Structures of the cGMP-dependent protein kinase in malaria parasites reveal a unique structural relay mechanism for activation

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 fromPlasmodium falciparumandPlasmodium 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 keepPlasmodiumPKG 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.

cGMP: a unique 2nd messenger molecule – recent developments in cGMP research and development

“…Further data showed that CNB-A and -B modulated kinase activity whereas CNB-C had no obvious function. Additional crystallographic data confirming these results and revealing further information on the regulation of PKG activation by AIS and the different CBDs have just been published (El Bakkouri et al 2019).…”

Mechanism of allosteric inhibition in the Plasmodium falciparum cGMP-dependent protein kinase

“…To simplify the analysis, we focused on the C-terminal helices that are directly linked to the catalytic domain and are one of the allosteric elements most critical in the inhibition and activation of the kinase (14,21,24). The C-terminal helices span two regions with clearly distinct average X values (<X>) in the PfD:8-NBD-cGMP complex: residues 515-530, denoted as the pre-lid with <X> = -0.48, and residues 534-535, denoted as the lid with <X> = -0.88 ( Fig.…”

“…The structure of 8-NBD-cGMP in a syn conformation was generated using RDKit (47). A model of the mixed intermediate state sampled by the PfD:8-NBD-cGMP complex was then built using residues 401-533 from the active PfD:cGMP crystal structure (PDB code: 4OFG) (14), and lid region residues 534-542 from the inactive apo PfD crystal structure (PDB code: 5DYK) (24). The 8-NBD-cGMP ligand was docked into the resulting mixed structure of PfD using AutoDock4 with default parameters generated by AutoDockTools (48).…”

“…Additionally, R528 in the αC plays a pivotal role in regulating PfPKG activity by controlling the αC conformation through a hydrogen-bond with Y480, which stabilizes the active conformation ( Fig. 1D) (14,21,24).…”

An Update on Development of Small-Molecule Plasmodial Kinase Inhibitors

“…Thus, PKG inhibitors have the potential to be active against multiple malaria species. Although human PKG orthologues (cytosolic PKG-Iα and PKG-Iβ, membrane-associated PKG-II) exist, selective inhibitor development is still possible as there is significant structural divergence between plasmodial and mammalian PKGs [74].…”