Negative charge of the AC-to-Hly linking segment modulates calcium-dependent membrane activities of Bordetella adenylate cyclase toxin
Identifikátory výsledku
Kód výsledku v IS VaVaI
<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61388963%3A_____%2F20%3A00531086" target="_blank" >RIV/61388963:_____/20:00531086 - isvavai.cz</a>
Nalezeny alternativní kódy
RIV/61388971:_____/20:00531086 RIV/00216208:11310/20:10414158
Výsledek na webu
<a href="https://www.sciencedirect.com/science/article/pii/S0005273620301413" target="_blank" >https://www.sciencedirect.com/science/article/pii/S0005273620301413</a>
DOI - Digital Object Identifier
<a href="http://dx.doi.org/10.1016/j.bbamem.2020.183310" target="_blank" >10.1016/j.bbamem.2020.183310</a>
Alternativní jazyky
Jazyk výsledku
angličtina
Název v původním jazyce
Negative charge of the AC-to-Hly linking segment modulates calcium-dependent membrane activities of Bordetella adenylate cyclase toxin
Popis výsledku v původním jazyce
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.
Název v anglickém jazyce
Negative charge of the AC-to-Hly linking segment modulates calcium-dependent membrane activities of Bordetella adenylate cyclase toxin
Popis výsledku anglicky
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.
Klasifikace
Druh
J<sub>imp</sub> - Článek v periodiku v databázi Web of Science
CEP obor
—
OECD FORD obor
10608 - Biochemistry and molecular biology
Návaznosti výsledku
Projekt
Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.
Návaznosti
I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace
Ostatní
Rok uplatnění
2020
Kód důvěrnosti údajů
S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů
Údaje specifické pro druh výsledku
Název periodika
Biochimica Et Biophysica Acta-Biomembranes
ISSN
0005-2736
e-ISSN
—
Svazek periodika
1862
Číslo periodika v rámci svazku
9
Stát vydavatele periodika
NL - Nizozemsko
Počet stran výsledku
14
Strana od-do
183310
Kód UT WoS článku
000540854800012
EID výsledku v databázi Scopus
2-s2.0-85083746018