Genomic characterization of equine influenza A subtype H3N8 viruses by long read sequencing and functional analyses of the PB1-F2 virulence factor of A/equine/Paris/1/2018.

Equine influenza virus (EIV) remains a threat to horses, despite the availability of vaccines. Strategies to monitor the virus and prevent potential vaccine failure revolve around serological assays, RT-qPCR amplification, and sequencing the viral hemagglutinin (HA) and neuraminidase (NA) genes. These approaches overlook the contribution of other viral proteins in driving virulence. This study assesses the potential of long-read nanopore sequencing for fast and precise sequencing of circulating equine influenza viruses. Therefore, two French Florida Clade 1 strains, including the one circulating in winter 2018-2019 exhibiting more pronounced pathogenicity than usual, as well as the two currently OIE-recommended vaccine strains, were sequenced. Our results demonstrated the reliability of this sequencing method in generating accurate sequences. Sequence analysis of HA revealed a subtle antigenic drift in the French EIV strains, with specific substitutions, such as T163I in A/equine/Paris/1/2018 and the N188T mutation in post-2015 strains; both substitutions were in antigenic site B. Antigenic site E exhibited modifications in post-2018 strains, with the N63D substitution. Segment 2 sequencing also revealed that the A/equine/Paris/1/2018 strain encodes a longer variant of the PB1-F2 protein when compared to other Florida clade 1 strains (90 amino acids long versus 81 amino acids long). Further biological and biochemistry assays demonstrated that this PB1-F2 variant has enhanced abilities to abolish the mitochondrial membrane potential ΔΨm and permeabilize synthetic membranes. Altogether, our results highlight the interest in rapidly characterizing the complete genome of circulating strains with next-generation sequencing technologies to adapt vaccines and identify specific virulence markers of EIV.

Dynamics and structural changes of calmodulin upon interaction with the antagonist calmidazolium.

BACKGROUND: Calmodulin (CaM) is an evolutionarily conserved eukaryotic multifunctional protein that functions as the major sensor of intracellular calcium signaling. Its calcium-modulated function regulates the activity of numerous effector proteins involved in a variety of physiological processes in diverse organs, from proliferation and apoptosis, to memory and immune responses. Due to the pleiotropic roles of CaM in normal and pathological cell functions, CaM antagonists are needed for fundamental studies as well as for potential therapeutic applications. Calmidazolium (CDZ) is a potent small molecule antagonist of CaM and one the most widely used inhibitors of CaM in cell biology. Yet, CDZ, as all other CaM antagonists described thus far, also affects additional cellular targets and its lack of selectivity hinders its application for dissecting calcium/CaM signaling. A better understanding of CaM:CDZ interaction is key to design analogs with improved selectivity. Here, we report a molecular characterization of CaM:CDZ complexes using an integrative structural biology approach combining SEC-SAXS, X-ray crystallography, HDX-MS, and NMR. RESULTS: We provide evidence that binding of a single molecule of CDZ induces an open-to-closed conformational reorientation of the two domains 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 physiological protein targets, despite their distinct chemical natures. CaM residues in close contact with CDZ and involved in the stabilization of the CaM:CDZ complex have been identified. CONCLUSION: Our results provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists. 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 provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists.

Dissecting the Structural and Chemical Determinants of the "Open-to-Closed" Motion in the Mannosyltransferase PimA from Mycobacteria.

The phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential peripheral membrane glycosyltransferase that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. PimA undergoes functionally important conformational changes, including (i) α-helix-to-ß-strand and ß-strand-to-α-helix transitions and (ii) an "open-to-closed" motion between the two Rossmann-fold domains, a conformational change that is necessary to generate a catalytically competent active site. In previous work, we established that GDP-Man and GDP stabilize the enzyme and facilitate the switch to a more compact active state. To determine the structural contribution of the mannose ring in such an activation mechanism, we analyzed a series of chemical derivatives, including mannose phosphate (Man-P) and mannose pyrophosphate-ribose (Man-PP-RIB), and additional GDP derivatives, such as pyrophosphate ribose (PP-RIB) and GMP, by the combined use of X-ray crystallography, limited proteolysis, circular dichroism, isothermal titration calorimetry, and small angle X-ray scattering methods. Although the ß-phosphate is present, we found that the mannose ring, covalently attached to neither phosphate (Man-P) nor PP-RIB (Man-PP-RIB), does promote the switch to the active compact form of the enzyme. Therefore, the nucleotide moiety of GDP-Man, and not the sugar ring, facilitates the "open-to-closed" motion, with the ß-phosphate group providing the high-affinity binding to PimA. Altogether, the experimental data contribute to a better understanding of the structural determinants involved in the "open-to-closed" motion not only observed in PimA but also visualized and/or predicted in other glycosyltransfeases. In addition, the experimental data might prove to be useful for the discovery and/or development of PimA and/or glycosyltransferase inhibitors.

Post-translational acylation controls the folding and functions of the CyaA RTX toxin.

The adenylate cyclase (CyaA) toxin is a major virulence factor of Bordetella pertussis, the causative agent of whooping cough. CyaA is synthetized as a pro-toxin, pro-CyaA, and converted into its cytotoxic form upon acylation of two lysines. After secretion, CyaA invades eukaryotic cells and produces cAMP, leading to host defense subversion. To gain further insights into the effect of acylation, we compared the functional and structural properties of pro-CyaA and CyaA proteins. HDX-MS results show that the refolding process of both proteins upon progressive urea removal is initiated by calcium binding to the C-terminal RTX domain. We further identified a critical hydrophobic segment, distal from the acylation region, that folds at higher urea concentration in CyaA than in pro-CyaA. Once refolded into monomers, CyaA is more compact and stable than pro-CyaA, due to a complex set of interactions between domains. Our HDX-MS data provide direct evidence that the presence of acyl chains in CyaA induces a significant stabilization of the apolar segments of the hydrophobic domain and of most of the acylation region. We propose a refolding model dependent on calcium and driven by local and distal acylation-dependent interactions within CyaA. Therefore, CyaA acylation is not only critical for cell intoxication, but also for protein refolding into its active conformation. Our data shed light on the complex relationship between post-translational modifications, structural disorder and protein folding. Coupling calcium-binding and acylation-driven folding is likely pertinent for other repeat-in-toxin cytolysins produced by many Gram-negative bacterial pathogens.-O'Brien, D. P., Cannella, S. E., Voegele, A., Raoux-Barbot, D., Davi, M., Douché, T., Matondo, M., Brier, S., Ladant, D., Chenal, A. Post-translational acylation controls the folding and functions of the CyaA RTX toxin.

Calmodulin fishing with a structurally disordered bait triggers CyaA catalysis.

Once translocated into the cytosol of target cells, the catalytic domain (AC) of the adenylate cyclase toxin (CyaA), a major virulence factor of Bordetella pertussis, is potently activated by binding calmodulin (CaM) to produce supraphysiological levels of cAMP, inducing cell death. Using a combination of small-angle X-ray scattering (SAXS), hydrogen/deuterium exchange mass spectrometry (HDX-MS), and synchrotron radiation circular dichroism (SR-CD), we show that, in the absence of CaM, AC exhibits significant structural disorder, and a 75-residue-long stretch within AC undergoes a disorder-to-order transition upon CaM binding. Beyond this local folding, CaM binding induces long-range allosteric effects that stabilize the distant catalytic site, whilst preserving catalytic loop flexibility. We propose that the high enzymatic activity of AC is due to a tight balance between the CaM-induced decrease of structural flexibility around the catalytic site and the preservation of catalytic loop flexibility, allowing for fast substrate binding and product release. The CaM-induced dampening of AC conformational disorder is likely relevant to other CaM-activated enzymes.

Development of Conformational Antibodies to Detect Bcl-xL's Amyloid Aggregates in Metal-Induced Apoptotic Neuroblastoma Cells.

Bcl-xL, a member of the Bcl-2 family, is a pro-survival protein involved in apoptosis regulation. We have previously reported the ability of Bcl-xL to form various types of fibers, from native to amyloid conformations. Here, we have mimicked the effect of apoptosis-induced caspase activity on Bcl-xL by limited proteolysis using trypsin. We show that cleaved Bcl-xL (ΔN-Bcl-xL) forms fibers that exhibit the features of amyloid structures (BclxLcf37). Moreover, three monoclonal antibodies (mAbs), produced by mouse immunization and directed against ΔN-Bcl-xL or Bcl-xL fibers, were selected and characterized. Our results show that these mAbs specifically target ΔN-Bcl-xL in amyloid fibers in vitro. Upon metal-stress-induced apoptosis, these mAbs are able to detect the presence of Bcl-xL in amyloid aggregates in neuroblastoma SH-SY5Y cell lines. In conclusion, these specific mAbs directed against amyloidogenic conformations of Bcl-xL constitute promising tools for studying, in vitro and in cellulo, the contribution of Bcl-xL in apoptosis. These mAbs may further help in developing new diagnostics and therapies, considering Bcl-xL as a strategic target for treating brain lesions relevant to stroke and neurodegenerative diseases.

Exposure to Bordetella pertussis adenylate cyclase toxin affects integrin-mediated adhesion and mechanics in alveolar epithelial cells.

BACKGROUND INFORMATION: The adenylate cyclase (CyaA) toxin is a major virulent factor of Bordetella pertussis, the causative agent of whooping cough. CyaA toxin is able to invade eukaryotic cells where it produces high levels of cyclic adenosine monophosphate (cAMP) affecting cellular physiology. Whether CyaA toxin can modulate cell matrix adhesion and mechanics of infected cells remains largely unknown. RESULTS: In this study, we use a recently proposed multiple bond force spectroscopy (MFS) with an atomic force microscope to assess the early phase of cell adhesion (maximal detachment and local rupture forces) and cell rigidity (Young's modulus) in alveolar epithelial cells (A549) for toxin exposure <1 h. At 30 min of exposure, CyaA toxin has a minimal effect on cell viability (>95%) at CyaA concentration of 0.5 nM, but a significant effect (≈81%) at 10 nM. MFS performed on A549 for three different concentrations (0.5, 5 and 10 nM) demonstrates that CyaA toxin significantly affects both cell adhesion (detachment forces are decreased) and cell mechanics (Young's modulus is increased). CyaA toxin (at 0.5 nM) assessed at three indentation/retraction speeds (2, 5 and 10 µm/s) significantly affects global detachment forces, local rupture events and Young modulus compared with control conditions, while an enzymatically inactive variant CyaAE5 has no effect. These results reveal the loading rate dependence of the multiple bonds newly formed between the cell and integrin-specific coated probe as well as the individual bond kinetics which are only slightly affected by the patho-physiological dose of CyaA toxin. Finally, theory of multiple bond force rupture enables us to deduce the bond number N which is reduced by a factor of 2 upon CyaA exposure (N ≈ 6 versus N ≈ 12 in control conditions). CONCLUSIONS: MFS measurements demonstrate that adhesion and mechanical properties of A549 are deeply affected by exposure to the CyaA toxin but not to an enzymatically inactive variant. This indicates that the alteration of cell mechanics triggered by CyaA is a consequence of the increase in intracellular cAMP in these target cells. SIGNIFICANCE: These results suggest that mechanical and adhesion properties of the cells appear as pertinent markers of cytotoxicity of CyaA toxin.

SEC-SAXS and HDX-MS: A powerful combination. The case of the calcium-binding domain of a bacterial toxin.

Small-angle X-ray scattering (SAXS) is a relatively simple experimental technique that provides information on the global conformation of macromolecules in solution, be they fully structured, partially, or extensively unfolded. Size exclusion chromatography in line with a SAXS measuring cell considerably improves the monodispersity and ideality of solutions, the two main requirements of a "good" SAXS sample. Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) offers a wealth of information regarding the solvent accessibility at the local (peptide) level. It constitutes a sensitive probe of local flexibility and, more generally, of structural dynamics. The combination of both approaches presented here is very powerful, as illustrated by the case of RD, a calcium-binding protein that is part of a bacterial virulence factor.

Molecular Basis of Membrane Association by the Phosphatidylinositol Mannosyltransferase PimA Enzyme from Mycobacteria.

Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannoside, lipomannan, and lipoarabinomannan, which are key glycolipids/lipoglycans of the mycobacterial cell envelope. PimA belongs to a large family of membrane-associated glycosyltransferases for which the understanding of the molecular mechanism and conformational changes that govern substrate/membrane recognition and catalysis remains a major challenge. Here, we determined that PimA preferentially binds to negatively charged phosphatidyl-myo-inositol substrate and non-substrate membrane model systems (small unilamellar vesicle) through its N-terminal domain, inducing an important structural reorganization of anionic phospholipids. By using a combination of single-point mutagenesis, circular dichroism, and a variety of fluorescence spectroscopy techniques, we determined that this interaction is mainly mediated by an amphipathic α-helix (α2), which undergoes a substantial conformational change and localizes in the vicinity of the negatively charged lipid headgroups and the very first carbon atoms of the acyl chains, at the PimA-phospholipid interface. Interestingly, a flexible region within the N-terminal domain, which undergoes ß-strand-to-α-helix and α-helix-to-ß-strand transitions during catalysis, interacts with anionic phospholipids; however, the effect is markedly less pronounced to that observed for the amphipathic α2, likely reflecting structural plasticity/variability. Altogether, we propose a model in which conformational transitions observed in PimA might reflect a molten globule state that confers to PimA, a higher affinity toward the dynamic and highly fluctuating lipid bilayer.

MEMHDX: an interactive tool to expedite the statistical validation and visualization of large HDX-MS datasets.

MOTIVATION: With the continued improvement of requisite mass spectrometers and UHPLC systems, Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) workflows are rapidly evolving towards the investigation of more challenging biological systems, including large protein complexes and membrane proteins. The analysis of such extensive systems results in very large HDX-MS datasets for which specific analysis tools are required to speed up data validation and interpretation. RESULTS: We introduce a web application and a new R-package named 'MEMHDX' to help users analyze, validate and visualize large HDX-MS datasets. MEMHDX is composed of two elements. A statistical tool aids in the validation of the results by applying a mixed-effects model for each peptide, in each experimental condition, and at each time point, taking into account the time dependency of the HDX reaction and number of independent replicates. Two adjusted P-values are generated per peptide, one for the 'Change in dynamics' and one for the 'Magnitude of ΔD', and are used to classify the data by means of a 'Logit' representation. A user-friendly interface developed with Shiny by RStudio facilitates the use of the package. This interactive tool allows the user to easily and rapidly validate, visualize and compare the relative deuterium incorporation on the amino acid sequence and 3D structure, providing both spatial and temporal information. AVAILABILITY AND IMPLEMENTATION: MEMHDX is freely available as a web tool at the project home page http://memhdx.c3bi.pasteur.fr CONTACT: marie-agnes.dillies@pasteur.fr or sebastien.brier@pasteur.frSupplementary information: Supplementary data is available at Bioinformatics online.

Secondary structure reshuffling modulates glycosyltransferase function at the membrane.

Secondary structure refolding is a key event in biology as it modulates the conformation of many proteins in the cell, generating functional or aberrant states. The crystal structures of mannosyltransferase PimA reveal an exceptional flexibility of the protein along the catalytic cycle, including ß-strand-to-α-helix and α-helix-to-ß-strand transitions. These structural changes modulate catalysis and are promoted by interactions of the protein with anionic phospholipids in the membrane.

The catalytic domains of Clostridium sordellii lethal toxin and related large clostridial glucosylating toxins specifically recognize the negatively charged phospholipids phosphatidylserine and phosphatidic acid.

Clostridium sordellii lethal toxin (TcsL) is a potent virulence factor belonging to the large clostridial glucosylating toxin family. TcsL enters target cells via receptor-mediated endocytosis and delivers the N-terminal catalytic domain (TcsL-cat) into the cytosol upon an autoproteolytic process. TcsL-cat inactivates small GTPases including Rac and Ras by glucosylation with uridine-diphosphate (UDP)-glucose as cofactor leading to drastic changes in cytoskeleton and cell viability. TcsL-cat was found to preferentially bind to phosphatidylserine (PS)-containing membranes and to increase the glucosylation of Rac anchored to lipid membrane. We here report binding affinity measurements of TcsL-cat for brain PS-containing membranes by surface plasmon resonance and enzyme-linked immunosorbent assay (ELISA). In addition, TcsL-cat bound to phosphatidic acid (PA) and, to a lesser extent, to other anionic lipids, but not to neutral lipids, sphingolipids or sterol. We further show that the lipid unsaturation status influenced TcsL-cat binding to phospholipids, PS with unsaturated acyl chains and PA with saturated acyl chains being the preferred bindingsubstrates. Phospholipid binding site is localized at the N-terminal four helical bundle structure (1-93 domain). However, TcsL-1-93 bound to a broad range of substrates, whereas TcsL-cat, which is the active domain physiologically delivered into the cytosol, selectively bound to PS and PA. Similar findings were observed with the other large clostridial glucosylating toxins from C. difficile, C. novyi and C. perfringens.

From bulk to plasmonic nanoparticle surfaces: the behavior of two potent therapeutic peptides, octreotide and pasireotide.

Octreotide and pasireotide are two cyclic somatostatin analogues with an important clinical use in the treatment and diagnosis of neuroendocrine tumors. Herein, by the combined use of several techniques (UV-visible absorption, fluorescence, circular dichroism, ζ-potential, transmission electron microscopy, Raman scattering, surface-enhanced Raman scattering, and quantum mechanical calculations) we have followed the structural dynamics of these analogues in the bulk, as well as their binding sites on plasmonic (gold and silver) colloids. In contrast to the previously derived conclusions, the two peptides seem to possess completely different conformational features. Octreotide, a cyclic octapeptide, is formed by a moderately flexible type-II'ß-turn maintained by a deformable disulfide linkage. Pasireotide, in which the cyclic character is made possible by peptide bonds, manifests a rigid backbone formed by two oppositely placed tight turns of different types, i.e.γ-turn and type-I ß-turn. Owing to their cationic character, both analogues induce aggregation of negatively charged gold and silver colloids. Nevertheless, despite their notable structural differences, both peptides bind onto gold nanoparticles through their unique d-Trp residue. In contrast, their binding to silver colloids seems to be of electrostatic nature, as formed through monodentate or bidentate ionic pairs.

Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer.

Numerous bacterial toxins can cross biological membranes to reach the cytosol of mammalian cells, where they exert their cytotoxic effects. Our model toxin, the adenylate cyclase (CyaA) from Bordetella pertussis, is able to invade eukaryotic cells by translocating its catalytic domain directly across the plasma membrane of target cells. To characterize its original translocation process, we designed an in vitro assay based on a biomimetic membrane model in which a tethered lipid bilayer (tBLM) is assembled on an amine-gold surface derivatized with calmodulin (CaM). The assembled bilayer forms a continuous and protein-impermeable boundary completely separating the underlying calmodulin (trans side) from the medium above (cis side). The binding of CyaA to the tBLM is monitored by surface plasmon resonance (SPR) spectroscopy. CyaA binding to the immobilized CaM, revealed by enzymatic activity, serves as a highly sensitive reporter of toxin translocation across the bilayer. Translocation of the CyaA catalytic domain was found to be strictly dependent on the presence of calcium and also on the application of a negative potential, as shown earlier in eukaryotic cells. Thus, CyaA is able to deliver its catalytic domain across a biological membrane without the need for any eukaryotic components besides CaM. This suggests that the calcium-dependent CyaA translocation may be driven in part by the electrical field across the membrane. This study's in vitro demonstration of toxin translocation across a tBLM provides an opportunity to explore the molecular mechanisms of protein translocation across biological membranes in precisely defined experimental conditions.

Allosteric activation of Bordetella pertussis adenylyl cyclase by calmodulin: molecular dynamics and mutagenesis studies.

Adenylyl cyclase (AC) toxin is an essential toxin that allows Bordetella pertussis to invade eukaryotic cells, where it is activated after binding to calmodulin (CaM). Based on the crystal structure of the AC catalytic domain in complex with the C-terminal half of CaM (C-CaM), our previous molecular dynamics simulations (Selwa, E., Laine, E., and Malliavin, T. (2012) Differential role of calmodulin and calcium ions in the stabilization of the catalytic domain of adenyl cyclase CyaA from Bordetella pertussis. Proteins 80, 1028­1040) suggested that three residues (i.e. Arg(338), Asn(347), and Asp(360)) might be important for stabilizing the AC/CaM interaction. These residues belong to a loop-helix-loop motif at the C-terminal end of AC, which is located at the interface between CaM and the AC catalytic loop. In the present study, we conducted the in silico and in vitro characterization of three AC variants, where one (Asn(347); ACm1A), two (Arg(338) and Asp(360); ACm2A), or three residues (Arg(338), Asn(347), and Asp(360); ACm3A) were substituted with Ala. Biochemical studies showed that the affinities of ACm1A and ACm2A for CaM were not affected significantly, whereas that of ACm3A was reduced dramatically. To understand the effects of these modifications, molecular dynamics simulations were performed based on the modified proteins. The molecular dynamics trajectories recorded for the ACm3AC-CaM complex showed that the calcium-binding loops of C-CaM exhibited large fluctuations, which could be related to the weakened interaction between ACm3A and its activator. Overall, our results suggest that the loop-helix-loop motif at the C-terminal end of AC is crucial during CaM binding for stabilizing the AC catalytic loop in an active configuration.

Calcium, acylation, and molecular confinement favor folding of Bordetella pertussis adenylate cyclase CyaA toxin into a monomeric and cytotoxic form.

The adenylate cyclase (CyaA) toxin, a multidomain protein of 1706 amino acids, is one of the major virulence factors produced by Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic target cells in which it produces high levels of cAMP, thus altering the cellular physiology. Although CyaA has been extensively studied by various cellular and molecular approaches, the structural and functional states of the toxin remain poorly characterized. Indeed, CyaA is a large protein and exhibits a pronounced hydrophobic character, making it prone to aggregation into multimeric forms. As a result, CyaA has usually been extracted and stored in denaturing conditions. Here, we define the experimental conditions allowing CyaA folding into a monomeric and functional species. We found that CyaA forms mainly multimers when refolded by dialysis, dilution, or buffer exchange. However, a significant fraction of monomeric, folded protein could be obtained by exploiting molecular confinement on size exclusion chromatography. Folding of CyaA into a monomeric form was found to be critically dependent upon the presence of calcium and post-translational acylation of the protein. We further show that the monomeric preparation displayed hemolytic and cytotoxic activities suggesting that the monomer is the genuine, physiologically active form of the toxin. We hypothesize that the structural role of the post-translational acylation in CyaA folding may apply to other RTX toxins.

Characterization of a membrane-active peptide from the Bordetella pertussis CyaA toxin.

Bordetella pertussis, the pathogenic bacteria responsible for whooping cough, secretes several virulence factors, among which is the adenylate cyclase toxin (CyaA) that plays a crucial role in the early stages of human respiratory tract colonization. CyaA invades target cells by translocating its catalytic domain directly across the plasma membrane and overproduces cAMP, leading to cell death. The molecular process leading to the translocation of the catalytic domain remains largely unknown. We have previously shown that the catalytic domain per se, AC384, encompassing residues 1-384 of CyaA, did not interact with lipid bilayer, whereas a longer polypeptide, AC489, spanning residues 1-489, binds to membranes and permeabilizes vesicles. Moreover, deletion of residues 375-485 within CyaA abrogated the translocation of the catalytic domain into target cells. Here, we further identified within this region a peptidic segment that exhibits membrane interaction properties. A synthetic peptide, P454, corresponding to this sequence (residues 454-485 of CyaA) was characterized by various biophysical approaches. We found that P454 (i) binds to membranes containing anionic lipids, (ii) adopts an α-helical structure oriented in plane with respect to the lipid bilayer, and (iii) permeabilizes vesicles. We propose that the region encompassing the helix 454-485 of CyaA may insert into target cell membrane and induce a local destabilization of the lipid bilayer, thus favoring the translocation of the catalytic domain across the plasma membrane.

Identification of a region that assists membrane insertion and translocation of the catalytic domain of Bordetella pertussis CyaA toxin.

The adenylate cyclase (CyaA) toxin, one of the virulence factors secreted by Bordetella pertussis, the pathogenic bacteria responsible for whooping cough, plays a critical role in the early stages of respiratory tract colonization by this bacterium. The CyaA toxin is able to invade eukaryotic cells by translocating its N-terminal catalytic domain directly across the plasma membrane of the target cells, where, activated by endogenous calmodulin, it produces supraphysiological levels of cAMP. How the catalytic domain is transferred from the hydrophilic extracellular medium into the hydrophobic environment of the membrane and then to the cell cytoplasm remains an unsolved question. In this report, we have characterized the membrane-interacting properties of the CyaA catalytic domain. We showed that a protein covering the catalytic domain (AC384, encompassing residues 1-384 of CyaA) displayed no membrane association propensity. However, a longer polypeptide (AC489), encompassing residues 1-489 of CyaA, exhibited the intrinsic property to bind to membranes and to induce lipid bilayer destabilization. We further showed that deletion of residues 375-485 within CyaA totally abrogated the toxin's ability to increase intracellular cAMP in target cells. These results indicate that, whereas the calmodulin dependent enzymatic domain is restricted to the amino-terminal residues 1-384 of CyaA, the membrane-interacting, translocation-competent domain extends up to residue 489. This thus suggests an important role of the region adjacent to the catalytic domain of CyaA in promoting its interaction with and its translocation across the plasma membrane of target cells.

Molecular crowding stabilizes both the intrinsically disordered calcium-free state and the folded calcium-bound state of a repeat in toxin (RTX) protein.

Macromolecular crowding affects most chemical equilibria in living cells, as the presence of high concentrations of macromolecules sterically restricts the available space. Here, we characterized the influence of crowding on a prototypical RTX protein, RC(L). RTX (Repeat in ToXin) motifs are calcium-binding nonapeptide sequences that are found in many virulence factors produced by Gram-negative bacteria and secreted by dedicated type 1 secretion systems. RC(L) is an attractive model to investigate the effect of molecular crowding on ligand-induced protein folding, as it shifts from intrinsically disordered conformations (apo-form) to a stable structure upon calcium binding (holo-form). It thus offers the rare opportunity to characterize the crowding effects on the same polypeptide chain under two drastically distinct folding states. We showed that the crowding agent Ficoll70 did not affect the structural content of the apo-state and holo-state of RC(L) but increased the protein affinity for calcium. Moreover, Ficoll70 strongly stabilized both states of RC(L), increasing their half-melting temperature, without affecting enthalpy changes. The power law dependence of the melting temperature increase (ΔT(m)) on the volume fraction (φ) followed theoretical excluded volume predictions and allowed the estimation of the Flory exponent (ν) of the thermally unfolded polypeptide chain in both states. Altogether, our data suggest that, in the apo-state as found in the crowded bacterial cytosol, RTX proteins adopt extended unfolded conformations that may facilitate protein export by the type I secretion machinery. Subsequently, crowding also enhances the calcium-dependent folding and stability of RTX proteins once secreted in the extracellular milieu.

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 classically used to measure protein partitioning into lipid bilayers, such as fluorescence and circular dichroism, are hardly usable when the peptides or proteins do not exhibit significant polarity and/or conformational changes upon membrane binding. Here, we describe binding to lipid vesicles (B2LiVe), a simple, robust, and widely applicable nuclear magnetic resonance (NMR) method to determine the solution-to-membrane partitioning of unlabeled proteins or peptides. B2LiVe relies on previously described proton 1D-NMR fast-pulsing techniques. Membrane partitioning induces a large line broadening, leading to a loss of protein signals; therefore, the decrease of the NMR signal directly measures the fraction of membrane-bound protein. The method uses low polypeptide concentrations and has been validated on several membrane-interacting polypeptides, ranging from 3 to 54 kDa, with membrane vesicles of different sizes and various lipid compositions.