title: The use of microcalorimetry to characterize tetanus neurotoxin, pertussis toxin and filamentous haemagglutinin.
abstract: Tetanus neurotoxin (TeNT), pertussis toxin (PT) and pertussis filamentous haemagglutinin (FHA) are major virulence factors of Clostridium tetani and Bordetella pertussis, which are the causative agents of tetanus and whooping cough respectively. Inactivated forms of these virulence factors are the protein components of vaccines against these diseases. Here we report microcalorimetric studies to characterize these proteins. The microcalorimetric titration curves of TeNT with micelles of gangliosides GD1b, GT1b and GQ1b were biphasic. For these gangliosides a high-affinity binding site (KD 45-277 nM) can be distinguished from a lower-affinity binding event (KD 666-1190 nM). This is direct evidence for multiple binding sites for gangliosides of the 1b series at TeNT as proposed by Emsley et al. [Emsley, Fotinou, Black, Fairweather, Charles, Watts, Hewitt and Isaacs (2000) J. Biol. Chem. 275, 8889-8894]. In agreement with previous reports, no binding was observed for gangliosides GM1, GM2, GM3 and GD2. The thermal denaturation of TeNT was characterized by two unfolding transitions centred around 57.4 and 62.4 degrees C. The conversion of TeNT into the toxoid form by formaldehyde treatment was accompanied by a large increase in Tm (the midpoint of protein unfolding transition, that is, the temperature at which half the protein is denatured and the other half is still present in its native form). Fetuin and asialofetuin bound to PT with similar affinities (KD 420 and 335 nM respectively). Binding was largely enthalpy-driven and counterbalanced by an unfavourable entropy change, indicating a loss of conformational flexibility. The latter could account for the observed inhibition of ATP binding after binding to fetuin. Furthermore, the molecular limits of mature PT subunit S5 were defined by MS and N-terminal peptide sequencing. The differential-scanning-calorimetry thermogram of FHA shows four well-resolved unfolding transitions, a finding consistent with the sequential denaturation of four structural domains.
authors.affiliation: Aventis Pasteur, 1541 avenue Marcel Mérieux, 69280 Marcy l'etoile, France. tino.krell@aventis.com
authors.forename: Tino
authors.initials: T
authors.lastname: Krell
authors.forename: Frédéric
authors.initials: F
authors.lastname: Greco
authors.forename: Marie-Claire
authors.initials: MC
authors.lastname: Nicolaï
authors.forename: Jean
authors.initials: J
authors.lastname: Dubayle
authors.forename: Geneviève
authors.initials: G
authors.lastname: Renauld-Mongénie
authors.forename: Nadège
authors.initials: N
authors.lastname: Poisson
authors.forename: Isabelle
authors.initials: I
authors.lastname: Bernard
mesh_terms: D018829:Adhesins, Bacterial / Q000032:analysis / Q000737:chemistry*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002151:Calorimetry / Q000379:methods*; D005732:Gangliosides / Q000032:analysis / Q000737:chemistry*; D006388:Hemagglutinins / Q000032:analysis / Q000737:chemistry*; D007700:Kinetics; D008666:Metalloendopeptidases / Q000032:analysis / Q000737:chemistry*; D008832:Microchemistry / Q000379:methods*; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D037342:Pertussis Toxin / Q000032:analysis / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D013744:Tetanus Toxin / Q000032:analysis / Q000737:chemistry*; D017186:Titrimetry / Q000379:methods*; D010566:Virulence Factors, Bordetella / Q000032:analysis / Q000737:chemistry*
chemical_list: D018829:Adhesins, Bacterial; D005732:Gangliosides; D006388:Hemagglutinins; D013744:Tetanus Toxin; D010566:Virulence Factors, Bordetella; C065273:filamentous hemagglutinin adhesin, Bordetella pertussis; C035979:tetanospasmin; D037342:Pertussis Toxin; D008666:Metalloendopeptidases