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Negative charge of the AC-to-Hly linking segment modulates calcium-dependent membrane activities of Bordetella adenylate cyclase toxin
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SYSNO ASEP 0531086 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Negative charge of the AC-to-Hly linking segment modulates calcium-dependent membrane activities of Bordetella adenylate cyclase toxin Author(s) Suková, Anna (MBU-M) ORCID
Bumba, Ladislav (MBU-M) RID, ORCID
Srb, Pavel (UOCHB-X) RID, ORCID
Veverka, Václav (UOCHB-X) RID, ORCID
Staněk, Ondřej (MBU-M) RID, ORCID
Holubová, Jana (MBU-M) RID, ORCID
Chmelík, Josef (MBU-M) RID, ORCID
Fišer, Radovan (MBU-M) RID, ORCID
Šebo, Peter (MBU-M) RID, ORCID
Mašín, Jiří (MBU-M) RID, ORCIDArticle number 183310 Source Title Biochimica Et Biophysica Acta-Biomembranes. - : Elsevier - ISSN 0005-2736
Roč. 1862, č. 9 (2020)Number of pages 14 s. Language eng - English Country NL - Netherlands Keywords Adenylate cyclase toxin ; AC-to-Hly linking segment ; Membrane penetration Subject RIV CE - Biochemistry OECD category Biochemistry and molecular biology Subject RIV - cooperation Institute of Organic Chemistry and Biochemistry - Biochemistry R&D Projects GA19-04607S GA ČR - Czech Science Foundation (CSF) GA19-15175S GA ČR - Czech Science Foundation (CSF) GA18-20621S GA ČR - Czech Science Foundation (CSF) LO1509 GA MŠMT - Ministry of Education, Youth and Sports (MEYS) LM2018133 GA MŠMT - Ministry of Education, Youth and Sports (MEYS) LO1304 GA MŠMT - Ministry of Education, Youth and Sports (MEYS) Method of publishing Limited access Institutional support MBU-M - RVO:61388971 ; UOCHB-X - RVO:61388963 UT WOS 000540854800012 EID SCOPUS 85083746018 DOI 10.1016/j.bbamem.2020.183310 Annotation 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. Workplace Institute of Microbiology Contact Eliška Spurná, eliska.spurna@biomed.cas.cz, Tel.: 241 062 231 Year of Publishing 2020 Electronic address https://www.sciencedirect.com/science/article/pii/S0005273620301413
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