Authors

A Mühleip¹;  ¹ Helsinki Institute of Life Science (HILIFE), University of Helsinki, Finland

Discussion

The apicomplexan mitochondrial electron transport chain is essential for parasite survival and displays a divergent subunit composition. We report cryo-electron microscopy structures of an apicomplexan III2–IV supercomplex and of the drug target complex III2. The Toxoplasma gondii supercomplex structure reveals how clade-specific subunits form an apicomplexan-conserved III2–IV interface with a unique, kinked architecture, suggesting that supercomplexes evolved independently in different eukaryotic lineages. A knockout resulting in supercomplex disassembly challenges the proposed role of III2–IV in electron transfer efficiency as suggested for mammals. Nevertheless, III2–IV assembly is critical for parasite fitness. High-resolution cryo-EM structures of the apicomplexan CIII with antimalarial cytochrome-b inhibitors reveal the basis for high-affinity binding. Notably, in the apicomplexan binding site, the preclinical compound ELQ-300 is flipped compared with related compounds in the mammalian enzyme. On the basis of the binding modes and parasite-specific interactions discovered, we designed more potent ELQs with subnanomolar activity against T. gondii. Furthermore, cryo-EM structures with dual-site inhibitors visualise the basis for simultaneous binding of the Qi and Qo catalytic sites, which is a key strategy in overcoming the emergence of drug resistance. Our findings reveal critical evolutionary differences in the role of supercomplexes in mitochondrial biology and provide insight into cytochrome b inhibition, informing future drug discovery.