation, and fibrinolysis are serum protein cascades that need strict regulation to maintain human health. Serum glycoprotein, C1-inhibitor (C1-INH) is a key regulator (inhibitor) of serine proteases of all the above-mentioned pathways. Recently, an autotransporter protein, Virulence Associated Gene 8 (Vag8) produced by the whopping cough causing pathogen, Bordetella pertussis has been shown to bind and interfere with C1-INH function. Here we present the structure of Vag8: C1-INH complex determined using cryo-electron microscopy at 3.6 [A] resolution. The structure shows a unique mechanism of C1-INH inhibition not employed by other pathogens where Vag8 sequesters the Reactive Centre Loop of the C1-INH preventing its interaction with the target proteases. ImportanceThe structure 105 kDa protein complex is one of the smallest to be determined using cryo-electron microscopy at high resolution. The mechanism of disrupting C1-INH revealed by the structure is crucial to understand how pathogens by producing a single virulence factor can disturb several homeostasis pathways. Virulence mechanisms such as the one described here assume more importance given the emerging evidence about dysregulation of contact activation, coagulation and fibrinolysis leading to COVID-19 pneumonia.

microbiology↗

bioRxiv2020

Structure of a proton-powered molecular motor that drives protein transport and gliding motility

Ion-driven motors are rare in biology. The archetypes of the three classes identified to date are ATP synthase, the bacterial flagellar motor, and a proton-driven motor that powers gliding motility and protein secretion in Bacteroidetes bacteria. Whilst the molecular mechanism of ATP synthase is now well understood, structural information is lacking for the other two classes of motor. Here we present the structure of the Bacteroidetes gliding motility motor determined by cryo-electron microscopy. The motor is an asymmetric inner membrane protein complex in which the single transmembrane helices of two periplasm-spanning GldM proteins are positioned within a ring of five GldL proteins. Combining mutagenesis and single-molecule tracking, we identify protonatable amino acid residues within the transmembrane domain of the complex that are important for motor function. Our data imply a mechanism in which proton flow leads the periplasm-spanning GldM dimer to rotate with respect to the intra-membrane GldL ring to drive processes at the bacterial outer membrane. This work provides a molecular basis for understanding how the gliding motility motor is able to transduce the energy of the inner membrane protonmotive force across the bacterial cell envelope.

biochemistry↗

bioRxiv2020

Structures of the stator complex that drives rotation of the bacterial flagellum

The bacterial flagellum is the proto-typical protein nanomachine and comprises a rotating helical propeller attached to a membrane-embedded motor complex1. The motor consists of a central rotor surround by stator units that couple ion flow across the cytoplasmic membrane to torque generation. Here we present the structures of stator complexes from multiple bacterial species, allowing interpretation of the extensive body of data on stator mechanism. The structures reveal an unexpected asymmetric A5B2 subunit assembly in which the five A subunits enclose the two B subunits. Comparison to novel structures of other ion-driven motors indicates that this A5B2 architecture is fundamental to bacterial systems that couple energy from ion-flow to generate mechanical work at a distance, and suggests that such events involve rotation in the motor structures.

biochemistry↗

bioRxiv2019

Structure of the bacterial flagellar rotor MS-ring: a minimum inventory/maximum diversity system

The bacterial flagellum is a complex, self-assembling, nanomachine that confers motility on the cell. Despite great variation across species, all flagella are ultimately constructed from a helical propellor attached to a motor embedded in the inner membrane. The motor consists of a series of stator units surrounding a central rotor made up of two ring complexes, the MS-ring and the C-ring. Despite many studies, high resolution structural information is still completely lacking for the MS-ring of the rotor, and proposed mismatches in stoichiometry between the two rings have long provided a source of confusion for the field. We here present structures of the Salmonella MS-ring, revealing an unprecedented level of inter- and intra-chain symmetry variation that provides a structural explanation for the ability of the MS-ring to function as a complex and elegant interface between the two main functions of the flagellum, protein secretion and rotation.

microbiology↗

bioRxiv2019

The flagellar substrate specificity switch protein FlhB assembles onto the extra-membrane export gate to regulate type three secretion

Export of proteins through type three secretion systems (T3SS) is critical for motility and virulence of many major bacterial pathogens. Proteins are transported through an export gate complex consisting of three proteins (FliPQR in flagellar systems, SctRST in virulence systems) that were initially annotated as membrane proteins, but which we have recently shown assemble into an extra-membranous helical assembly. A fourth putative membrane protein (FlhB/SctU) is essential to the export process, and also functions to \"switch\" secretion substrate specificity once the growing hook/needle structures reach their determined length. Here we present the structure of an export gate containing the switch protein from a Vibrio polar flagellar system at 3.2 [A] resolution by cryo-electron microscopy. The structure reveals that the FlhB/SctU further extends the helical export gate assembly with its four putative transmembrane helices adopting an out-of-membrane location, wrapped around the other export gate components at the base of the structure. The unusual topology of the switch protein helices creates a loop that wraps around the bottom of the closed export gate complex. Structure-informed mutagenesis suggests that this loop is critical in gating secretion and we propose that a series of conformational changes in the type 3 secretion system trigger opening of the export gate through the interactions between FlhB/SctU and FliPQR/SctRST.

microbiology↗

bioRxiv2019

A novel inhibitor of complement C5 provides structural insights into activation

The complement system is a crucial part of innate immune defences against invading pathogens. The blood-meal of the tick Rhipicephalus pulchellus lasts for days, and the tick must therefore rely on inhibitors to counter complement activation. We have identified a novel class of inhibitors from tick saliva, the CirpT family, and generated detailed structural data revealing their mechanism of action. We show direct binding of a CirpT to complement C5 and have determined the structure of the C5-CirpT complex by cryo-electron microscopy. This reveals an interaction with the peripheral macro globulin domain 4 (C5_MG4) of C5. To achieve higher resolution detail, the structure of the C5_MG4-CirpT complex was solved by X-ray crystallography (at 2.7 [A]). We thus present the novel fold of the CirpT protein family, and provide detailed mechanistic insights into its inhibitory function. Analysis of the binding interface reveals a novel mechanism of C5 inhibition, and provides information to expand our biological understanding of the activation of C5, and thus the terminal complement pathway.

immunology↗

bioRxiv2019

The structure of an injectisome export gate demonstratesconservation of architecture in the core export gatebetween flagellar and virulence type three secretionsystems

Export of proteins through type three secretion systems (T3SS) is critical for motility and virulence of many major bacterial pathogens. Proteins are exported though a genetically defined export gate complex consisting of three proteins. We have recently shown at 4.2 [A] that the flagellar complex of these three putative membrane proteins (FliPQR in flagellar systems, SctRST in virulence systems) assemble into an extra-membrane helical assembly that likely seeds correct assembly of the rod above. Here we present the structure of an equivalent complex from the more fragile Shigella virulence system at 3.5 [A] by cryo-electron microscopy. This higher resolution structure reveals further detail and confirms the prediction of structural conservation in this core complex. Analysis of particle heterogeneity also reveals details of how the SctS/FliQ subunits sequentially assemble in the complex.

biochemistry↗

bioRxiv2018

Structure of the Core of the Type Three Secretion System ExportApparatus

Export of proteins through type three secretion systems is critical for bacterial motility and virulence of many major bacterial pathogens. Three putative integral membrane proteins (FliP/FliQ/FliR) are suggested to form the core of an export gate in the inner membrane, but their structure, assembly and location within the final nanomachine remain unclear. We here present the structure of this complex at 4.2 [A] by cryo-electron microscopy. None of the subunits adopt canonical integral membrane protein topologies and common helix-turn-helix structural elements allow them to form a helical assembly with 5:4:1 stoichiometry. Fitting of the structure into reconstructions of intact secretion systems localize the export gate as a core component of the periplasmic portion of the machinery, and cross-linking experiments confirm this observation. This study thereby identifies the export gate as a key element of the secretion channel and implies that it primes the helical architecture of the components assembling downstream.\n\nOne Sentence SummaryThe core of the T3SS export gate forms a supra-membrane helical assembly

microbiology↗