Inhibition of Plasmodium falciparum CDPK1 by conditional expression of its J-domain demonstrates a key role in schizont development

Abstract

PfCDPK1 [Plasmodium falciparum CDPK1 (calcium-dependent protein kinase 1)] is highly expressed in parasite asexual blood and mosquito stages. Its role is still poorly understood, but unsuccessful gene knockout attempts suggest that it is essential for parasite replication and/or RBC (red blood cell) invasion. In the present study, by tagging endogenous CDPK1 with GFP (green fluorescent protein), we demonstrate that CDPK1 localizes to the parasite plasma membrane of replicating and invasive forms as well as very young intracellular parasites and does not appear to be exported into RBCs. Although a knockdown of endogenous CDPK1 was achieved using a destabilization domain, parasites tolerated reduced expression without displaying a phenotype. Because of this, the PfCDPK1 auto-inhibitory J (junction) domain was explored as a means of achieving inducible and specific inhibition. Under in vitro conditions, a fusion protein comprising a J-GFP fusion specifically bound to PfCDPK1 and inhibited its activity. This fusion protein was conditionally expressed in P. falciparum asexual blood stages under the regulation of a DD (destabilization domain) (J-GFP-DD). We demonstrate that J-GFP-DD binds to CDPK1 and that this results in the arrest of parasite development late in the cell cycle during early schizogony. These data point to an early schizont function for PfCDPK1 and demonstrate that conditionally expressing auto-inhibitory regions can be an effective way to address the function of Plasmodium enzymes.

Analysis of Noncanonical Calcium-Dependent Protein Kinases in Toxoplasma gondii by Targeted Gene Deletion Using CRISPR/Cas9

“…In the absence of calcium, CDPKs are autoinhibited due to the fact that the CAD occludes the active face of the kinase domain. Peptides from the junctional domain are capable of inhibiting kinase activity of several CDPKs in vitro (13,14), consistent with the finding that the junctional domain interacts with the kinase domain in the autoinhibited structure (11,12). Upon binding to calcium, the CAD domain completely reorganizes and flips to the backside of the kinase domain, opening the nucleotide and substrate binding pockets to activate the enzyme (11,12).…”

Allosteric activation of apicomplexan calcium-dependent protein kinases

“…We identify a vulnerability in this family of parasite kinases, exposed by their need for stabilization. Through the use of 1B7, we provide proof of principle that this feature can be exploited to inhibit TgCDPK1 and related kinases, including PfCDPK1, a kinase essential for the erythrocytic stages of the malaria parasite (6). Conservation of the residues that mediate allosteric activation suggests that this mechanism may extend to Cryptosporidium CDPK1 and Plasmodium CDPK4.…”

“…A similar autoinhibitory region is part of the CAD of CDPKs (11). Peptide mimetics of this region inhibit both plant (7,8) and parasite kinases (6,21). Based on observations of plant CDPK truncations that appeared constitutively active and Ca 2+ -independent, CDPK KDs-like those of CaMKs-were thought to be intrinsically active (7,21).…”

“…Inhibition of TgCDPK1 (T. gondii CDPK1) phenocopies a conditional knock-down of the kinase by blocking Ca 2+ -regulated secretion (2). The closest paralog of TgCDPK1, TgCDPK3, regulates the initiation of T. gondii egress from host cells (3)(4)(5), and its homolog in Plasmodium falciparum, PfCDPK1, is essential (6).…”

Chemical Genetics to Study Plasmodium Kinases

“…PfCDPK1 has been demonstrated to play critical role in egress of merozoites from mature schizonts using a pharmacological inhibitor, purfalcamine and conditional over-expression of the junction domain [16,17]. Interestingly, PfCDPK1 has also been implicated in microneme secretion and invasion of red blood cells by Plasmodium merozoites [18].…”