Structural consequences of divalent metal binding by the adenylyl cyclase toxin of Bordetella pertussis

Adenylyl cyclase toxin of Bordetella pertussis has been shown by several in vestigators to require Ca2+ for its actions on target cells, but little is known about the nature and specificity of divalent metal binding to this no vel toxin. Calcium is the preferred divalent metal since toxic actions are markedly reduced in the presence of divalent species other than calcium. Mn 2+ EPR was used to quantitate and characterize divalent metal binding and r evealed that the toxin contains approximately 40 divalent metal sites, cons isting of at least one class of high-affinity sites that bind Mn2+ with a K -D of 0.05 to 0.35 muM and one or more classes of lower affinity sites. Wat er proton relaxation data indicate that approximately 30 of these sites are completely inaccessible to bulk solvent. Our observations, together with t he sequence homology between adenylyl cyclase toxin and the alkaline protea se of Pseudomonas aeruginosa, indicate that the formation of five beta -she et helices within the repeat domain of the toxin upon binding C2+ is requir ed for cell intoxication. (C) 2001 Academic Press.