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Publication Abstract from PubMed

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 CyaA411-490 polypeptide in dodecylphosphocholine micelles and show that it consists of two alpha-helices linked by an unrestrained loop. The N-terminal alpha-helix (Gly418 to His439) remained solvent accessible, while the C-terminal alpha-helix (His457 to Phe485) was fully enclosed within detergent micelles. CyaA411-490 weakly bound Ca(2+) ions (apparent KD 2.6 mM) and permeabilized negatively charged lipid vesicles. At high concentrations (10 muM) the CyaA411-490 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.

Negative charge of the AC-to-Hly linking segment modulates calcium-dependent membrane activities of Bordetella adenylate cyclase toxin.,Sukova A, Bumba L, Srb P, Veverka V, Stanek O, Holubova J, Chmelik J, Fiser R, Sebo P, Masin J Biochim Biophys Acta Biomembr. 2020 Sep 1;1862(9):183310. doi:, 10.1016/j.bbamem.2020.183310. Epub 2020 Apr 22. PMID:32333856^[2]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.