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

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 (Tg PKG) 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 Tg PKG using a bacterial source. A phylogenetic analysis further reveals that Tg PKG2 belongs to an evolutionarily distinct sub-group of the AGC-kinase family. The overall domain composition of Tg PKG2 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, Tg PKG2 illustrated a remarkable level of cooperativity (nH = 2.9) accompanied by a 200-400 fold cGMP-mediated activation utilizing the common PKG substrate TQAKRKKSLAMA (K m = 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, Tg PKG2 was completely insensitive to cAMP (K a » 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 Tg PKG2 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

  1. Nare B, Allocco JJ, Liberator PA, Donald RG: Evaluation of a cyclic GMP- dependent protein kinase inhibitor in treatment of murine toxoplasmosis: gamma interferon is required for efficacy. Antimicrob Agents Chemother. 2002, 46: 300-307. 10.1128/AAC.46.2.300-307.2002.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  2. Gurnett AM, Liberator PA, Dulski PM, Salowe SP, Donald RG, Anderson JW, Wiltsie J, Diaz CA, Harris G, Chang B, Darkin-Rattray SJ, Nare B, Crumley T, Blum PS, Misura AS, Tamas T, Sardana MK, Yuan J, Biftu T, Schmatz DM: Purification and molecular characterization of cGMP- dependent protein kinase from Apicomplexan parasites. A novel chemotherapeutic target. J Biol Chem. 2002, 277: 15913-15922. 10.1074/jbc.M108393200.

    Article  CAS  PubMed  Google Scholar 

  3. Donald RG, Allocco J, Singh SB, Nare B, Salowe SP, Wiltsie J, Liberator PA: Toxoplasma gondii cyclic GMP-dependent kinase: chemotherapeutic targeting of an essential parasite protein kinase. Eukaryot Cell. 2002, 1: 317-328. 10.1128/EC.1.3.317-328.2002.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Donald RG, Zhong T, Wiersma H, Nare B, Yao D, Lee A, Allocco J, Liberator PA: Anticoccidial kinase inhibitors: identification of protein kinase targets secondary to cGMP-dependent protein kinase. Mol Biochem Parasitol. 2006, 149: 86-98. 10.1016/j.molbiopara.2006.05.003.

    Article  CAS  PubMed  Google Scholar 

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Correspondence to Caitlin J McFarland.

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This article is published under license to 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.

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McFarland, C.J., Nickl, C.K., Osborne, B.W. et al. cGMP-dependent protein kinase from Toxoplasma gondii: functional expression in E. coli and molecular characterization. BMC Pharmacol 11 (Suppl 1), P45 (2011). https://doi.org/10.1186/1471-2210-11-S1-P45

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  • DOI: https://doi.org/10.1186/1471-2210-11-S1-P45

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