Biology subjects

Desfosses, A.

Publications and source records attributed to Desfosses, A..

14 recordsLinked to original sources

bioRxiv2026

Distinct atypical chemokine receptor 1 determinants underlie bacterial toxins recognition and pore formation

Atypical chemokine receptor 1 (ACKR1) is one of the most promiscuous receptors in the human chemokine system, engaging structurally diverse chemokines through a conformationally flexible N-terminal tail. This same interface is exploited by pathogens, including Plasmodium vivax and Staphylococcus aureus (SA), via a compact sulfotyrosine code. Among pathogenic proteins recognizing ACKR1, the SA leukocidin pair HlgAB is a notable exception. HlgAB-mediated pore formation is only weakly competed by chemokines, the Duffy binding protein, or antibodies targeting the receptor's N-terminus, leaving open how HlgAB engage ACKR1. Combining structural biology approaches with cell-based assays, we show that HlgA and HlgB engage ACKR1 sulfated N-terminus with distinct affinities and site hierarchies, with a single higher-affinity site for sulfated tyrosine 41 present in HlgA but absent in HlgB. Unexpectedly, this N-terminal engagement is dispensable for pore formation; productive lysis instead requires a separate interaction between the toxins and ACKR1 extracellular vestibule. These results define a two-step recognition mechanism, toxin capture by the sulfotyrosine N-terminus followed by vestibule-dependent pore formation, and extend the view that ACKR1 promiscuity arises from distributed, ligand-specific use of multiple receptor surfaces rather than a single adaptable interface.

biochemistry↗

bioRxiv2026

A histidine switch controls the pH-responsive self-assembly of a helical protein filament

Self-assembling helical protein filaments underlie diverse biological processes, from signaling pathways to cell motility. Encoding tunable self-assembly into the sequences of filamentous proteins remains a major challenge. Here, we discovered that the caspase-9 CARD can natively self-assemble into helical filaments in a pH-regulated manner. We defined the determinants of filament assembly using an integrative structural, biophysical, and computational approach. Using NMR spectroscopy, we found that the protonation of a single histidine residue near an N-terminal helix dipole, H38, regulates the pH-dependent self-assembly process. Charge-altering mutations at this site tune thermodynamic stability and filament self-assembly across solution and pH conditions. We solved 3.3- and 3.5-[A] cryo-EM structures of the wild-type and H38R filaments, respectively, which show H38 positioned directly at a filament interface. Molecular dynamics simulations show that H38 functions as a molecular switch, whereby protonation rotates its positively charged side-chain toward solvent and away from the partial-positive charge at the N-terminal helix dipole. This reflects a fine balance between stabilizing intermolecular association and a destabilizing intramolecular electrostatic clash at the helix dipole. More broadly, across 350 helix-containing protein domains we identified electrostatic contributions to protein stability near helix dipoles by integrating AlphaFold2 predictions with deep mutational scanning data. Together, our results identify a native, pH-sensitive histidine switch that regulates a self-assembling helical protein filament. Our results establish a mechanism by which charge-altering mutations near helical N-termini can be engineered to control side-chain rotamers, protein stability, and self-assembly.

biophysics↗

bioRxiv2026

Structural basis of Arf1-driven membrane tubulation

Membrane tubules form at Golgi compartments to facilitate membrane and cargo flow in intracellular trafficking. Here we show that the small GTPase Arf1, an inducer of membrane curvature and key regulator of trafficking, is able to form strongly curved tubules in the presence of lipids and GTP{gamma}S in vitro without the need for further coat components. Using cryo-electron microscopy, we determined the structures of tubular Arf1-scaffolds with diameters of 195 and 215 [A] at 3.1 and 3.8 [A] resolutions, respectively. The nucleotide-bound globular domains of Arf1 form polar helical lattices (i.e. directional assemblies with distinct start/finish orientations), with conserved interfaces and a consistent back-to-face orientation along the filaments. The rigid coat is tethered to the membrane by a flexible linker and anchored by an amphipathic helix (AH) that is free to diffuse and make space within the leaflet, allowing for accommodation of transmembrane cargo. The diversity of tubular diameters observed would allow various cargo sizes to be accommodated in the lumen, while maintaining the local coat architecture. Apart from serving as tubular transport intermediates, Arf1-scaffolds may also play a role at the neck of COPI vesicle on the route to scission.

biophysics↗

bioRxiv2026

A single methylation site regulates HRSV nucleocapsid architecture and replication

The nucleoprotein N of the human respiratory syncytial virus (HRSV) encases the viral genome, forming a flexible N-RNA nucleocapsid helix that serves as template for the viral polymerase L. Recent structural analysis revealed a non-canonical helical nucleocapsid arrangement that modulates RNA accessibility, yet its impact on polymerase function remains unknown. Here, we identified symmetric dimethylation of residue R27 of N as a critical modulator of nucleocapsid architecture and viral replication. We also showed that the methylase PRMT5 interacts with N and likely catalyses R27 methylation. Molecular dynamics simulations of RNA-free N dimers indicate that R27 methylation enhances opening and closing of the RNA-binding cavity, whereas a methylation-mimicking R27M substitution favours a closed state. Cryo-electron microscopy reveals a canonical, markedly straight R27M helix with increased rise and pitch. These findings demonstrate that post-translational modifications fine-tune interactions between N protomers, shaping nucleocapsid assembly, structure and dynamics, and thereby controlling HRSV replication.

microbiology↗

bioRxiv2025

Structures of the LPOR-Chlide Complexes Imply the Basis of Membrane Remodeling and a Photocatalytic Mechanism

Flowering plants rely on the photocatalytic enzyme light-dependent protochlorophyllide oxidoreductase (LPOR) to synthesize chlorophyllide (Chlide), a chlorophyll intermediate, and at the same time to remodel lipid membranes into the cubic phase required for chloroplast development. Yet neither the mechanism of this light-driven catalysis nor the structural basis of its membrane remodeling activity is well understood, largely due to the lack of high-resolution structural information. To address these questions, we analyzed Chlide:LPOR:NADPH oligomeric assemblies by cryo-electron microscopy. Eleven maps were obtained, enabling the reconstruction of nine distinct LPOR oligomer models. Most assemblies adopt helical or stacked-ring forms, whereas one displays a segmented string-of-dimers architecture, representing a previously unobserved structural organization. Strings of LPOR dimers induce distinct periodic deformations of the lipid bilayer that stabilize multiple complex architectures. These architectures are maintained by three different inter-string interfaces occurring in various combinations. The plasticity of these interactions suggest that the filaments are capable of twisting and sliding relative to each other. Our highest-resolution map (2.55 [A]) provided a detailed view of the pigment-binding site, revealing a water channel that connects the central magnesium ion of the pigment to the bulk solvent. Unexpectedly, the propionate group of the pigment protrudes out of the porphyrin plane and arches back toward NADPH, ideally positioned for hydride transfer. These structural insights allowed us to propose a novel reaction mechanism for LPOR photocatalysis. Together, our data highlight the critical role of Chlide:LPOR:NADPH complexes in delaying prolamellar body disassembly and point to their possible regulatory function in mature leaves.

biochemistry↗

bioRxiv2025

A histidine switch regulates pH-dependent filament formation by the caspase-9 CARD

The caspase activation and recruitment domain (CARD) mediates protein-protein interactions in apoptotic and inflammatory signaling pathways. In humans, more than 30 proteins contain a CARD, several of which have been reported to polymerize into helical filaments. Here we found that the CARD from the apoptotic protease caspase-9 (C9CARD) self assembles into filaments in vitro at physiological pH and salt concentrations. The C9CARD more readily polymerizes under low-salt or mildly acidic conditions, suggesting a significant role for electrostatic interactions in mediating filament formation. Using NMR spectroscopy, we determined the pKa of the lone histidine residue, H38, which supports a role for histidine protonation in enhancing filament formation. Indeed, mutation of H38 to introduce a positive (H38R) or negative (H38D) charge, or to remove the pH-dependence of the side chain at this site altogether (H38N), dramatically alters the filament propensity of the domain. Using cryo-election microscopy, we determined 3.4- and 3.2-[A] structures of the wild-type and H38R C9CARD filaments, respectively, which provide new insights into the molecular basis of C9CARD polymerization and its pH dependence via H38.

biophysics↗

bioRxiv2025

Expanding the Tubulin Code: TTLL11 Polyglutamylase Drives Elongation of Primary Tubulin Chains

Microtubules (MTs) undergo diverse post-translational modifications that regulate their structural and functional properties. Among these, polyglutamylation - a dominant and conserved modification targeting the unstructured tubulin C-terminal tails - plays a pivotal role in defining the tubulin code. Here, we uncovered a novel mechanism by which tubulin tyrosine ligase-like 11 (TTLL11) expands and diversifies the code. Cryo-electron microscopy revealed a unique bipartite MT recognition strategy wherein TTLL11s binding and catalytic domains engage adjacent MT protofilaments. Biochemical assays identified previously unknown polyglutamylation patterns, showing that TTLL11 directly extends the primary polypeptide chains of - and {beta}-tubulin, challenging the prevailing paradigms emphasizing lateral branching. Moreover, cell-based and in vivo data firmly established a crosstalk between TTLL11-mediated polyglutamylation and other tubulin-modifying processes, notably the detyrosination/tyrosination cycle. This discovery unveils an unrecognized layer of complexity within the tubulin code and offers new insights into the molecular basis of functional specialization of cytoskeleton across diverse cellular contexts.

biochemistry↗

bioRxiv2023

CryoEM architecture of a native stretch-sensitive membrane microdomain

Biological membranes are partitioned into functional zones containing specific lipids and proteins, termed membrane microdomains. Their composition and organization remain controversial owing to a paucity of techniques that can visualize lipids in situ without disrupting their native behavior1,2. The yeast eisosome, a membrane compartment scaffolded by the BAR-domain proteins Pil1 and Lsp1, senses and responds to mechanical stress by flattening and releasing sequestered factors3-7. Here, we isolated native eisosomes as helical filaments of Pil1/Lsp1 lattice bound to plasma membrane lipids and solved their structures by helical reconstruction. We observe remarkable organization within the lipid bilayer density from which we could assign headgroups of PI(4,5)P2 and phosphatidylserine bound to Pil1/Lsp1 and a pattern of membrane voids, signatures of sterols, beneath an amphipathic helix. We verified these assignments using in vitro reconstitutions and molecular dynamics simulations. 3D variability analysis of the native eisosomes revealed a dynamic stretching of the Pil1/Lsp1 lattice that affects functionally important lipid sequestration, supporting a mechanism in which membrane stretching liberates lipids otherwise anchored by the Pil1/Lsp1 coat. Our results provide mechanistic insight into how eisosome BAR-domain proteins create a mechanosensitive membrane microdomain and, more globally, resolve long-standing controversies about the architecture and nature of lipid microdomains.

biophysics↗

bioRxiv2023

The structural mechanism of MCIA complex assembly links mitochondrial redox pathways

The mitochondrial Complex I assembly (MCIA) complex is an essential player in the biogenesis of respiratory Complex I (CI), the multiprotein complex responsible for the initiation of oxidative phosphorylation (OXPHOS). It is not well understood how MCIA facilitates the assembly of CI. Here we report the structural basis of the complex formation between the MCIA subunits ECSIT and ACAD9. ECSIT binding induces a major conformational change in the FAD-binding loop of ACAD9, resulting in efflux of the FAD cofactor and redeployment of ACAD9 from fatty acid {beta}-oxidation (FAO) to CI assembly. We identify an adjacent -helix as a key structural element that specifically enables the CI assembly functionality of ACAD9, distinguishing it from its closely related VLCAD counterpart. Furthermore, we show that ECSIT is phosphorylated in vitro and ex cellulo and provide evidence that phosphorylation downregulates its association with ACAD9. Interestingly, ECSIT has previously been linked to the pathogenesis of Alzheimers disease and here we show that ECSIT phosphorylation in neuronal cells is reduced upon exposure to amyloid-{beta} (A{beta}) oligomers. These findings shed light on the assembly of the MCIA complex and implicate ECSIT as a potential reprogrammer of bioenergetic metabolic pathways that can be altered when mitochondria are affected by A{beta} toxicity, a hallmark of Alzheimers disease.

molecular biology↗

bioRxiv2023

Structural landscape of the Respiratory Syncytial Virus nucleocapsids

Human Respiratory Syncytial Virus (RSV) is a prevalent cause of severe respiratory infections in children and the elderly. The viral genome, enwrapped by the nucleoprotein N into a helical nucleocapsid (NC), is a template for the viral RNA synthesis and a scaffold for the virion assembly. Although the structures of NC filaments representative of the other major families of the Mononegavirales order have been solved, a detailed understanding of the RSV NCs is missing. This cryo-electron microscopy (cryo-EM) analysis highlights the polymorphism of the RSV nucleocapsid-like assemblies. We reveal in particular the non-canonical arrangement of the RSV NC helix, composed of 16 N per asymmetric unit, and the resulting systematic variations in the RNA accessibility. We demonstrate that this unique helical symmetry originates from recurring longitudinal interactions by the C-terminal arm of the RSV N, whose truncation abrogates the inter-turn contacts. We report the cryo-EM structures of the full-length helical NC filaments, double-headed NCs, ring-capped NCs and double-decameric N-RNA rings, as well as those of the alternative assemblies formed by a C-terminally truncated N mutant. In addition, we demonstrate the functional importance of the interface involved in the formation of the double-headed and the ring-capped interactions. We put all these findings in the context of the RSV RNA synthesis machinery and delineate the structural basis for its further investigation.

microbiology↗

bioRxiv2022

Structural basis of CHMP2A-CHMP3 ESCRT-III polymer assembly and membrane cleavage

The endosomal sorting complex required for transport (ESCRT) is a highly conserved protein machinery that drives a divers set of physiological and pathological membrane remodeling processes. However, the structural basis of ESCRT-III polymers stabilizing, constricting and cleaving negatively curved membranes is yet unknown. Here we present cryo electron microscopy structures of membrane-coated CHMP2A-CHMP3 filaments of two different diameters at 3.3 and 3.6 [A] resolution. The structures reveal helical filaments assembled by CHMP2A-CHMP3 heterodimers in the open ESCRT-III conformation, which generates a partially positive charged membrane interaction surface, positions short N-terminal motifs for membrane interaction and the C-terminal VPS4 target sequence towards the tube interior. Inter-filament interactions are electrostatic, which facilitate filament sliding upon VPS4-mediated polymer remodeling. Fluorescence microscopy as well as high speed atomic force microscopy imaging corroborate that CHMP2A-CHMP3 polymers and VPS4 can constrict and cleave narrow membrane tubes, thus acting as a minimal membrane fission machinery.

cell biology↗

bioRxiv2020

Supramolecular assembly of the E. coli LdcI upon acid stress

Pathogenic and commensal bacteria often have to resist the harsh acidity of the host stomach. The inducible lysine decarboxylase LdcI buffers the cytosol and the local extracellular environment to ensure enterobacterial survival at low pH. Here, we investigate the acid-stress response regulation of E. coli LdcI by combining biochemical and biophysical characterisation with negative stain and cryo-electron microscopy, and wide-field and super-resolution fluorescence imaging. Due to deleterious effects of fluorescent protein fusions on native LdcI decamers, we opt for three-dimensional localisation of nanobody-labelled endogenous wild-type LdcI in acid-stressed E. coli cells, and show that it organises into distinct patches at the cell periphery. Consistent with recent hypotheses that in vivo clustering of metabolic enzymes often reflects their polymerisation as a means of stimulus-induced regulation, we show that LdcI assembles into filaments in vitro at physiologically relevant low pH. We solve the structures of these filaments and of the LdcI decamer formed at neutral pH by cryo-electron microscopy, and reveal the molecular determinants of LdcI polymerisation, confirmed by mutational analysis. Finally, we propose a model for LdcI function inside the enterobacterial cell, providing a structural and mechanistic basis for further investigation of the role of its supramolecular organisation in the acid stress response.Significance statement Bacteria possess a sophisticated arsenal of defence mechanisms that allow them to survive in adverse conditions. Adaptation to acid stress and hypoxia is crucial for the enterobacterial transmission in the gastrointestinal tract of their human host. When subjected to low pH, E. coli and many other enterobacteria activate a proton-consuming resistance system based on the acid-stress inducible lysine decarboxylase LdcI. Here we develop generally-applicable tools to uncover the spatial localisation of LdcI inside the cell by super-resolution fluorescence microscopy, and investigate the in vitro supramolecular organisation of this enzyme by cryo-EM. We build on these results to propose a mechanistic model for LdcI function and offer tools for further in vivo investigations.Competing Interest StatementThe authors have declared no competing interest.View Full Text

microbiology↗

bioRxiv2019

Complete structure of the core signalling unit of the E. coli chemosensory array in an optimised minicell strain

Motile bacteria sense chemical gradients with transmembrane receptors organised in supramolecular signalling arrays.1,2 Understanding stimulus detection and transmission at the molecular level requires precise structural characterisation of the array building block known as a core signalling unit (CSU). Here we introduce a novel E. coli strain that forms small minicells possessing extended and highly ordered chemosensory arrays. We provide a three-dimensional (3D) map of a complete CSU at ~16 [A] resolution by cryo-electron tomography (cryo-ET) and subtomogram averaging. This map, combined with previously determined high resolution structures and molecular dynamics simulations, yields an atomistic model of the membrane-bound CSU and enables spatial localisation of its signalling domains. Our work thus offers a solid structural basis for interpretation of existing data and design of new experiments to elucidate signalling mechanisms within the CSU and larger array.

cell biology↗

bioRxiv2017

In vitro assembly of the Rous Sarcoma Virus capsid protein into hexamer tubes under physiological conditions.

During a proteolytically-driven maturation process, the ortho-retroviral capsid protein (CA) assembles to form the convex shell that surrounds the viral genome. In some orthoretroviruses, including Rous Sarcoma Virus (RSV), CA carries a short and hydrophobic spacer peptide (SP) at its C-terminus early in the maturation process, which is progressively removed as maturation proceeds. In this work, we show that RSV CA assembles in vitro at physiological temperatures, forming hexamer tubes that effectively model the mature capsid surface. Tube assembly is strongly influenced by electrostatic effects, and is a nucleated process that remains thermodynamically favored at lower temperatures, but is effectively arrested by the large Gibbs energy barrier associated with nucleation. RSV CA tubes are multi-layered, being formed by nested and concentric tubes of capsid hexamers. However the spacer peptide acts as a layering determinant during tube assembly. If only a minor fraction of CA-SP is present, multi-layered tube formation is blocked, and single-layered tubes predominate. This likely prevents formation of biologically aberrant multi-layered capsids in the virion. The generation of single-layered hexamer tubes facilitated 3D helical image reconstruction from cryo-electron microscopy data, revealing the basic tube architecture.

biochemistry↗