Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium

Abstract

Calcium-dependent protein kinases (CDPKs) play pivotal roles in the calcium-signaling pathway in plants, ciliates and apicomplexan parasites, and comprise a CaMK-like kinase domain regulated by a calcium-binding domain in the C-terminus. To understand this intramolecular mechanism of activation, we solved the structures of the autoinhibited (apo) and activated (calcium-bound) conformations of CDPKs from the apicomplexan parasites Toxoplasma gondii and Cryptosporidium parvum. In the apo form, the C-terminal CDPK activation domain (CAD) resembles a calmodulin protein with an unexpected long helix in the N-terminus that inhibits the kinase domain in the same manner as CaMKII. Calcium binding triggers the reorganization of the CAD into a highly intricate fold, leading to its relocation around the base of the kinase domain to a site remote from the substrate-binding site. This large conformational change constitutes a distinct mechanism in calcium signal transduction pathways.

The kinomes of apicomplexan parasites

“…As shown with the T. gondii CDPK1 orthologue (TgCDPK1), activation of the enzyme upon calcium binding occurs through a large conformational change. The binding of calcium reorganizes the C-terminal CDPK activation domain (CAP) causing its relocation to a site distant from the substrate binding site [54]. Moreover, TgCDPK1 has been shown to be an essential regulator of calcium-dependent exocytosis by controlling the calcium-dependent secretion of micronemes, and blocking TgCDPK1 inhibits parasite motility, host-cell invasion and egress [9].…”

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

“…However, instead of the kinase domain being regulated by a separate calmodulin protein, CDPKs have a fused domain structure between an N-terminal serine threonine protein kinase and a C-terminal calcium activation domain (CAD) that consists of a calmodulin-like module linked to the kinase domain by a junctional domain (11,12). A number of CDPKs have been crystallized, and analysis of their structures has led to an understanding of their unique activation mechanism (11,12). In the absence of calcium, CDPKs are autoinhibited due to the fact that the CAD occludes the active face of the kinase domain.…”

Calcium/Calmodulin-Dependent Protein Kinase Is Negatively and Positively Regulated by Calcium, Providing a Mechanism for Decoding Calcium Responses during Symbiosis Signaling

“…This is based on in vitro studies that have shown that autophosphorylation of Thr-271 is Ca 2+ dependent (Takezawa et al, 1996) and by analogy to structural studies of CDPK (Wernimont et al, 2010(Wernimont et al, 2011) that reveal that Ca 2+ binding can cause a large conformational change, moving the C-terminal domain away from the active site enabling ATP and substrate access. Following Shimoda et al (2012), we assumed that the phosphorylatable species is in the non-Ca 2+ -activated form.…”

“…These are structures of CDPKs (Wernimont et al, 2010(Wernimont et al, , 2011 with and without Ca 2+ that had a high structural similarity (C-a root mean square deviation [RMSD] of 0.81 Å) to the top ab initio-predicted structure from I-Tasser (Zhang, 2008;Roy et al, 2010). Despite some key differences, such as the lack of a CaM binding domain, CDPKs have some striking features in common with CCaMK, such as the kinase domain, a linker region, and an EF-hand domain.…”