The Cryptosporidium parvum kinome

Abstract

Background: Hundreds of millions of people are infected with cryptosporidiosis annually, with immunocompromised individuals suffering debilitating symptoms and children in socioeconomically challenged regions at risk of repeated infections. There is currently no effective drug available. In order to facilitate the pursuit of anti-cryptosporidiosis targets and compounds, our study spans the classification of the Cryptosporidium parvum kinome and the structural and biochemical characterization of representatives from the CDPK family and a MAP kinase.

Results: The C. parvum kinome comprises over 70 members, some of which may be promising drug targets. These C. parvum protein kinases include members in the AGC, Atypical, CaMK, CK1, CMGC, and TKL groups; however, almost 35% could only be classified as OPK (other protein kinases). In addition, about 25% of the kinases identified did not have any known orthologues outside of Cryptosporidium spp. Comparison of specific kinases with their Plasmodium falciparum and Toxoplasma gondii orthologues revealed some distinct characteristics within the C. parvum kinome, including potential targets and opportunities for drug design. Structural and biochemical analysis of 4 representatives of the CaMK group and a MAP kinase confirms features that may be exploited in inhibitor design. Indeed, screening CpCDPK1 against a library of kinase inhibitors yielded a set of the pyrazolopyrimidine derivatives (PP1-derivatives) with IC₅₀ values of < 10 nM. The binding of a PP1-derivative is further described by an inhibitor-bound crystal structure of CpCDPK1. In addition, structural analysis of CpCDPK4 identified an unprecedented Zn-finger within the CDPK kinase domain that may have implications for its regulation.

Conclusions: Identification and comparison of the C. parvum protein kinases against other parasitic kinases shows how orthologue- and family-based research can be used to facilitate characterization of promising drug targets and the search for new drugs.

Figure 1

Maximum likelihood tree from a rapid bootstrap analysis describing the classification of the 73 C. parvum protein kinases. The families are colour-coded as follows: AGC (yellow), CK1 (orange), Atypical (pink), CaMK (blue), CMGC (green), TKL (red), and OPK (grey).

Figure 2

Maximum likelihood tree from a rapid bootstrap analysis for C. parvum CaMK family, also including select representatives of the CaMK families from human, P. falciparum, and T. gondii. The subfamilies are as follows: CDPK (green), CaMK (blue), SNF1/AMPK (orange), and potential apicomplexan CaM-binding kinases (pink). Note that although PFB0815w is annotated as CDPK1, it is the orthologue to TgCDPK3 (TGME40_105860) and PF07_0072 is annotated as PfCDPK4, but is actually the orthologue to TgCDPK1 (TGME49_101440).

Figure 3

Overall structures of CpCDPK enzymes. The green circles highlight important features within the structures. In the CpCDPK1 structure, the binding of the PP1 inhibitor is shown. The α-helix D orientation is highlighted in the apo CpCDPK2 structure. For the CpCDPK4 structure, the Zn-finger and β-mesh position are shown. In the CpMAP-1 structure, the unusually long α-L16 helix is shown.

Figure 4

Comparison of CpCDPK1 structures. (A) Overall comparison of CpCDPK1 in apo form [PDB: 3DFA] (beige) and CpCDPK1- PP1-derivative bound [PDB: 2WEI] (green). (B) Active sites of CpCDPK1 structures showing some of the residues (numbering from the PP1-bound structure PDB: 2WEI) involved in binding a PP1-derivative.

Figure 5

Comparison of CpCDPK2 structures. CpCDPK2 in apo form [PDB: 2QG5] (yellow) and CpCDPK2- indirubin E804 bound [PDB: 3F3Z] (orange) shows the shift in the glycine-rich loop.

Figure 6

Structural analysis of CpCDPK4. (A) Overall view of CpCDPK4 [PDB: 3HKO] with the zinc finger and β-mesh insert highlighted in lime. (B) Zn-finger of CpCDPK4 is shown with the residues involved in zinc binding noted. (C) Overlay of aligned views of CpCDPK4 with the C-terminus of the KD and entire CAD of TgCDPK1 (for visual clarity the N-terminal portion of the KD is not shown) in grey [PDB: 3KU2]. (D) An overall structural comparison of CpCDPK4 (teal/lime) and PKC-ι (salmon/magenta) [PDB:3AX8] with the C-terminal tail (from K531) highlighted in magenta and a circle indicating its overlap with CpCDPK4. (E) An overall structural comparison of CpCDPK4 (teal/lime) and human CLK1 (pale yellow) [PDB:1Z57]. (F) An overall structural comparison of CpCDPK4 (teal/lime) and human SRPK (beige) [PDB:1WBP].

Figure 7

Comparison of CpMAP-1 with the rat ERK2. CpMAP-1 [PDB: 3OZ6] is shown in blue, while ERK2 [PDB: 2ERK] is in beige.