Biology subjects

Leger, C.

Publications and source records attributed to Leger, C..

7 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

A conserved isoleucine gates the diffusion of small ligands to the active site of NiFe CO-dehydrogenase

CO dehydrogenases (CODH) are metalloenzymes that reversibly oxidize CO to CO2, at a buried NiFe4S4 active site. The substrates, CO and CO2, need therefore to be transported through the protein matrix to reach the active site. The most likely pathway for intra-protein diffusion is the hydrophobic channel identified in the crystal structures. We used site-directed mutagenesis in an extensive manner to study the role of the highly conserved isoleucine 563 of Thermococcus sp. AM4 CODH2. Certain substitutions significantly change the biochemical properties of the enzyme (KM for CO, catalytic efficiency, product inhibition constant, catalytic bias...), and increase its resistance to the inhibitor O2, showing that isoleucine 563 plays a key role in determining access to the active site. The mutations have the same effects on the rates of binding of CO and O2 showing that the two molecules follow the same pathway and are not discriminated by the protein matrix. The I563F mutation decreases the bimolecular rate constant of inhibition by O2 15-fold, and increases the IC50 20-fold, which is the strongest improvement in O2 resistance reported so far.

biochemistry↗

bioRxiv2026

Correspondence on "Fortification of FeS Clusters Reshapes Anaerobic CO Dehydrogenase into an Air-Viable Enzyme ThroughMultilayered Sealing of O2 Tunnels"

In their recent communication in Angewandte Chemie (10.1002/anie.202508565), Suk Min Kim and coworkers have described the effect of modifying the gas channels of the CO dehydrogenase II from Carboxydothermus hydrogenoformans, an enzyme that oxidizes reversibly CO into CO2. Their goal was to use mutagenesis to slow down the arrival of O2 at the active site. They reported a large increase in the resistance against oxygen, one of the major barriers to the application of this extremely fast and efficient enzyme in biotechnological devices, with an increase in the IC50 of more than two orders of magnitudes for some variants, with only a minor impact on the affinity of the enzyme for CO. We have produced the same variants, and characterized them in depth using Protein Film Electrochemistry. We used an approach that has proven very useful to learn and understand about the reactivity of CO dehydrogenases (and other redox enzymes like hydrogenases) with O2. We found that, contrary to the claims by Kim and coworkers, the A559W and the A559W/V610H mutants are not more resistant than the WT against oxygen.

biochemistry↗

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↗

bioRxiv2023

Catalytic bias and redox-driven inactivation of ancestral FeFe hydrogenases from group B

The biodiversity of hydrogenases, the enzymes that oxidize and produce H2, is only just beginning to be explored. Here we use direct electrochemistry to characterize two enzymes from a subgroup of ancestral FeFe hydrogenases, defined by the presence of three adjacent cysteine residues near the active site: the third FeFe hydrogenase from Clostridium pasteurianum (CpIII) and the second from Megasphaera elsdenii (MeII). To examine the functional role of the unusual TSCCCP motif, which defines the group B2 and is replaced with TSCCP in group A hydrogenases, we also produced a CpIII variant where the supernumerary cysteine is deleted. CpIII and MeII inactivate under oxidative conditions in a manner that is distinct from all other previously characterized hydrogenases from group A. Our results suggest that the supernumerary cysteine allows the previously observed sulfide-independent formation of the Hinact state in these enzymes. We also evidence a second reversible, oxidative inactivation process. Because of their inactivation under oxidative conditions, these enzymes are inefficient H2 oxidation catalysts, but their active site itself is not tuned to make them more active in one particular direction.

biochemistry↗

bioRxiv2022

Gallocin A, an atypical two-peptide bacteriocin secreted by Streptococcus gallolyticus subsp. gallolyticus

Streptococcus gallolyticus subsp. gallolyticus (SGG) is an opportunistic gut pathogen associated with colorectal cancer. We previously showed that colonization of the murine colon by SGG in tumoral conditions was strongly enhanced by the production of gallocin A, a two-peptide bacteriocin. Here, we aimed at characterizing the mechanisms of its action and resistance. Using a genetic approach, we demonstrated that gallocin A is composed of two peptides, GllA1 and GllA2, which are inactive alone and act together to kill "target" bacteria. We showed that gallocin A can kill phylogenetically close relatives. Importantly, we demonstrated that gallocin A peptides can insert into membranes and permeabilize lipid bilayer vesicles. Next, we showed that the third gene of the gallocin A operon named GIP, is necessary and sufficient to confer immunity to gallocin A. Structural modelling of GllA1 and GllA2 mature peptides suggested that both peptides form alpha-helical hairpins stabilized by intramolecular disulfide bridges. The presence of a disulfide bond in GllA1 and GllA2 was confirmed experimentally. Addition of disulfide reducing agents abrogated gallocin A activity. Likewise, deletion of a gene encoding a surface protein with a thioredoxin-like domain impaired gallocin A ability to kill Enterococcus faecalis. Structural modelling of GIP revealed a hairpin-like structure strongly resembling that of the GllA1 and GllA2 mature peptides, suggesting a mechanism of immunity by competition with GllA1/2. Finally, identification of other class IIb bacteriocins exhibiting a similar alpha-helical hairpin fold stabilized with an intramolecular disulfide bridge suggests the existence of a new subclass of class IIb bacteriocins. IMPORTANCEStreptococcus gallolyticus subsp. gallolyticus (SGG), previously named Streptococcus bovis biotype I, is an opportunistic pathogen responsible for invasive infections (septicemia, endocarditis) in elderly people and often associated with asymptomatic colon tumors. SGG is one of the first bacteria to be associated with the occurrence of colorectal cancer in humans. Previously, we showed that tumor-associated conditions in the colon provide to SGG with the ideal environment to proliferate at the expense of phylogenetically and metabolically closely related commensal bacteria such as enterococci (Aymeric et al., 2017). SGG takes advantage of CRC-associated conditions to outcompete and substitute commensal members of the gut microbiota using a specific bacteriocin named gallocin and renamed gallocin A recently following the discovery of gallocin D in a peculiar SGG isolate. Here, we showed that gallocin A is a two-peptide bacteriocin and that both GllA1 and GllA2 peptides are required for antimicrobial activity. Gallocin A was shown to permeabilize bacterial membranes and to kill phylogenetically closely related bacteria such as most streptococci, lactococci and enterococci, probably through membrane pore formation. GllA1 and GllA2 secreted peptides are unusually long (42 and 60 amino acids long) and with very few charged amino acids compared to well-known class IIb bacteriocins. In silico modelling revealed that both GllA1 and GllA2 exhibit a similar hairpin-like conformation stabilized by an intramolecular disulfide bond. We also showed that the GIP immunity peptide also forms a hairpin like structure like GllA1/GllA2. Thus, we hypothesize that GIP blocks the formation of the GllA1/GllA2 complex by interacting with GllA1 or GllA2. Gallocin A may constitute the first class IIb bacteriocin displaying disulfide bridges important for its structure and activity and the founding member of a subtype of class IIb bacteriocins.

microbiology↗

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↗