Abstract
Anthrax toxin complex consists of three different molecules, the binding component protective antigen (PA, 83 kDa), and the enzymatic components lethal factor (LF, 90 kDa) and edema factor (EF, 89 kDa). The 63-kDa N-terminal part of PA, PA(63), forms a heptameric channel that inserts at low pH in endosomal membranes and that is necessary to translocate EF and LF in the cytosol of the target cells. EF is an intracellular active enzyme, which is a calmodulin-dependent adenylate cyclase (89 kDa) that causes a dramatic increase of intracellular cAMP level. Here, the binding of full-length EF on heptameric PA(63) channels was studied in experiments with artificial lipid bilayer membranes. Full-length EF blocks the PA(63) channels in a dose, temperature, voltage, and ionic strength-dependent way with half-saturation constants in the nanomolar concentration range. EF only blocked the PA(63) channels when PA(63) and EF were added to the same side of the membrane, the cis side. Decreasing ionic strength and increasing transmembrane voltage at the cis side of the membranes resulted in a strong decrease of the half-saturation constant for EF binding. This result suggests that ion-ion interactions are involved in EF binding to the PA heptamer. Increasing temperature resulted in increasing half-saturation constants for EF binding to the PA(63) channels. The binding characteristics of EF to the PA(63) channels are compared with those of LF binding. The comparison exhibits similarities but also remarkable differences between the bindings of both toxins to the PA(63) channel.
Highlights
The plasmid-encoded tripartite anthrax toxin comprises a receptor binding moiety termed protective antigen (PA)2 and two enzymatically active components, edema factor (EF) and lethal factor (LF) [1,2,3]
Evidence has been presented that the PA63 channel represents the pathway of LF transport into the cytosol of the target cells driven by voltage and pH gradient [24, 26]
It has been demonstrated that EF binds to the PA63 channel in vivo and in vitro but the binding process has not been studied in such detail as the interaction between full-length LF and the PA63 channel [10, 11, 25, 27]
Summary
Anthrax Protective Antigen PA63—Nicked anthrax protein PA63 from Bacillus anthracis was obtained from List Biological Laboratories Inc., Campbell, CA. 1 mg of lyophilized protein was dissolved in 1 ml of 5 mM HEPES, 50 mM NaCl, pH 7.5 complemented with 1.25% trehalose. PA63 was added from concentrated protein solutions either immediately before membrane formation or after the membranes had turned black to the cis side of the membrane. Membrane conductance was measured after application of a fixed membrane potential with a pair of silver/silver chloride electrodes with salt bridges inserted into the aqueous solutions on both sides of the membrane. The results of the titration experiments, i.e. the block of the PA63 channels, were analyzed using Langmuir adsorption isotherms [11, 31]. This means that the conductance as a function of the EF concentration was analyzed using Lineweaver-Burk plots. The half-saturation constant, Ks ( known as Kd) of its binding is given by the inverse stability constant 1/K
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
