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This article is part of the supplement: 5th International Conference on cGMP: Generators, Effectors and Therapeutic Implications

Open Access Poster presentation

cGMP-dependent protein kinase from Toxoplasma gondii: functional expression in E. coli and molecular characterization

Caitlin J McFarland1*, Christian K Nickl1, Brent W Osborne1, Indra Neil Sarkar2 and Wolfgang R Dostmann1

Author Affiliations

1 Department of Pharmacology, College of Medicine, University of Vermont, Burlington, VT 05405, USA

2 Center for Clinical & Translational Science, Department of Microbiology & Molecular Genetics and Department of Computer Science, University of Vermont, Burlington, VT 05405, USA

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BMC Pharmacology 2011, 11(Suppl 1):P45  doi:10.1186/1471-2210-11-S1-P45


The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1471-2210/11/S1/P45


Published:1 August 2011

© 2011 McFarland et al; licensee BioMed Central Ltd.

This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background

The apicomplexan parasite Toxoplasma gondii is an obligate intracellular human pathogen causing toxoplasmosis predominantly in immune-compromised hosts such as cancer and transplant patients as well as patients with AIDS [1]. A specific cGMP-dependent protein kinase (TgPKG) which appears to be crucial for host invasion has been identified in T. gondii and related coccidial protozoa [2]. However, detailed structural and biochemical analyses have been hampered due to the inability to functionally express these kinases in high yields in systems other than their parasite host organisms.

Results

Here we describe the expression, purification and initial characterization of the 911 amino acid (103 kDa) His-tagged type II isoform of TgPKG using a bacterial source. A phylogenetic analysis further reveals that TgPKG2 belongs to an evolutionarily distinct sub-group of the AGC-kinase family. The overall domain composition of TgPKG2 substantially deviates from its mammalian cousins at two regions. First, the 196 amino acid N-terminal/auto-inhibitory domain bears no resemblance with any other PKG subfamily, and secondly the kinase consists of three cGMP binding sites with the third binding sites separated from the others by 135 amino acids. Consequently, TgPKG2 illustrated a remarkable level of cooperativity (nH = 2.9) accompanied by a 200-400 fold cGMP-mediated activation utilizing the common PKG substrate TQAKRKKSLAMA (Km = 9 µM). The associated activation constant was 1.7 µM which is in full agreement with the isoforms obtained from T. gondii parasite extract [3]. Interestingly, TgPKG2 was completely insensitive to cAMP (Ka » 100 µM). Recently, the trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H pyrrol-3-yl] pyridine (Compound 1) was shown to exhibit anticoccidial kinase activity [3,4]. Compound 1 blocked kinase activity of TgPKG2 with high potency (IC50 = 59 nM) and high selectivity; the mammalian type Iα PKG showed an approximately 1000-fold reduced IC50 of 45 µM.

Conclusion

This work demonstrates the first catalytically active expression of any cGMP-dependent protein kinase from E. coli and may provide a new platform for the functional and structural analysis, as well as evolutionary history, of PKG isoforms from apicomplexan parasites.

References

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