The parasitic protists which comprise the phylum Apicomplexa are responsible for human diseases of global importance, such as malaria (caused by Plasmodium falciparum
and other members of the Plasmodium
genus), cryptosporidiosis (Cryptosporidium
species) and toxoplasmosis (Toxoplasma gondii
), as well as the agricultural diseases babesiosis (Babesia bovis
in cattle) and coccidiosis (Eimeria tenella
in chickens) [1
]. In recent years, understanding of the molecular biology and evolution of this phylum has improved dramatically; yet effective treatments for these diseases are still elusive, and there remains an urgent need for deeper research into the basic biology of apicomplexans [2
Several traits make these pathogens difficult to target therapeutically. As eukaryotes, they share a number of pathways with their mammalian and avian hosts; as intracellular parasites, they have been observed to quickly develop resistance to pharmaceutical treatments [3
]. The identification of distinctive protein features which appear conserved across apicomplexan species, but not in their hosts, however, will aid the search for potential new targets for selective inhibition that are more likely to be safe and effective [4
]. As protein kinases have been successfully targeted for inhibition in cancer, this diverse protein superfamily warrants consideration as a target for parasitic diseases as well [2
Recent whole-genome sequencing efforts have targeted a number of apicomplexan species [6
]. Several analyses of protein kinases in these organisms, in particular, have pointed out key signaling pathways [18
], instances of expansion and loss of kinase gene families [21
], and emergence of novel protein kinase families [21
], thus providing important insights into biological functions. These comparative studies have furthermore proposed hypotheses which have subsequently been validated by functional and structural studies [19
The eukaryotic protein kinase (ePK) superfamily is classified into several major groups, corresponding to broad functional categories with distinguishing sequence and structural features [27
]. The presence of specific ePK groups and families in a genome is a key indicator of biological functions critical for an organism; likewise, missing groups or families indicate functions less critical for an organism's survival and reproduction. These proteins, and the fundamental cell processes in which they participate, are well characterized in humans and several model organisms [28
Previous efforts to perform detailed comparative analysis of apicomplexan kinases have largely focused on the kinomes of individual species within the genera Plasmodium
]. Thus, there is no global overview of the sequence and structural features that distinguish apicomplexan kinases collectively from their metazoan counterparts.
Sequence data from 15 apicomplexan species and several crystallographic structures of a variety of apicomplexan protein kinases are now available. We can use these data to perform a systematic comparison of protein kinases in apicomplexans and model eukaryotes to identify broadly conserved orthologous groups and distinctive residue-level differences.
In this study we use a bioinformatics approach to comprehensively analyze genomic and structural data sets. We perform an exhaustive comparison of apicomplexan kinomes, providing broad coverage of the phylum. We also perform a quantitative, residue-level analysis of the differences between kinases within the Apicomplexa and those in model eukaryotes, in particular humans. We use a Bayesian method [33
] to rigorously quantify sequence differences between homologous protein kinases in apicomplexans and other eukaryotes, and reveal contrastingly conserved features that were not apparent previously. Where possible, we then place these sequence features in structural context to postulate specific hypotheses for experimental testing.
Our specific findings include: (i) a detailed accounting of the lineages in which the apicomplexan-specific kinase families FIKK and ROPK appear; (ii) a unique apicomplexan-specific subfamily of cyclin-dependent kinases (CDK), orthologous to P. falciparum crk-5, and the motifs that distinguish it; (iii) a hypothesized mechanism of activation by phosphorylation, resembling that of MAP kinases, in a chromalveolate-specific subfamily of calcium-dependent protein kinases (CDPK); and (iv) a description of the adaptation of the substrate-recognition and docking sites in the CLK kinase family in a clade including apicomplexans and other chromalveolates, revealed by the co-evolution of a small set of key residues.