title: Mutation in the β-hairpin of the Bordetella pertussis adenylate cyclase toxin modulates N-lobe conformation in calmodulin.
abstract: Bordetella pertussis, causative agent of whooping cough, produces an adenylate cyclase toxin (CyaA) that is an important virulence factor. In the host cell, the adenylate cyclase domain of CyaA (CyaA-ACD) is activated upon association with calmodulin (CaM), an EF-hand protein comprised of N- and C-lobes (N-CaM and C-CaM, respectively) connected by a flexible tether. Maximal CyaA-ACD activation is achieved through its binding to both lobes of intact CaM, but the structural mechanisms remain unclear. No high-resolution structure of the intact CaM/CyaA-ACD complex is available, but crystal structures of isolated C-CaM bound to CyaA-ACD shed light on the molecular mechanism by which this lobe activates the toxin. Previous studies using molecular modeling, biochemical, and biophysical experiments demonstrate that CyaA-ACD's β-hairpin participates in site-specific interactions with N-CaM. In this study, we utilize nuclear magnetic resonance (NMR) spectroscopy to probe the molecular association between intact CaM and CyaA-ACD. Our results indicate binding of CyaA-ACD to CaM induces large conformational perturbations mapping to C-CaM, while substantially smaller structural changes are localized primarily to helices I, II, and IV, and the metal-binding sites in N-CaM. Site-specific mutations in CyaA-ACD's β-hairpin structurally modulate N-CaM, resulting in conformational perturbations in metal binding sites I and II, while no significant structural modifications are observed in C-CaM. Moreover, dynamic light scattering (DLS) analysis reveals that mutation of the β-hairpin results in a decreased hydrodynamic radius (Rh) and reduced thermal stability in the mutant complex. Taken together, our data provide new structural insights into the β-hairpin's role in stabilizing interactions between CyaA-ACD and N-CaM.
authors.affiliation: Department of Microbiology, Miami University, Oxford, OH 45056, USA.
authors.forename: Tzvia I
authors.initials: TI
authors.lastname: Springer
authors.affiliation: Department of Microbiology, Miami University, Oxford, OH 45056, USA.
authors.forename: Erich
authors.initials: E
authors.lastname: Goebel
authors.affiliation: Department of Microbiology, Miami University, Oxford, OH 45056, USA.
authors.forename: Dinesh
authors.initials: D
authors.lastname: Hariraju
authors.affiliation: Department of Microbiology, Miami University, Oxford, OH 45056, USA; Cell, Molecular, and Structural Biology Program, Miami University, Oxford, OH 45056, USA. Electronic address: finleynl@miamioh.edu.
authors.forename: Natosha L
authors.initials: NL
authors.lastname: Finley
mesh_terms: D037361:Adenylate Cyclase Toxin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019943:Amino Acid Substitution; D001665:Binding Sites; D001886:Bordetella pertussis / Q000201:enzymology* / Q000235:genetics* / Q000472:pathogenicity; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D054884:Host-Pathogen Interactions; D006801:Humans; D008958:Models, Molecular; D008968:Molecular Conformation; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D037361:Adenylate Cyclase Toxin; D002147:Calmodulin; D046912:Multiprotein Complexes; D011994:Recombinant Proteins
keywords: Adenylate cyclase; CaM; Calmodulin; CyaA; Molecular mechanism; NMR spectroscopy