Structure-function relationships of kinesin molecular motors in Plasmodium parasites

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Strategic Research Priority: World Class BioscienceAbstract Kinesins are ATP-dependent microtubule-based motors with multiple essential roles in eukaryotes, including parasitic protozoa. The intracellular Plasmodium parasite infects >200 million people annually, killing nearly 1 million. The complex parasite life cycle presents numerous challenges to text-book concepts of cell biology. The few drugs available and emerging drug resistance also mean that novel drug targets are urgently needed. Mitotic kinesins are targets for cancer therapeutics and could form attractive targets for anti-malarials. Little is known about kinesins in Plasmodium, so the aim of this project is to combine structural, biochemical and parasitology approaches to mechanistically and functionally characterize these motors. Project This project will elucidate the structure-function relationship of Plasmodium kinesins, with a focus on kinesin-13 and kinesin-8 motors. Their molecular mechanisms, biological roles, and sensitivity to inhibition, primarily in P. falciparum, will be investigated. Microtubules in all eukaryotes, including Plasmodium, undergo dramatic cell cycle-dependent reorganisation, particularly during cell division. A subset of kinesins - principally kinesin-13s and kinesin-8s - are implicated in microtubule remodeling and reflect an important, but poorly understood, divergence of the typical kinesin stepping mechanism. Kinesin-13s are known to be microtubule depolymerisers, using ATP to shorten microtubules; we have shown that recombinant subdomains of kinesin-13 motors from both P. falciparum and humans depolymerise microtubules. However, the specifics of their activities suggest there are mechanistic differences between motors from each organism. The kinesin-8 mechanism is more controversial. It is essential to discover how sequence homology/divergence between Plasmodium kinesins and those from higher organisms is reflected mechanistically. Differences potentially render parasite enzymes susceptible to specific inhibition by anti-malaria treatments. We will use cryo-electron microscopy to structurally characterize motor mechanisms. Another central question is what role individual kinesins play in the parasite. Novel technologies for manipulating the otherwise genetically challenging P. falciparum - including Zinc Finger Nuclease Technology - will enable efficient assessment of the role of kinesins in Plasmodium. The objectives of the project are: 1) Purify recombinant P. falciparum kinesin-13 and kinesin-8 motor domains; 2) Biochemically/structurally characterize them and their microtubule interaction using cryo-electron microscopy; 3) Screen for inhibitors using small molecule libraries; 4) Determine expression profiles of kinesin-13/8 to understand their biological roles; 5) Compare sequences of kinesin-13/8 across Plasmodium species; 6) Generate transgenic parasite lines of modified kinesin-13/8 in P. falciparum to specify their functions. Thus, this project will involve the study of both the molecular mechanisms and functional contribution of kinesins in intracellular parasites.