A Budding-Defective M2 Mutant Exhibits Reduced Membrane Interaction, Insensitivity To Cholesterol, And Perturbed Interdomain Coupling

Influenza A M2 is a membrane-associated protein with a C-terminal amphipathic helix that plays a cholesterol-dependent role in viral budding. An M2 mutant with alanine substitutions in the C-terminal amphipathic helix is deficient in viral scission. With the goal of providing atomic-level understanding of how the wild-type protein functions, we used a multipronged site-directed spin labeling electron paramagnetic resonance spectroscopy (SDSL-EPR) approach to characterize the conformational properties of the alanine mutant. We spin-labeled sites in the transmembrane (TM) domain and the C-terminal amphipathic helix (AH) of wild-type (WT) and mutant M2, and collected information on line shapes, relaxation rates, membrane topology, and distances within the homotetramer in membranes with and without cholesterol. Our results identify marked differences in the conformation and dynamics between the WT and the alanine mutant. Compared to WT, the dominant population of the mutant AH is more dynamic, shallower in the membrane, and has altered quaternary arrangement of the C-terminal domain. While the AH becomes more dynamic, the dominant population of the TM domain of the mutant is immobilized. The presence of cholesterol changes the conformation and dynamics of the WT protein, while the alanine mutant is insensitive to cholesterol. These findings provide new insight into how M2 may facilitate budding. We propose the AH–membrane interaction modulates the arrangement of the TM helices, effectively stabilizing a conformational state that enables M2 to facilitate viral budding. Antagonizing the properties of the AH that enable interdomain coupling within M2 may therefore present a novel strategy for anti-influenza drug design

Screening the Toxoplasma kinome with high-throughput tagging identifies a regulator of invasion and egress

Protein kinases regulate fundamental aspects of eukaryotic cell biology, making them attractive chemotherapeutic targets in parasites like Plasmodium spp. and Toxoplasma gondii. To systematically examine the parasite kinome, we developed a high-throughput tagging (HiT) strategy to endogenously label protein kinases with an auxin-inducible degron and fluorophore. Hundreds of tagging vectors were assembled from synthetic sequences in a single reaction and used to generate pools of mutants to determine localization and function. Examining 1,160 arrayed clones, we assigned 40 protein localizations and associated 15 kinases with distinct defects. The fitness of tagged alleles was also measured by pooled screening, distinguishing delayed from acute phenotypes. A previously unstudied kinase, associated with a delayed phenotype, was shown to be a regulator of invasion and egress. We named the kinase Store Potentiating/Activating Regulatory Kinase (SPARK), based on its impact on intracellular Ca2+ stores. Despite homology to mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1), SPARK lacks a lipid-binding domain, suggesting a rewiring of the pathway in parasites. HiT screening extends genome-wide approaches into complex cellular phenotypes, providing a scalable and versatile platform to dissect parasite biology

Genetic screens reveal a central role for heme metabolism in artemisinin susceptibility

© 2020, The Author(s). Artemisinins have revolutionized the treatment of Plasmodium falciparum malaria; however, resistance threatens to undermine global control efforts. To broadly explore artemisinin susceptibility in apicomplexan parasites, we employ genome-scale CRISPR screens recently developed for Toxoplasma gondii to discover sensitizing and desensitizing mutations. Using a sublethal concentration of dihydroartemisinin (DHA), we uncover the putative transporter Tmem14c whose disruption increases DHA susceptibility. Screens performed under high doses of DHA provide evidence that mitochondrial metabolism can modulate resistance. We show that disrupting a top candidate from the screens, the mitochondrial protease DegP2, lowers porphyrin levels and decreases DHA susceptibility, without significantly altering parasite fitness in culture. Deleting the homologous gene in P. falciparum, PfDegP, similarly lowers heme levels and DHA susceptibility. These results expose the vulnerability of heme metabolism to genetic perturbations that can lead to increased survival in the presence of DHA