Exchange characteristics of calcium ions bound to anthrax protective antigen☆
Section snippets
Materials and methods
Protein purification. An expression vector pET22b containing the DNA fragment encoding PA was transformed into Escherichia coli (BL21-DE3) (Novagen) and PA was expressed as described previously [12]. The protein was purified to at least 90% homogeneity as judged by SDS–PAGE and stored at −80 °C.
Preparation of heptameric PA63prepore. Nicked PA (nPA) was prepared by incubating PA with trypsin (Sigma) at a ratio of 1000:1 (w:w) at room temperature for 30 min followed by addition of a 10-fold excess
Results
Quantification of Ca2+ in native PA by atomic absorption gave a value of 1.84±0.06 mol Ca2+/mol protein, consistent with 2 Ca2+ seen in domain 1 by X-ray crystallography [14]. The Ca2+ was not removed by extensive dialysis against 5 mM EGTA alone, but treating the protein with 5 mM EGTA in the presence of 3 M urea, followed by extensive dialysis against 1 mM EGTA, yielded a product devoid of Ca2+ (apo-PA), as determined by atomic absorption.
The fluorescence emission λmax of holo-PA, with excitation
Discussion
The results presented are consistent with the notion that the two Ca2+ ions within domain 1′ of PA play principally a structural role, maintaining that domain in a functional conformation. These ions are tightly bound in both native PA and the heptameric PA63 prepore; exchange with free Ca2+ in the medium is slow, with a t1/2 of 4 h. No evidence of heterogeneity in behavior of the two ions was detected in our measurements of the kinetics of exchange with free Ca2+. This is consistent with the
Acknowledgements
This work was supported by NIH Grant AI-22021. One of the authors (R.J.C.) holds equity in PharmAthene, Inc.
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2015, Handbook of Toxicology of Chemical Warfare Agents: Second EditionBiophysical characterisation of thermal-induced precipitates of recombinant anthrax protective antigen: Evidence for kinetically trapped unfolding domains in solid-state
2012, European Journal of Pharmaceutics and BiopharmaceuticsCitation Excerpt :The domain d1 is stabilized through an extensive interface with domain d2 and internally by two Ca2+ ions [14]. The Ca2+ ions are critical for proper folding of the protein and maintaining structural order [41,42]. In addition to extensive interaction with domain d1, the pore forming domain d2 is stabilized by the domain d3.
Comparison of the structural stability and dynamic properties of recombinant anthrax protective antigen and its 2-fluorohistidine-labeled analogue
2012, Journal of Pharmaceutical SciencesCitation Excerpt :The prepore subsequently combines with edema factor (EF) and lethal factor (LF) to create a number of different, but functionally similar, complex toxins such as edema toxin (EdTx) and lethal toxin (LeTx). After receptor-mediated endocytosis, and a subsequent conformational change in PA from a prepore to a pore in acidified endosomes (pH 5–6), actively toxic proteins EF and LF can be translocated through the pore into the cytoplasm of target cells, which results in cellular toxicity causing cell killing and host death.5,8–10 Since PA plays an important role in the pathology of anthrax, yet is a nontoxic and a strongly immunogenic molecule, it is used as a primary antigen in anthrax vaccines.
Anthrax sub-unit vaccine: The structural consequences of binding rPA83 to Alhydrogel®
2012, European Journal of Pharmaceutics and BiopharmaceuticsCitation Excerpt :PA contains two calcium ions in domain 1, essential for its structural stability and correct folding [20,21]. In native rPA83, these ions are stably bound [40]. We showed that rPA83 retained both calcium ions upon binding to Alhydrogel®.
Membrane translocation by anthrax toxin
2009, Molecular Aspects of MedicineAnthrax
2009, Handbook of Toxicology of Chemical Warfare Agents
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Abbreviations: PA, protective antigen; LF, lethal factor; EF, edema factor; LFn, amino-terminal domain of LF.