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

0531086 - MBÚ 2020 RIV NL eng J - Článek v odborném periodiku
Suková, Anna - Bumba, Ladislav - Srb, Pavel - Veverka, Václav - Staněk, Ondřej - Holubová, Jana - Chmelík, Josef - Fišer, Radovan - Šebo, Peter - Mašín, Jiří
Negative charge of the AC-to-Hly linking segment modulates calcium-dependent membrane activities of Bordetella adenylate cyclase toxin.
Biochimica Et Biophysica Acta-Biomembranes. Roč. 1862, č. 9 (2020), č. článku 183310. ISSN 0005-2736. E-ISSN 1879-2642
Grant CEP: GA ČR(CZ) GA19-04607S; GA ČR(CZ) GA19-15175S; GA ČR(CZ) GA18-20621S; GA MŠMT(CZ) LO1509; GA MŠMT(CZ) LM2018133; GA MŠMT(CZ) LO1304
Institucionální podpora: RVO:61388971 ; RVO:61388963
Klíčová slova: Adenylate cyclase toxin * AC-to-Hly linking segment * Membrane penetration
Obor OECD: Biochemistry and molecular biology; Biochemistry and molecular biology (UOCHB-X)
Impakt faktor: 3.747, rok: 2020
Způsob publikování: Omezený přístup
https://www.sciencedirect.com/science/article/pii/S0005273620301413

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 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. CyaA(411-490) weakly bound Ca2+ ions (apparent K-D 2.6 mM) and permeabilized negatively charged lipid vesicles. At high concentrations (10 mu M) 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.
Trvalý link: http://hdl.handle.net/11104/0309834