Exchange characteristics of calcium ions bound to anthrax protective antigen

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Abstract

Protective antigen (PA), the receptor-binding moiety of anthrax toxin, contains two calcium atoms buried within domain 1 (amino acid residues 168–258). We showed that these ions are stably bound and exchange with free 45Ca2+ only slowly (t1/2∼4.0 h). Dissociation is the rate-limiting step. PA63, the heptameric prepore form of PA, showed a slightly higher exchange rate than the monomeric intact protein. Exchange by this form was retarded by binding of the enzymatic moieties of the toxin, but was unaffected by reducing the pH to 5.0, a condition known to trigger conversion of the prepore to the pore form. These results are consistent with the hypothesis that bound Ca2+ within PA plays primarily a structural role, maintaining domain 1 in a conformation that allows PA63 to oligomerize and bind the enzymatic moieties of the toxin.

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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.

References (17)

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Abbreviations: PA, protective antigen; LF, lethal factor; EF, edema factor; LFn, amino-terminal domain of LF.

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