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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 amore » 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.« less

The mammalian ortholog of the retroviral oncogene v-Eyk, and a receptor tyrosine kinase upstream ofantiapoptotic and transforming signals, Mer (MerTK) is a mediator of the phagocytic process, beinginvolved in retinal and immune cell clearance and platelet aggregation. Mer knockout mice are viableand are protected from epinephrine-induced pulmonary thromboembolism and ferric chloride-inducedthrombosis. Mer overexpression, on the other hand, is associated with numerous carcinomas. AlthoughMer adaptor proteins and signaling pathways have been identified, it remains unclear how Mer initiatesphagocytosis. When bound to its nucleotide cofactor, the high-resolution structure of Mer shows an auto-inhibitedaC-Glu-out conformation with insertion of an activation loop residue intomore » the active site. Mercomplexed with compound-52 (C52: 2-(2-hydroxyethylamino)-6-(3-chloroanilino)-9-isopropylpurine),a ligand identified from a focused library, retains its DFG-Asp-in andaC-Glu-out conformation, butacquires other conformational changes. TheaC helix and DFGL region is closer to the hinge region andthe ethanolamine moiety of C52 binds in the groove formed between Leu593 and Val601 of the P-loop,causing a compression of the active site pocket. These conformational states reveal the mechanisms ofautoinhibition, the pathophysiological basis of disease-causing mutations, and a platform for the devel-opment of chemical probes.« less