Alain Chaffotte

Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay.

A simple, general procedure is described for the determination of the dissociation constant (KD) of antigen-antibody equilibria in solution. First the monoclonal antibody is incubated in solution with the antigen until the equilibrium is reached; then the proportion of antibody which remains unsaturated at equilibrium is measured by a classical indirect ELISA. The experimental values of KD found by this ELISA procedure for 2 monoclonal antibodies are shown to be very close to those obtained by conventional methods (immunoprecipitation of the radiolabeled antigen, or fluorescence transfer). Moreover, it is shown that, provided the measurements are made under conditions where the total antigen concentration is in large excess over the total antibody concentration, the dissociation constant of antibody-antigen complexes can be determined even with crude preparations of monoclonal antibody. The sensitivity of the ELISA used permits the detection of very small concentrations of antibody and the determination of KD values as small as 10(-9) M. This method also offers the great advantage of dealing with unmodified molecules since no labeling of either the antigen or the antibody is required.

Ponericins, New Antibacterial and Insecticidal Peptides from the Venom of the Ant Pachycondyla goeldii

The antimicrobial, insecticidal, and hemolytic properties of peptides isolated from the venom of the predatory ant Pachycondyla goeldii, a member of the subfamily Ponerinae, were investigated. Fifteen novel peptides, named ponericins, exhibiting antibacterial and insecticidal properties were purified, and their amino acid sequences were characterized. According to their primary structure similarities, they can be classified into three families: ponericin G, W, and L. Ponericins share high sequence similarities with known peptides: ponericins G with cecropin-like peptides, ponericins W with gaegurins and melittin, and ponericins L with dermaseptins. Ten peptides were synthesized for further analysis. Their antimicrobial activities against Gram-positive and Gram-negative bacteria strains were analyzed together with their insecticidal activities against cricket larvae and their hemolytic activities. Interestingly, within each of the three families, several peptides present differences in their biological activities. The comparison of the structural features of ponericins with those of well-studied peptides suggests that the ponericins may adopt an amphipathic α-helical structure in polar environments, such as cell membranes. In the venom, the estimated peptide concentrations appear to be compatible with an antibacterial activity in vivo. This suggests that in the ant colony, the peptides exhibit a defensive role against microbial pathogens arising from prey introduction and/or ingestion.

Structural and functional characterization of an essential RTX subdomain of Bordetella pertussis adenylate cyclase toxin

The adenylate cyclase toxin (CyaA) is one of the major virulence factors of Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic cells by a unique mechanism that consists in a calcium-dependent, direct translocation of the CyaA catalytic domain across the plasma membrane of the target cells. CyaA possesses a series of a glycine- and aspartate-rich nonapeptide repeats (residues 1006–1613) of the prototype GGXG(N/D)DX(L/I/F)X (where X represents any amino acid) that are characteristic of the RTX (repeat in toxin) family of bacterial cytolysins. These repeats are arranged in a tandem fashion and may fold into a characteristic parallel β-helix or β-roll motif that constitutes a novel type of calcium binding structure, as revealed by the three-dimensional structure of the Pseudomonas aeruginosa alkaline protease. Here we have characterized the structure-function relationships of various fragments from the CyaA RTX subdomain. Our results indicate that the RTX functional unit includes both the tandem repeated nonapeptide motifs and the adjacent polypeptide segments, which are essential for the folding and calcium responsiveness of the RTX module. Upon calcium binding to the RTX repeats, a conformational rearrangement of the adjacent non-RTX sequences may act as a critical molecular switch to trigger the CyaA entry into target cells.

Thymidylate kinase of Mycobacterium tuberculosis :A chimera sharing properties common to eukaryotic and bacterial enzymes

We have overexpressed in Escherichia coli the thymidylate kinase of Mycobacterium tuberculosis (TMPKmt). Biochemical and physico‐chemical characterization of TMPKmt revealed distinct structural and catalytic features when compared to its counterpart from yeast (TMPKy) or E. coli (TMPKec). Denaturation of the dimeric TMPKmt by urea under equilibrium conditions was studied by intrinsic fluorescence and circular dichroism (CD) spectroscopy. It suggested a three‐state unfolding mechanism with a monomeric intermediate. On the other hand, 3′‐azido‐3′‐deoxythymidine monophosphate (AZT‐MP), which is substrate for TMPKy and TMPKec acts as a potent competitive inhibitor for TMPKmt. We propose a structural model of TMPKmt in which the overall fold described in TMPKy and TMPKec is conserved and slight differences at the level of primary and 3D‐structure explain strong variations in the phosphorylation rate of substrate analogs. According to the model, we synthesized dTMP analogs acting either as substrates or specific inhibitors of TMPKmt. This approach based on slight structural differences among similar proteins could be applied to other essential enzymes for the design of new species‐specific antimicrobials.

Carbon Black Nanoparticles Impair Acetylation of Aromatic Amine Carcinogens through Inactivation of Arylamine N-Acetyltransferase Enzymes

Carbon black nanoparticles (CB NPs) and their respirable aggregates/agglomerates are classified as possibly carcinogenic to humans. In certain industrial work settings, CB NPs coexist with aromatic amines (AA), which comprise a major class of human carcinogens. It is therefore crucial to characterize the interactions of CB NPs with AA-metabolizing enzymes. Here, we report molecular and cellular evidence that CB NPs interfere with the enzymatic acetylation of carcinogenic AA by rapidly binding to arylamine N-acetyltransferase (NAT), the major AA-metabolizing enzyme. Kinetic and biophysical analyses showed that this interaction leads to protein conformational changes and an irreversible loss of enzyme activity. In addition, our data showed that exposure to CB NPs altered the acetylation of 2-aminofluorene in intact lung Clara cells by impairing the endogenous NAT-dependent pathway. This process may represent an additional mechanism that contributes to the carcinogenicity of inhaled CB NPs. Our results add to recent data suggesting that major xenobiotic detoxification pathways may be altered by certain NPs and that this can result in potentially harmful pharmacological and toxicological effects.

Undistorted structural analysis of soluble proteins by attenuated total reflectance infrared spectroscopy.

Water from the solvent very strongly absorbs light in the frequency range of interest for studying protein structure by infrared (IR) spectroscopy. This renders handling of the observation cells painstaking and time consuming, and limits the reproducibility of the measurements when IR spectroscopy is applied to proteins in aqueous solutions. These difficulties are circumvented by the use of an Attenuated Total Reflectance (ATR) accessory. However, when protein solutions are studied, ATR spectroscopy suffers from several drawbacks, the most severe being nonproportionality of the observed absorbance with the protein concentration and spectral distortions that vary from protein to protein and from sample to sample. In this study, we show (1) that the nonproportionality is due to adsorption of the protein on the ATR crystal surface; (2) that the contribution of the crystal‐adsorbed protein can easily be taken into account, rendering the corrected absorbance proportional to the protein concentration; (3) that the observed variable base line distortions, likely due to changes in the penetration depth of the light beam in solutions with the refractive index that depends on the protein concentration, can be easily eliminated; and (4) that ATR IR spectra thus corrected for protein adsorption and light penetration can be used to properly analyze the secondary structure of proteins in solution.

Antifolding Activity of the SecB Chaperone Is Essential for Secretion of HasA, a Quickly Folding ABC Pathway Substrate

We have previously shown that SecB, the ATP-independent chaperone of the Sec pathway, is required for the secretion of the HasA hemophore from Serratia marcescens via its type I secretion pathway, both in the reconstituted system in Escherichia coli and in the original host. The refolding of apo-HasA after denaturation with guanidine HCl was followed by stopped-flow measurements of fluorescence of its single tryptophan, both in the absence and presence of SecB. In the absence of SecB, HasA folds very quickly with one main phase (45 s–1) accounting for 92% of the signal. SecB considerably slows down HasA folding. At stoichiometric amounts of SecB and HasA, a single phase (0.014 s–1) of refolding is observed. Two double point mutants of HasA were made, abolishing two hydrogen bonds between N-terminal and C-terminal side chain residues. In both cases, the mutants essentially maintained the same secondary and tertiary structure as wild-type HasA and were fully functional. Refolding of both mutants was much slower than that of wild-type HasA and they were secreted essentially independently of SecB. We conclude that SecB has mainly an antifolding function in the HasA ABC secretion pathway.

Substrate-induced conformational changes in the essential peripheral membrane-associated mannosyltransferase PimA from mycobacteria. Implications for catalysis

Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase (GT) involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs), which are key components of the mycobacterial cell envelope. PimA is the paradigm of a large family of peripheral membrane-binding GTs for which the molecular mechanism of substrate/membrane recognition and catalysis is still unknown. Strong evidence is provided showing that PimA undergoes significant conformational changes upon substrate binding. Specifically, the binding of the donor GDP-Man triggered an important interdomain rearrangement that stabilized the enzyme and generated the binding site for the acceptor substrate, phosphatidyl-myo-inositol (PI). The interaction of PimA with the β-phosphate of GDP-Man was essential for this conformational change to occur. In contrast, binding of PI had the opposite effect, inducing the formation of a more relaxed complex with PimA. Interestingly, GDP-Man stabilized and PI destabilized PimA by a similar enthalpic amount, suggesting that they formed or disrupted an equivalent number of interactions within the PimA complexes. Furthermore, molecular docking and site-directed mutagenesis experiments provided novel insights into the architecture of the myo-inositol 1-phosphate binding site and the involvement of an essential amphiphatic α-helix in membrane binding. Altogether, our experimental data support a model wherein the flexibility and conformational transitions confer the adaptability of PimA to the donor and acceptor substrates, which seems to be of importance during catalysis. The proposed mechanism has implications for the comprehension of the peripheral membrane-binding GTs at the molecular level.

A point mutation in NEMO associated with anhidrotic ectodermal dysplasia with immunodeficiency pathology results in destabilization of the oligomer and reduces lipopolysaccharide- and tumor necrosis factor-mediated NF-kappa B activation.

The NEMO (NF-κB essential modulator) protein plays a crucial role in the canonical NF-κB pathway as the regulatory component of the IKK (IκB kinase) complex. The human disease anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) has been recently linked to mutations in NEMO. We investigated the effect of an alanine to glycine substitution found in the NEMO polypeptide of an EDA-ID patient. This pathogenic mutation is located within the minimal oligomerization domain of the protein, which is required for the IKK activation in response to diverse stimuli. The mutation does not dramatically change the native-like state of the trimer, but temperature-induced unfolding studied by circular dichroism showed that it leads to an important loss in the oligomer stability. Furthermore, fluorescence studies showed that the tyrosine located in the adjacent zinc finger domain, which is possibly required for NEMO ubiquitination, exhibits an alteration in its spectral properties. This is probably due to a conformational change of this domain, providing evidence for a close interaction between the oligomerization domain and the zinc finger. In addition, functional complementation assays using NEMO-deficient pre-B and T lymphocytes showed that the pathogenic mutation reduced TNF-α and LPS-induced NF-κB activation by altering the assembly of the IKK complex. Altogether, our findings provide understanding as to how a single point mutation in NEMO leads to the observed EDA-ID phenotype in relation to the NEMO-dependent mechanism of IKK activation.

Interference with the PTEN-MAST2 Interaction by a Viral Protein Leads to Cellular Relocalization of PTEN

The G protein of rabies virus manipulates the cellular localization of PTEN, which may promote cell survival. Rabies Virus Relocalizes PTEN Virulent strains of rabies virus infect neurons and promote survival of the infected cells to favor viral replication. Among the host factors that inhibit neuronal survival are the phosphatase PTEN and one of its binding partners, the kinase MAST2. PTEN and MAST2 interact through the PDZ domain of MAST2 and the PDZ domain–binding site (PDZ-BS) of PTEN. Terrien et al. found that the rabies virus glycoprotein (G protein), which contains a PDZ-BS, disrupted the MAST2-PTEN complex in infected cells. Structural analysis showed that the surfaces of PTEN and G protein that interacted with MAST2 were similar and contained previously uncharacterized PDZ-binding regions. Finally, disruption of the MAST2-PTEN complex by viral G protein resulted in the relocalization of PTEN from the nucleus to the cytoplasm. Together, these data suggest that competition between viral G protein and MAST2 for binding to PTEN plays a role in the survival of infected cells. PTEN (phosphatase and tensin homolog deleted on chromosome 10) and MAST2 (microtubule-associated serine and threonine kinase 2) interact with each other through the PDZ domain of MAST2 (MAST2-PDZ) and the carboxyl-terminal (C-terminal) PDZ domain–binding site (PDZ-BS) of PTEN. These two proteins function as negative regulators of cell survival pathways, and silencing of either one promotes neuronal survival. In human neuroblastoma cells infected with rabies virus (RABV), the C-terminal PDZ domain of the viral glycoprotein (G protein) can target MAST2-PDZ, and RABV infection triggers neuronal survival in a PDZ-BS–dependent fashion. These findings suggest that the PTEN-MAST2 complex inhibits neuronal survival and that viral G protein disrupts this complex through competition with PTEN for binding to MAST2-PDZ. We showed that the C-terminal sequences of PTEN and the viral G protein bound to MAST2-PDZ with similar affinities. Nuclear magnetic resonance structures of these complexes exhibited similar large interaction surfaces, providing a structural basis for their binding specificities. Additionally, the viral G protein promoted the nuclear exclusion of PTEN in infected neuroblastoma cells in a PDZ-BS–dependent manner without altering total PTEN abundance. These findings suggest that formation of the PTEN-MAST2 complex is specifically affected by the viral G protein and emphasize how disruption of a critical protein-protein interaction regulates intracellular PTEN trafficking. In turn, the data show how the viral protein might be used to decipher the underlying molecular mechanisms and to clarify how the subcellular localization of PTEN regulates neuronal survival.