Talevich et al. BMC Evolutionary Biology 2011, 11:321 http://www.biomedcentral.com/1471-2148/11/321 RESEARCH ARTICLE Open Access Structural and evolutionary divergence of eukaryotic protein kinases in Apicomplexa Eric Talevich1, Amar Mirza2 and Natarajan Kannan1,2* Abstract Background: The Apicomplexa constitute an evolutionarily divergent phylum of protozoan pathogens responsible for widespread parasitic diseases such as malaria and toxoplasmosis. Many cellular functions in these medically important organisms are controlled by protein kinases, which have emerged as promising drug targets for parasitic diseases. However, an incomplete understanding of how apicomplexan kinases structurally and mechanistically differ from their host counterparts has hindered drug development efforts to target parasite kinases. Results: We used the wealth of sequence data recently made available for 15 apicomplexan species to identify the kinome of each species and quantify the evolutionary constraints imposed on each family of apicomplexan kinases. Our analysis revealed lineage-specific adaptations in selected families, namely cyclin-dependent kinase (CDK), calcium-dependent protein kinase (CDPK) and CLK/LAMMER, which have been identified as important in the pathogenesis of these organisms. Bayesian analysis of selective constraints imposed on these families identified the sequence and structural features that most distinguish apicomplexan protein kinases from their homologs in model organisms and other eukaryotes. In particular, in a subfamily of CDKs orthologous to Plasmodium falciparum crk-5, the activation loop contains a novel PTxC motif which is absent from all CDKs outside Apicomplexa. Our analysis also suggests a convergent mode of regulation in a subset of apicomplexan CDPKs and mammalian MAPKs involving a commonly conserved arginine in the aC helix. In all recognized apicomplexan CLKs, we find a set of co-conserved residues involved in substrate recognition and docking that are distinct from metazoan CLKs. Conclusions: We pinpoint key conserved residues that can be predicted to mediate functional differences from eukaryotic homologs in three identified kinase families. We discuss the structural, functional and evolutionary implications of these lineage-specific variations and propose specific hypotheses for experimental investigation. The apicomplexan-specific kinase features reported in this study can be used in the design of selective kinase inhibitors. Background and there remains an urgent need for deeper research The parasitic protists which comprise the phylum Api- into the basic biology of apicomplexans [2]. complexa are responsible for human diseases of global Several traits make these pathogens difficult to target importance, such as malaria (caused by Plasmodium fal- therapeutically. As eukaryotes, they share a number of ciparum and other members of the Plasmodium genus), pathways with their mammalian and avian hosts; as cryptosporidiosis (Cryptosporidium species) and toxo- intracellular parasites, they have been observed to plasmosis (Toxoplasma gondii), as well as the agricul- quickly develop resistance to pharmaceutical treatments tural diseases babesiosis (Babesia bovis in cattle) and [3]. The identification of distinctive protein features coccidiosis (Eimeria tenella in chickens) [1]. In recent which appear conserved across apicomplexan species, years, understanding of the molecular biology and evo- but not in their hosts, however, will aid the search for lution of this phylum has improved dramatically; yet potential new targets for selective inhibition that are effective treatments for these diseases are still elusive, more likely to be safe and effective [4]. As protein kinases have been successfully targeted for inhibition in cancer, this diverse protein superfamily warrants consid- * Correspondence: [email protected] eration as a target for parasitic diseases as well [2,5]. 1Institute of Bioinformatics, University of Georgia, Athens, GA 30602, USA Full list of author information is available at the end of the article © 2011 Talevich 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. Talevich et al. BMC Evolutionary Biology 2011, 11:321 Page 2 of 20 http://www.biomedcentral.com/1471-2148/11/321 Recent whole-genome sequencing efforts have targeted kinase families FIKK and ROPK appear; (ii) a unique a number of apicomplexan species [6-17]. Several ana- apicomplexan-specific subfamily of cyclin-dependent lyses of protein kinases in these organisms, in particular, kinases (CDK), orthologous to P. falciparum crk-5, and have pointed out key signaling pathways [18-20], the motifs that distinguish it; (iii) a hypothesized instances of expansion and loss of kinase gene families mechanism of activation by phosphorylation, resembling [21,22], and emergence of novel protein kinase families that of MAP kinases, in a chromalveolate-specific sub- [21,23,24], thus providing important insights into biolo- family of calcium-dependent protein kinases (CDPK); gical functions. These comparative studies have further- and (iv) a description of the adaptation of the substrate- more proposed hypotheses which have subsequently recognition and docking sites in the CLK kinase family been validated by functional and structural studies in a clade including apicomplexans and other chromal- [19,20,25,26]. veolates, revealed by the co-evolution of a small set of The eukaryotic protein kinase (ePK) superfamily is key residues. classified into several major groups, corresponding to broad functional categories with distinguishing sequence Results and Discussion and structural features [27,28]. The presence of specific We identified and classified the eukaryotic protein ePK groups and families in a genome is a key indicator kinases in a total of 17 genomes from 15 species, as well of biological functions critical for an organism; likewise, as the solved apicomplexan ePK structures in the Pro- missing groups or families indicate functions less critical tein Data Bank [34]. We used our classification to for an organism’s survival and reproduction. These pro- broadly describe the conserved ePK families in the Api- teins, and the fundamental cell processes in which they complexa and then performed a residue-level analysis of participate, are well characterized in humans and several the lineage-specific differences within several conserved model organisms [28]. families: CDK, CDPK and CLK. We place our findings Previous efforts to perform detailed comparative ana- in the context of the known evolutionary history of api- lysis of apicomplexan kinases have largely focused on complexans and their relatives. the kinomes of individual species within the genera Plasmodium, Toxoplasma and Cryptosporidium Kinome classification and composition: Variations within [10,11,20,29-32]. Thus, there is no global overview of the Apicomplexa the sequence and structural features that distinguish api- Recent published evolutionary relationships of eukar- complexan kinases collectively from their metazoan yotes provide the basis for our genomic comparison counterparts. [35]. In this study we have chosen model organisms Sequence data from 15 apicomplexan species and sev- representing major evolutionary splits — the emergence eral crystallographic structures of a variety of apicom- of Chromalveolata (a proposed super-kingdom of plas- plexan protein kinases are now available. We can use tid-containing eukaryotes [36]), Alveolata (the kingdom these data to perform a systematic comparison of pro- comprising ciliates, dinoflagellates and apicomplexans tein kinases in apicomplexans and model eukaryotes to [37]), and Apicomplexa — to illuminate the origin and identify broadly conserved orthologous groups and dis- divergence of the major ePK groups. For genomic com- tinctive residue-level differences. parison we use the parasitic dinoflagellate Perkinsus Inthisstudyweuseabioinformaticsapproachto marinus as an outgroup to the Apicomplexa, the photo- comprehensively analyze genomic and structural data synthetic diatom Thalassiosira pseudonana as an out- sets. We perform an exhaustive comparison of apicom- group to the Alveolata, and the yeast Saccharomyces plexan kinomes, providing broad coverage of the phy- cerevisiae as an outgroup to the Chromalveolata. lum. We also perform a quantitative, residue-level Detailed kinase annotations are given for each genome analysis of the differences between kinases within the in Additional File 1. Apicomplexa and those in model eukaryotes, in parti- Apicomplexan kinome sizes are comparable to those of cular humans. We use a Bayesian method [33] to rig- other unicellular protists orously quantify sequence differences between The number of ePKs identified in each of the surveyed homologous protein kinases in apicomplexans and apicomplexan genomes varies, with the coccidians (Tox- other eukaryotes, and reveal contrastingly conserved oplasma gondii, Neospora caninum and Eimeria tenella) features that were not apparent previously. Where pos- containing more ePKs than the haemosporidians (Plas- sible, we then place these sequence features in struc- modium spp.), and the piroplasms (Babesia bovis and tural context to postulate specific hypotheses for Theileria spp.) containing fewer (Table 1). Cryptospori-