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

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216208%3A11310%2F20%3A10414158" target="_blank" >RIV/00216208:11310/20:10414158 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/61388971:_____/20:00531086 RIV/61388963:_____/20:00531086

Výsledek na webu

<a href="https://verso.is.cuni.cz/pub/verso.fpl?fname=obd_publikace_handle&handle=VSxIfYOyv" target="_blank" >https://verso.is.cuni.cz/pub/verso.fpl?fname=obd_publikace_handle&handle=VSxIfYOyv</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>

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 &apos;AC-to-Hly-linking segment&apos; (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 &apos;AC-to-Hly-linking segment&apos; (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 &apos;AC-to-Hly-linking segment&apos; (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 &apos;AC-to-Hly-linking segment&apos; (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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10606 - Microbiology

Projekt

—

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2020

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

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