6RFM

Negative charge of the AC-to-Hly linking segment modulates calcium-dependent membrane activities of Bordetella adenylate cyclase toxin.

(2020) Biochim Biophys Acta Biomembr 1862: 183310-183310

• PubMed: 32333856 Search on PubMed
• DOI: https://doi.org/10.1016/j.bbamem.2020.183310
• Primary Citation of Related Structures:  
  6RFM


• PubMed Abstract: 
  

  Two distinct conformers of the adenylate cyclase toxin (CyaA) appear to accomplish its two parallel activities within target cell membrane. The translocating conformer would deliver the N-terminal adenylyl cyclase (AC) enzyme domain across plasma membrane into cytosol of cells, while the pore precursor conformer would assemble into oligomeric cation-selective pores and permeabilize cellular membrane. Both toxin activities then involve a membrane-interacting 'AC-to-Hly-linking segment' (residues 400 to 500). Here, we report the NMR structure of the corresponding CyaA _{411 - 490} polypeptide in dodecylphosphocholine micelles and show that it consists of two α-helices linked by an unrestrained loop. The N-terminal α-helix (Gly418 to His439) remained solvent accessible, while the C-terminal α-helix (His457 to Phe485) was fully enclosed within detergent micelles. CyaA _{411 - 490} weakly bound Ca ²⁺ ions (apparent K _D 2.6 mM) and permeabilized negatively charged lipid vesicles. At high concentrations (10 μM) the CyaA _{411 - 490} polypeptide formed stable conductance units in artificial lipid bilayers with applied voltage, suggesting its possible transmembrane orientation in the membrane-inserted toxin. Mutagenesis revealed that two clusters of negatively charged residues within the 'AC-to-Hly-linking segment' (Glu419 to Glu432 and Asp445 to Glu448) regulate the balance between the AC domain translocating and pore-forming capacities of CyaA in function of calcium concentration.

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Organizational Affiliation: 

Institute of Microbiology of the Czech Academy of Sciences, Videnska 1083, 142 20 Prague, Czech Republic.