Biology subjects

Deme, J. C.

Publications and source records attributed to Deme, J. C..

14 recordsLinked to original sources

bioRxiv2026

Structure of the proton-powered secretion motor at the heart of the bacterial flagellum

Bacterial type III secretion systems export proteins across the inner membrane using the proton-motive force (PMF), but how proton flow is coupled to opening of the export channel is unknownXX,XX. Here we present single-particle cryo-EM structures at 2.5-4.2 [A] resolution of the intact flagellar Export Apparatus from Salmonella enterica, obtained using optimized extraction conditions of the endogenous assembly that retain the complete transmembrane complex. The transmembrane domain of FlhA forms a nonameric funnel-shaped basket beneath the Export Gate, with each subunit harboring a buried pathway of conserved hydrophilic residues spanning the hydrophobic core of the membrane. FlhB is resolved for the first time within the intact gate, revealing helices that thread through the FlhA channel, completely sealing it at rest. A symmetry-free reconstruction captures one FlhA protomer hinged outward at the water-filled cavity, breaking the rotational symmetry. Together, these structures define the architecture of the proton-transducing element of the type III secretion system and suggest a rotary gating mechanism, with parallels to the F0 motor of ATP synthase, in which PMF-driven conformational cycling of FlhA drives regulated opening of the export channel.

microbiology↗

bioRxiv2025

Identification and Characterization of a Small Molecule Ligand for the Huntingtin-HAP40 Complex

Huntingtons disease (HD) is caused by a CAG repeat expansion mutation, giving rise to a polyglutamine expansion in the huntingtin (HTT). However, the explicit molecular functions of HTT and opportunities for direct pharmacological modulation remain incompletely understood. Here, we report the discovery of a small molecule ligand for the full-length HTT protein in complex with its partner, HAP40. Using affinity selection mass spectrometry (AS-MS), we identified a stereoselective binder, whose binding was characterized by surface plasmon resonance, hydrogen-deuterium exchange mass spectrometry, and cryo-electron microscopy at 2.3 [A] resolution. The ligand binds HTT-HAP40 in vitro with single-digit micromolar affinity and one-to-one stoichiometry at a druggable interface previously predicted computationally. In silico studies predicted and experimental analyses confirmed the (R)-enantiomer as the eutomer and initial structure activity relationship was established experimentally. This work details a structurally-validated chemical scaffold and highlights a ligandable pocket which could enable development of chemical probes for probing HTT biology, as well as therapeutics such as degraders and imaging agents for HD.

pharmacology and toxicology↗

bioRxiv2025

Mechanism of phosphoinositide regulation of lysosomal pH via inhibition of CLC-7

AbstractLysosomes process cellular waste and coordinate responses to metabolic challenge. Central to lysosomal homeostasis are phosphoinositide lipids, key signaling molecules which establish organelle identity, regulate membrane dynamics and are tightly linked to the pathophysiology and therapy of lysosomal storage disorders, neurodegeneration, and cancer. Phosphatidylinositol 3,5- bisphosphate (PI(3,5)P2) interacts with multiple lysosomal membrane proteins and plays a critical role in regulating lysosomal pH by directly inhibiting the chloride/proton antiporter ClC-7, though the molecular mechanism of this inhibition remains unclear. Here, using a combination of functional, structural, and computational analysis, we demonstrate that PI(3,5)P2 binding dramatically remodels the structure of ClC-7 by inducing close association between cytosolic and transmembrane domains. Disease-causing mutations show increased transport activity through loss of PI(3,5)P2 binding and subsequent inhibition. Conversely, ClC-7 activation is correlated with dissociation and increased disorder of the cytoplasmic domain along with novel transmembrane domain conformations, revealing a mechanistic link between specific lysosomal lipids, transporter regulation, and the enigmatic basis of the ClC-7 slow gate.

biochemistry↗

bioRxiv2025

Structure and substrate recognition by the Twin-arginine translocation (Tat) pathway core complex

The twin-arginine translocation (Tat) system is a mechanistically unique protein transport pathway moving folded proteins across membranes (1-4). It is found in all domains of life and is essential for plant photosynthesis and bacterial virulence(2, 5-7). A core complex composed of membrane proteins TatA, TatB, and TatC binds substrate proteins and then recruits additional TatA protomers to form the transport site(8-10). Here we present structures of resting and substrate-bound states of the Tat core complex together with biochemical and functional analysis. Substrate proteins associate with the core complex solely through their N-terminal signal peptides with their Tat targeting sequences making specific contacts with TatC and the peptide body clamped by TatB. The core complex contains highly tilted transmembrane helices that drive extreme local membrane thinning. Based on our results we propose a model for the early steps in Tat transport.

biochemistry↗

bioRxiv2025

High-Affinity, Structure-Validated and Selective Macrocyclic Peptide Tools for Chemical Biology Studies of Huntingtin

Huntingtons disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion in the Huntingtin (HTT) gene, with no disease-modifying therapies currently available. The precise molecular function of the HTT protein is unclear, and the lack of selective chemical tools has limited functional studies. We have identified and characterized macrocyclic peptide binders targeting HTT. These binders exhibit low-nanomolar affinity in vitro and engage distinct HTT and HTT-HAP40 interfaces, as revealed by hydrogen-deuterium exchange mass spectrometry and cryo-electron microscopy. Chemoproteomics confirmed selective binding in cell extracts from wildtype but not HTT-null cell lines. HAP40 consistently and stoichiometrically co-purified with HTT across cell lines, including with HTT variants containing different CAG repeat lengths, highlighting the broad presence of the HTT-HAP40 complex. Significance StatementHuntingtin (HTT) is a large, essential protein with conserved roles in development, intracellular trafficking, and protein homeostasis, yet its precise molecular functions remain incompletely defined. Here, we report the first high-affinity, selective, and structurally validated macrocyclic peptide ligands for HTT. These chemical tools bind HTT and its complex with HAP40 across polyglutamine repeat lengths, enabling direct interrogation of HTT structure and function in health and disease contexts. By overcoming longstanding barriers to studying HTT at the molecular level, these ligands open new avenues for discovery in neurobiology, cell biology, and offer opportunities for therapeutic development. This study delivers urgently needed tools to both the Huntingtons disease field and the broader scientific community seeking to understand this elusive and biologically fundamental protein.

biochemistry↗

bioRxiv2025

Structure of the conjugation surface exclusion protein TraT

Conjugal transfer of plasmids between bacteria is a major route for the spread of antimicrobial resistance. Many conjugative plasmids encode exclusion systems that inhibit redundant conjugation. In incompatibility group F (IncF) plasmids surface exclusion is mediated by the outer membrane protein TraT. Here we report the cryoEM structure of the TraT exclusion protein complex from the canonical F plasmid of Escherichia coli. TraT is a hollow homodecamer shaped like a chefs hat. In contrast to most outer membrane proteins, TraT spans the outer membrane using transmembrane helices. We use a novel microscopy-based conjugation assay to probe the effects of directed mutagenesis on TraT. Our analysis provides no support for the idea that TraT has specific interactions with partner proteins. Instead, we infer that TraT is most likely to function by physical interference with conjugation.

microbiology↗

bioRxiv2025

A central, shared role for disulfide bonds in Bacteroidota protein transport and gliding motility

Bacteria of the phylum Bacteroidota are major human commensals and pathogens in addition to being abundant members of the wider biosphere. Bacteroidota move by gliding and they export proteins using the Type 9 Secretion System (T9SS). Here we discover that gliding motility and the T9SS share a novel mechanism of energisation in which outer membrane proteins are covalently attached by disulfide bonds to a moving internal track structure which propels them through the membrane. We determined the structure of a representative Bacteriodota mobile track by obtaining the cryoEM structure of a 3 MDa circular mini-track from Porphyromonas gingivalis. Our discoveries identify a novel mechanistic and evolutionary link between gliding motility and T9SS-dependent protein transport.

microbiology↗

bioRxiv2025

A new structural paradigm for outer membrane protein biogenesis in the Bacteroidota

In Gram-negative bacteria the outer membrane (OM) is the first line of defence against antimicrobial agents and immunological attacks. A key part of OM biogenesis is insertion of OM proteins (OMPs) by the {beta}-barrel-assembly machinery (BAM). Here we report the cryo-electron microscopy (cryoEM) structure of a BAM complex isolated from Flavobacterium johnsoniae, a member of the phylum Bacteroidota that includes key human commensals and major anaerobic pathogens. This BAM complex is radically different from the canonical Escherichia coli system and includes an extensive extracellular canopy that overhangs the substrate folding site and a subunit that inserts into the BAM pore. We find that two of the novel subunits involved in forming the extracellular canopy are essential for BAM function. For one of these subunits isolation of a suppressor mutation allows the separation of their essential and non-essential functions. The need for a highly remodelled and enhanced BAM complex reflects the unusually complex membrane proteins found in the OM of the Bacteroidota.

microbiology↗

bioRxiv2024

Identification of SLC45A4 as a pain gene encoding a neuronal polyamine transporter.

Polyamines are regulatory metabolites with key roles in transcription, translation, cell signalling and autophagy1. They are implicated in multiple neurological disorders including stroke, epilepsy and neurodegeneration and can regulate neuronal excitability through interactions with ion channels2. Polyamines have been linked to pain showing altered levels in human persistent pain states and modulation of pain behaviour in animal models3. However, the systems governing polyamine transport within the nervous system remain unclear. In undertaking a Genome Wide Association Study (GWAS) of chronic pain intensity in the UK-Biobank we found significant association with variants mapping to the SLC45A4 gene locus. In the mouse nervous system SLC45A4 expression is enriched in all sensory neuron sub-types within the dorsal root ganglion including nociceptors. Cell-based assays show that SLC45A4 is a selective plasma membrane polyamine transporter, whilst the cryo-EM structure reveals a novel regulatory domain and basis for polyamine recognition. Mice lacking SLC45A4 show normal mechanosensitivity but reduced sensitivity to noxious heat and algogen induced tonic pain that is associated with reduced excitability of peptidergic nociceptors. Our findings thus establish a role for neuronal polyamine transport in pain perception and identify a new target for therapeutic intervention in pain treatment.

neuroscience↗

bioRxiv2023

EsxB, EsxC and EsxD facilitate export of the antibacterialnuclease EsaD by the Staphylococcus aureus Type VII secretionsystem

The type VIIb protein secretion system (T7SSb) plays a role in interbacterial competition in Gram-positive Firmicute bacteria and secretes various toxic effector proteins. The mechanism of secretion and the roles of numerous conserved genes within T7SSb gene clusters remain unknown. EsaD is a nuclease toxin secreted by the Staphylococcus aureus T7SSb, which forms a complex with its cognate immunity protein, EsaG, and chaperone EsaE. Encoded upstream of EsaD are three small secreted proteins, EsxB, EsxC and EsxD. Here we show that EsxBCD bind to the transport domain of EsaD and function as EsaD export factors. We report the first structural information for a complete T7SSb substrate pre-secretion complex. Cryo-EM of the EsaDEG trimer and the EsaDEG-EsxBCD hexamer shows that incorporation of EsxBCD confers a conformation comprising a flexible globular cargo domain attached to a long narrow shaft that is likely to be crucial for efficient toxin export.

microbiology↗

bioRxiv2022

Expanding the Huntingtons disease research toolbox; validated huntingtin subdomain constructs for biochemical and structural investigation of the huntingtin protein

Huntingtons disease is characterised by CAG expansion in the huntingtin gene above a critical threshold of ~35 repeats, resulting in polyglutamine expansion of the huntingtin protein (HTT). The biological role of wildtype HTT and the associated mechanisms of disease pathology caused by expanded HTT remain incompletely understood, in part, due to challenges characterising interactions between HTT and putative binding partners. Here we describe a biochemical toolkit of rationally designed, high-quality recombinant HTT subdomains; one spanning the N-terminal HEAT and bridge domains (NTD) and the second spanning the C-terminal HEAT domain (CTD). Using biophysical methods and cryo-electron microscopy, we show these smaller subdomains are natively folded and can associate to reconstitute a functional full-length HTT structure capable of forming a near native-like complex with 40 kDa HTT-associated protein (HAP40). We report biotin-tagged variants of these subdomains, as well as full-length HTT, that permit immobilisation of each protein for quantitative biophysical assays without impacting protein quality. We demonstrate the CTD alone can form a stable complex when co-expressed with HAP40, which can be structurally resolved. The CTD-HAP40 complex binds the NTD, with a dissociation constant of approximately 10 nM as measured by bio-layer interferometry. We validate the interaction between the CTD and HAP40 using a luciferase two-hybrid assay and use subdomain constructs to demonstrate their respective stabilization of HAP40 in cells. These open-source biochemical tools will enable the wider HD community to study fundamental HTT biology, discover new macromolecular or small-molecule binding partners and map interaction sites across this very large protein.

biochemistry↗

bioRxiv2022

Structures of the Type IX Secretion/gliding motility motor from across the phylum Bacteroidetes

Gliding motility using cell surface adhesins and export of proteins by the Type IX Secretion System (T9SS) are two phylum-specific features of the Bacteroidetes. Both of these processes are energized by the GldLM motor complex which transduces the protonmotive force at the inner membrane into mechanical work at the outer membrane. We previously used cryo-electron microscopy to solve the structure of the GldLM motor core from Flavobacterium johnsoniae at 3.9 [A] resolution (Nat Microbiol (2021) 6: 221-233). Here we present structures of homologous complexes from a range of pathogenic and environmental Bacteroidetes species at up to 3.0 [A] resolution. These structures show that the architecture of the GldLM motor core is conserved across the Bacteroidetes phylum although there are species-specific differences at the N-terminus of GldL. The resolution improvements reveal a cage-like structure that ties together the membrane-proximal cytoplasmic region of GldL and influences gliding function. These findings add detail to our structural understanding of bacterial ion-driven motors that drive the T9SS and gliding motility. ImportanceMany bacteria in the Bacteroidetes phylum use the Type IX Secretion System to secrete proteins across their outer membrane. Most of these bacteria can also glide across surfaces using adhesin proteins that are propelled across the cell surface. Both secretion and gliding motility are driven by the GldLM protein complex which forms a nanoscale electrochemical motor. We used cryo-electron microscopy to study the structure of the GldLM protein complex from different species including the human pathogens Porphyromonas gingivalis and Capnocytophaga canimorsus. The organisation of the motor is conserved across species, but we find species-specific structural differences and resolve motor features at higher resolution. This work improves our understanding of the Type IX Secretion System, which is a virulence determinant in human and animal diseases.

biochemistry↗

bioRxiv2021

Molecular basis for redox control by the human cystine/glutamate antiporter System xc-.

Cysteine plays an essential role in cellular redox homeostasis as a key constituent of the tripeptide glutathione (GSH). A rate limiting step in cellular GSH synthesis is the availability of cysteine. However, circulating cysteine exists in the blood as the oxidised di-peptide cystine, requiring specialised transport systems for its import into the cell. System xc- is a dedicated cystine transporter, importing cystine in exchange for intracellular glutamate. To counteract elevated levels of reactive oxygen species in cancerous cells system xc- is frequently upregulated, making it an attractive target for anticancer therapies. However, the molecular basis for ligand recognition remains elusive, hampering efforts to specifically target this transport system. Here we present the cryo-EM structure of system xc- in both the apo and glutamate bound states. Structural comparisons reveal an allosteric mechanism for ligand discrimination, supported by molecular dynamics and cell-based assays, establishing a mechanism for cystine transport in human cells.

biochemistry↗

bioRxiv2021

HAP40 orchestrates huntingtin structure for differential interaction with polyglutamine expanded exon 1

Huntingtons disease results from expansion of a glutamine-coding CAG tract in the huntingtin (HTT) gene, producing an aberrantly functioning form of HTT. Both wildtype and disease-state HTT form a hetero-dimer with HAP40 of unknown functional relevance. We demonstrate in vivo that HTT and HAP40 cellular abundance are coupled. Integrating data from a 2.6 [A] cryo-electron microscopy structure, cross-linking mass spectrometry, small-angle X-ray scattering, and modeling, we provide a near-atomic-level view of HTT, its molecular interaction surfaces and compacted domain architecture, orchestrated by HAP40. Native mass-spectrometry reveals a remarkably stable hetero-dimer, potentially explaining the cellular inter-dependence of HTT and HAP40. The polyglutamine tract containing N-terminal exon 1 region of HTT is dynamic, but shows greater conformational variety in the mutant than wildtype exon 1. By providing novel insight into the structural consequences of HTT polyglutamine expansion, our data provide a foundation for future functional and drug discovery studies targeting Huntingtons disease.

biochemistry↗