Abstract
The cyclic guanosine-3',5'-monophosphate (cGMP)-dependent protein kinase (PKG) was identified >25 y ago; however, efforts to obtain a structure of the entire PKG enzyme or catalytic domain from any species have failed. In malaria parasites, cooperative activation of PKG triggers crucial developmental transitions throughout the complex life cycle. We have determined the cGMP-free crystallographic structures of PKG from Plasmodium falciparum and Plasmodium vivax, revealing how key structural components, including an N-terminal autoinhibitory segment (AIS), four predicted cyclic nucleotide-binding domains (CNBs), and a kinase domain (KD), are arranged when the enzyme is inactive. The four CNBs and the KD are in a pentagonal configuration, with the AIS docked in the substrate site of the KD in a swapped-domain dimeric arrangement. We show that although the protein is predominantly a monomer (the dimer is unlikely to be representative of the physiological form), the binding of the AIS is necessary to keep Plasmodium PKG inactive. A major feature is a helix serving the dual role of the N-terminal helix of the KD as well as the capping helix of the neighboring CNB. A network of connecting helices between neighboring CNBs contributes to maintaining the kinase in its inactive conformation. We propose a scheme in which cooperative binding of cGMP, beginning at the CNB closest to the KD, transmits conformational changes around the pentagonal molecule in a structural relay mechanism, enabling PKG to orchestrate rapid, highly regulated developmental switches in response to dynamic modulation of cGMP levels in the parasite.
Highlights
The cyclic guanosine-3′,5′-monophosphate-dependent protein kinase (PKG) was identified >25 y ago; efforts to obtain a structure of the entire protein kinase G (PKG) enzyme or catalytic domain from any species have failed
Mammalian PKGs are classified into types Iα, Iβ, and II, all featuring a single polypeptide comprising a regulatory domain of two cyclic nucleotide-binding domains (CNBs) fused to the Nterminal flank of a single catalytic kinase domain (KD)
In all apicomplexan PKGs examined, one of the four CNBs is similar in sequence to canonical cGMP-binding domains but lacks one or more critical residues [23]
Summary
The cyclic guanosine-3′,5′-monophosphate (cGMP)-dependent protein kinase (PKG) was identified >25 y ago; efforts to obtain a structure of the entire PKG enzyme or catalytic domain from any species have failed. Regulation of cGMP has been associated with calcium flux and egress in the related apicomplexan pathogens Toxoplasma and Eimeria [10] These lines of evidence support PKG as a promising target for antiparasitic drug discovery, as well as a gateway to a deeper understanding of parasite signaling. The regulatory and catalytic domains of PKG coexist within a single polypeptide encoded by a single gene In mammals, this assembly contains two cyclic nucleotide-binding (CNB) sites, compared with the four CNB domains in apicomplexan PKG isoforms [14]. Even though PKA and PKG were both among the earliest kinases identified, understanding of how the structural divergence of PKG from PKA affects cGMPmediated allostery and cooperativity [15] in different organisms is at best fragmentary
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call