Biology subjects

Ladant, D.

Publications and source records attributed to Ladant, D..

11 recordsLinked to original sources

bioRxiv2026

Covalently linked peptides and membrane potential enable CyaA segment translocation

The adenylate cyclase toxin (CyaA) from Bordetella pertussis intoxicates host cells by directly translocating its N-terminal catalytic domain across the plasma membrane; however, the forces driving this unique process remain poorly defined. Here, we dissect the membrane translocation mechanisms of two peptide segments derived from CyaA: P233 and P454 from the catalytic domain and the translocation region, respectively. Both P454 and P233 are calmodulin-binding segments that are sequentially involved in the translocation and activation of the catalytic domain. Using a newly developed Droplet Interface Bilayer (DIB) approach, called DIB-Pipette, which enables direct visualization of peptide transport under controlled membrane potentials, we show that P454 translocates across membranes independently of membrane potential, whereas P233 translocation requires a negative electric membrane potential. Strikingly, covalent coupling of P233 and P454 enables efficient translocation of the resulting peptide even in the absence of a membrane potential. Together, these results suggest that two distinct membrane-active segments within CyaA act cooperatively to promote translocation at the peptide level, revealing an intrinsic mechanism that may contribute to membrane potential-dependent translocation. These findings provide new mechanistic insights into CyaA cell intoxication process and reveal a multifunctional strategy for protein delivery across membranes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/716334v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@750ab3org.highwire.dtl.DTLVardef@11a980org.highwire.dtl.DTLVardef@18f2b27org.highwire.dtl.DTLVardef@5a3a59_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2026

Structure-guided generative design of peptides targeting the FtsQBL divisome complex inhibit Escherichia coli cell division.

The discovery of antibiotics targeting Gram-negative bacteria remains limited in part by the difficulty of pharmacologically modulating protein-protein interactions essential for bacterial physiology. The divisome complex formed by FtsQ, FtsB and FtsL represents an attractive but challenging target, as its assembly relies on {beta}-strand-mediated interface interactions within the bacterial periplasm. Here, we combined interpretable interface mapping using InDeep with hotspot-constrained RFdiffusion design to generate peptides targeting the FtsB-binding site of Escherichia coli FtsQ. The designed peptides mimic the native {beta}-augmentation interaction and selectively engage the FtsQ interface both in bacterial cells and in vitro. X-ray crystallography of one of these peptides in complex with FtsQ reveals that it accurately adopts the native binding geometry while introducing additional stabilizing interactions within a hydrophobic pocket. Complementary NMR analyses further show that optimized peptides adopt pre-organized {beta}-hairpin conformations in solution consistent with the bound state. Several of these peptides disrupt bacterial cell division and inhibit growth in an E. coli strain exhibiting increased outer membrane permeability. Together, these results establish a structurally validated framework in which predictive interface analysis and generative design can be combined to target cooperative protein-protein interfaces in bacteria and provide a foundation for the development of divisome-targeting antibacterial strategies.

microbiology↗

bioRxiv2025

Post-translational acylation drives folding and activity of the CyaA bacterial toxin.

Post-translational modifications critically shape protein conformation and function, yet how they regulate bacterial toxins remains elusive. The adenylate cyclase (CyaA) toxin is a major virulence factor of Bordetella pertussis, the causative agent of whooping cough. CyaA is produced as an inactive precursor, proCyaA, which is activated by acylation of two lysine residues within the bacterium. Once acylated and secreted, CyaA invades innate immune cells and disrupts their phagocytic functions. High-resolution structural characterization of CyaA has remained elusive due to its size, multi-domain organization, flexibility, and aggregation propensity. Here, we overcome these challenges and generate the first structural ensembles of both non-acylated and acylated CyaA in solution by combining experimental data with integrative modeling. Coarse-grained molecular dynamics simulations reveal that acylation is critical to stabilize the native fold and to favorably orient CyaA on the target membrane. Overall, our findings reveal how post-translational acylation triggers native folding and provide mechanistic insights into the early steps of host cell intoxication.

biophysics↗

bioRxiv2025

Interplay between T3SS effectors, ExoY activation, and cGMP signaling in Pseudomonas aeruginosa infection

ExoY is a nucleotidyl cyclase, secreted by the Pseudomonas aeruginosa type 3 secretion system (T3SS), which generates various cyclic nucleotides within host cells, predominantly cGMP. How ExoY-catalyzed cNMPs production contributes to the infection process remains unclear. Using recombinant P. aeruginosa strains expressing ExoY variants with different substrate specificities, we identified that ExoY-derived cGMP modulates the cytotoxicity of ExoT, a co-injected T3SS exotoxin. Our results demonstrated that dephosphorylation of the host CrkII adaptor protein, a downstream effect of the ADP-ribosyltransferase activity of ExoT, is limited by ExoY-derived cGMP, therefore antagonizing ExoT-induced cell retraction. We also revealed a reciprocal interaction between T3SS effectors, showing that ExoT and ExoS, can in turn, suppress ExoY activity and its production of cGMP in certain cell types, thereby limiting their regulation by ExoY-synthesized cGMP. These findings highlight the intricate interplay between T3SS effectors and uncover a novel host cell-dependent regulation within P. aeruginosa infections.

microbiology↗

bioRxiv2024

In vivo detection of protein-protein interactions with single molecule resolution

Protein-protein interactions are central in all biological processes. Methods capable of detecting interactions within living, intact cells have been particularly useful to identify and characterize protein interaction networks. We describe here an exquisitely sensitive regulatory circuit that can detect in bacteria, protein-protein interaction with single molecule sensitivity. This approach involves the interaction-mediated reconstitution of a cyclic AMP signaling cascade in Escherichia coli taking advantage of the high catalytic activity of the adenylate cyclase (AC) from Bordetella pertussis upon activation by its natural activator, calmodulin (CaM). We show that a single complex of interacting hybrid proteins per cell is enough to confer a selectable trait to the host. This exquisitely sensitive adenylate cyclase hybrid (ESACH) system allows for direct in vivo selection of ligands exhibiting high affinity for given targets or for studying interactions involving toxic proteins. The extreme sensitivity of the AC/CaM/cAMP signaling cascade may thus be harnessed to interrogate biological processes with single molecule resolution in live bacteria and could be exploited to design novel synthetic regulatory networks operating at, or even below, the theoretical threshold limit of one molecule per cell.

microbiology↗

bioRxiv2023

Functional and structural insights into the interaction and multi-step activation of bacterial ExoY nucleotidyl cyclases toxins by profilin-bound G-actin

ExoY virulence factors are members of a family of bacterial nucleotidyl cyclases (NCs) that are activated by specific eukaryotic cofactors and overproduce cyclic purine and pyrimidine nucleotides in host cells. ExoYs are actin-activated NC toxins. Here, we investigate the Vibrio nigripulchritudo Multifunctional-Autoprocessing Repeats-in-ToXin (MARTX) ExoY effector domain (Vn-ExoY) as a model for ExoY-type members that interact with monomeric (G-actin) rather than filamentous (F-actin) actin. Vn-ExoY binds with only modest affinity to free or profilin-bound G-actin, but can capture the G-actin:profilin complex for its own activation by preventing the spontaneous or VASP- or formin-mediated assembly of G-actin:profilin at the barbed ends of F-actin in vitro. This may prolong the lifetime of the cofactor-bound state of Vn-ExoY at sites of active actin cytoskeleton remodelling. A series of high-resolution crystal structures of nucleotide-free, 3-deoxy-ATP- or 3-deoxy-CTP-bound Vn-ExoY, activated by free or profilin-bound G-actin-ATP/-ADP show that the cofactor only partially stabilises the nucleotide-binding pocket (NBP) of all NC toxins. Substrate binding promotes a large, previously-unidentified, closure of their NBP. This confines catalytically important residues of the NC toxins and metal cofactors around the substrate and promotes the recruitment of two metal ions to tightly coordinate the triphosphate moiety of purine or pyrimidine nucleotide substrates. Residues that play an important role in both the purinyl and pyrimidinyl cyclase activity of NC toxins are validated in Vn-ExoY and the distantly-related ExoY from Pseudomonas aeruginosa that interact with F-actin. The data conclusively demonstrate that NC toxins employ a similar two-metal-ion mechanism for catalysing the cyclisation reaction of nucleotides of different sizes. These structural insights into the dynamics of the actin-binding interface of actin-activated ExoYs and the multi-step activation of all NC toxins open up new perspectives for identifying ways to specifically inhibit these bacterial NC enzymes. Author SummaryExoY toxins belong to a family of bacterial nucleotidyl cyclases (NCs) that are injected into eukaryotic cells and bind to specific host cofactors to trigger their toxic, potent NC enzymatic activity. They alter host cell signalling by overproducing purine and pyrimidine cyclic nucleotides, which act as canonical and non-canonical intracellular messengers, respectively. The molecular and mechanistic details underlying the activation and catalytic specificities of NC toxins are only partially understood. Here, we investigate ExoY-type members that are unable to interact with actin filaments for their activation. We show in vitro that such ExoYs capture the actin:profilin complex for activation by disrupting its association with the most dynamic ends of actin filaments. We have captured several structural snapshots along the Vn-ExoY activation pathway by G-actin or G-actin-profilin without or with purine or pyrimidine nucleotide analogues. Our structural data reveal unprecedented mechanistic details of how the active site of all NC toxins is sequentially remodelled by cofactor and substrate binding, how they can accommodate nucleotides of different sizes as substrates, and elucidate important features of their catalytic reaction. These structural insights into the multi-step activation of NC toxins provide new perspectives for identifying ways to specifically inhibit this class of NC enzymes.

biophysics↗

bioRxiv2022

B2LiVe, a label-free 1D-NMR method to quantify the binding of amphitropic peptides or proteins to membrane vesicles

Amphitropic proteins and peptides reversibly partition from solution to membrane, a key process that regulates their functions. Experimental approaches, such as fluorescence and circular dichroism, are classically used to measure the partitioning of amphitropic peptides and proteins into lipid bilayers, yet hardly usable when the peptides or proteins do not exhibit significant polarity and/or conformational changes upon membrane binding. Here, we describe B2LiVe (i.e., Binding to Lipid Vesicles), a simple, robust, and widely applicable NMR method to determine the solution-to-membrane partitioning of unlabeled proteins or peptides. The experimental strategy proposed here relies on previously described proton 1D NMR fast pulsing techniques with selective adiabatic pulses. Membrane partitioning induces a large line broadening leading to a progressive loss of protein signals, and therefore, the decrease of the NMR signal directly measures the fraction of membrane-bound protein. The B2LiVe method uses low polypeptide concentrations and has been validated on several membrane-interacting peptides and proteins, ranging from 3 to 54 kDa, with membrane vesicles of different sizes and various lipid compositions. MotivationCharacterization of the interaction of peptides and proteins with lipid membranes is involved in various biological processes and is often challenging for polypeptides which do not possess intrinsic fluorophores, do not exhibit significant structural content changes, as well as for those characterized by low affinities for membranes. To meet these challenges, we have developed a simple and robust label-free NMR-based experimental approach, named B2LiVe, to measure the binding of polypeptides to lipid vesicles. The experimental strategy relies on previously described proton 1D NMR fast pulsing techniques with selective adiabatic pulses to excite the amide resonances. B2LiVe is a label-free method based on the observation of amide hydrogen nuclei which are naturally present in all protein and peptide backbones. Our results validate the B2LiVe method and indicate that it compares well with established technics to quantify polypeptide-membrane interactions. Overall, B2LiVe should efficiently complement the arsenal of label-free biophysical assays available to characterize protein-membrane interactions. In briefWe describe a robust label-free NMR-based experimental approach (B2LiVe) to measure interactions between peptides or proteins with membranes. The validity of this approach has been established on several polypeptides and on various membrane vesicles. The B2LiVe method efficiently complements the arsenal of label-free biophysical techniques to characterize protein-membrane interactions. HighlightsB2LiVe is a simple and robust NMR-based method to quantify affinity of proteins and peptides for membranes B2LiVe is a label-free approach that relies on proton 1D NMR fast pulsing techniques with selective excitation of amide resonances B2LiVe has been validated on several membrane-interacting peptides and proteins B2LiVe coupled to DOSY can pinpoint the presence within a membrane-bound protein of polypeptide segments remaining in solution

biophysics↗

bioRxiv2022

Dynamics and structural changes of calmodulin upon interaction with its potent antagonist calmidazolium

Calmodulin (CaM) is a eukaryotic multifunctional, calcium-modulated protein that regulates the activity of numerous effector proteins involved in a variety of physiological processes. Calmidazolium (CDZ) is a potent small molecule antagonist of CaM and one the most widely used inhibitors of CaM in cell biology. Here, we report the structural characterization of CaM:CDZ complexes using combined SAXS, X-ray crystallography, HDX-MS and NMR approaches. Our results provide molecular insights into the CDZ-induced dynamics and structural changes of CaM leading to its inhibition. CDZ-binding induces an open-to-closed conformational change of CaM and results in a strong stabilization of its structural elements associated with a reduction of protein dynamics over a large time range. These CDZ-triggered CaM changes mimic those induced by CaM-binding peptides derived from protein targets, despite their distant chemical nature. CaM residues in close contact with CDZ and involved in the stabilization of the CaM:CDZ complex have been identified. These results open the way to rationally design new CaM-selective drugs. Figure and text for the Table of Contents (ToC) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/474921v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@b0c76org.highwire.dtl.DTLVardef@15f462forg.highwire.dtl.DTLVardef@1f8e57forg.highwire.dtl.DTLVardef@1a33575_HPS_FORMAT_FIGEXP M_FIG C_FIG Calmidazolium is a potent and widely used inhibitor of calmodulin, a major mediator of calcium-signaling in eukaryotic cells. Structural characterization of calmidazolium-binding to calmodulin reveals that it triggers open-to-closed conformational changes similar to those induced by calmodulin-binding peptides derived from enzyme targets. These results open the way to rationally design new and more selective inhibitors of calmodulin.

biophysics↗

bioRxiv2020

A high-affinity calmodulin-binding site in the CyaA toxin translocation domain is essential for invasion into eukaryotic cells

The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells have thus far remained elusive. The adenylate cyclase (CyaA) toxin from Bordetella pertussis displays a unique intoxication pathway in which its catalytic domain is directly translocated across target cell membranes. We have previously identified a translocation region in CyaA that contains a segment, P454 (residues 454-484), exhibiting membrane-active properties related to antimicrobial peptides. Herein, we show that this peptide is able to translocate across membranes and interact with calmodulin. Structural and biophysical analyses have revealed the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrated that these residues play a crucial role in CyaA translocation into target cells. We have also shown that calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. We propose that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of the CyaA polypeptide chain by the CaM:P454 interaction in the cytosol may assist the entry of the N-terminal catalytic domain by converting the stochastic process of protein translocation into an efficient vectorial chain transfer into host cells.

biophysics↗

bioRxiv2020

Direct Visualization of Protein Kinase A Activation on DNA Origami using Fast-Scan Atomic Force Microscopy

DNA origami nanostructures provide a unique platform for the direct observation of protein-protein interactions at the single-molecule level. Here, we used DNA origami in combination with fast-scan atomic force microscopy to observe the activation-induced dissociation of individual protein kinase A (PKA) holoenzymes. The PKA holoenzyme consists of two regulatory (R) and two catalytic (C) subunits. When cAMP binds to the R subunits it causes dissociation of the C subunits from the R subunit dimer and activation of the enzyme. Using a DNA origami platform, we were able to observe the activation of PKA in response to photolysis of caged cAMP. Furthermore, exploiting the potential of DNA origami for precise positioning of biomolecules, we were able to position the catalytic subunit of adenylyl cyclase in close proximity to PKA and to observe the activation of PKA in response to cAMP produced by adenylyl cyclase. We provide tools for the observation of signalling pathways at the single-molecule level and show that the C subunits of PKA dissociate from the holoenzyme but stay within ~10 nm of the R subunit dimer upon activation.

molecular biology↗

bioRxiv2020

Functional and structural consequences of epithelial cell invasion by Bordetella pertussis adenylate cyclase toxin

Bordetella pertussis, the causative agent of whopping cough, produces an adenylate cyclase toxin (CyaA) that plays a key role in the host colonization by targeting innate immune cells which express CD11b/CD18, the cellular receptor of CyaA. CyaA is also able to invade non-phagocytic cells, via a unique entry pathway consisting in a direct translocation of its catalytic domain across the cytoplasmic membrane of the cells. Within the cells, CyaA is activated by calmodulin to produce high levels of cyclic adenosine monophosphate (cAMP) and alter cellular physiology. In this study, we explored the effects of CyaA toxin on the cellular and molecular structure remodeling of A549 alveolar epithelial cells. Using classical imaging techniques, biochemical and functional tests, as well as advanced cell mechanics method, we quantify the structural and functional consequences of the massive increase of intracellular cyclic AMP induced by the toxin: cell shape rounding associated to adhesion weakening process, actin structure remodeling for the cortical and dense components, increase in cytoskeleton stiffness, and inhibition of migration and repair. We also show that, at the low concentrations that may be found in vivo during B. pertussis infection, CyaA impairs the migration and wound healing capacities of the intoxicated alveolar epithelial cells. Our results suggest that the CyaA, beyond its major role in disabling innate immune cells, might also contribute to the local alteration of the epithelial barrier of the respiratory tract, that is an hallmark of pertussis.

cell biology↗