The role of a parasite-specific allosteric site in the distinctive activation behavior of Eimeria tenella cGMP-dependent protein kinase

A cGMP-dependent protein kinase (PKG) was recently identified as an anticoccidial target for the apicomplexan parasite Eimeria tenella Gurnett, A., Liberator, P. A., Dulski, P., Salowe, S., Donald, R. G. K., Anderson, J., Wiltsie, J., Diaz, C., Harris, G., Chang, B., Darkin-Rattray, S. J., Nare, B., Crumley, T., Blum, P., Misura, A., Tamas, T., Sardana, M., Yuan, J., Biftu, T., and Schmatz, D. (2002) J. Biol. Chem. (in press)]. Unlike the PKGs of higher organisms that have two cGMP binding sites in their regulatory domain, the PKG from Eimeria tenella (Et-PKG) contains three putative cGMP binding sites and has distinctive activation properties, including a very large stimulation by cGMP ( approximately 1000-fold) with significant cooperativity (Hill coefficient of 1.7). During our investigation of Et-PKG activation, we found that 8-substituted cGMP analogues are weak partial activators. For example, 8-NBD-cGMP provides a maximal stimulation of activity of only 20-fold with little evident cooperativity, although cGMP can synergize with the analogue to provide full activation. The results suggest that partial activation is a consequence of restricted binding of 8-NBD-cGMP to a subset of cGMP sites in the enzyme. Site-directed mutagenesis of conserved arginine and glutamate residues in the parasite-specific third cGMP site confirms that this site is an important functional participant in the allosteric regulation of the kinase and that it exhibits very high selectivity against 8-NBD-cGMP. Since the results are consistent with full activation of Et-PKG requiring cyclic nucleotide binding in all three allosteric sites, one role for the additional cGMP site may be to establish a stricter regulatory mechanism for the kinase activity than is present in the PKGs of higher organisms containing only two allosteric sites.