Understanding the molecular mechanism of Plasmodium actin-based gliding motility

Malaria is one of the most devastating global diseases. The disease is caused by unicellular eukaryotic parasites that are transmitted to the human host by mosquitoes. These parasites use a particular form of motility, termed gliding, to get into their host cells. Force for gliding is generated by a molecular motor complex, the central players of which are the two proteins, actin and myosin. This project aims to understand the molecular basis of the function of this motor. The results will be important from many aspects. From the evolutionary point of view, the malaria parasites are far from humans in the evolutionary tree, and understanding their motility gives clues to the development of cell motility in general. Because of the evolutionary distance, the proteins studied here are attractive targets for drug and/or vaccine development against malaria. In addition, studying cell motility at the molecular level enhances our understanding of fundamental cellular processes from development to disease, including many infectious diseases, cardiac and skeletal muscle disorders, and cancer. In this project, we use most modern structural biology techniques complemented by biochemical and biophysical methods to gain a holistic picture of the motor complex of an important human pathogen. Our most important results from this project are high-resolution structures of the malaria parasite actin alone and decorated with the motor protein MyoA. These structures give new insight into how actin polymerization dynamics are regulated by ATP hydrolysis and phosphate release and how MyoA generates force for parasite motility.

The overall objective of this project was to understand how force is generated in the apicomplexan actin-based gliding motility. The specific goals were: 1. To resolve actin protomer conformations within the filament at different stages of polymerisation and elucidate the link between ATP hydrolysis and polymerization. 2. To establish recombinant expression systems for six Plasmodium myosins and characterize them structurally and functionally. 3. To characterize the physical interactions of Plasmodium F-actin with myosins. Goal no. 1 was fully reached and the work has been published (Kumpula et al. PNAS 2018 and Kumpula et al. PLoS Biol 2019). Goal no. 2 was partly reached, as we have established recombinant expression systems for four out f the six Plasmodium myosins. We have performed biochemical and biophysical characterization of three of these. Goal no. 3 was reached and we are in the process of getting the Plasmodium actin I - MyoA complex published.

Malaria is one of the most devastating global diseases. The disease is caused by unicellular eukaryotic parasites of the genus Plasmodium, which belong to the phylum Apicomplexa. Apicomplexan parasites use a particular form of motility, termed gliding, to traverse tissues and enter host cells. Force for gliding is generated by an unconventional actin-myosin motor, which is part of a large membrane-associated protein complex called the glideosome. Most components of the glideosome are known, but detailed molecular mechanisms of how force is generated and transformed into rapid and smooth gliding are not understood. Apicomplexa are early-branched organisms, and our work is aimed at shedding light on the evolutionary relationships of the cytoskeleton and motor proteins in these parasites and higher eukaryotes and comparing the ways in which force is produced for different forms of cell motility. Glideosome proteins, including actin and myosin, are either unique to Apicomplexa or highly divergent from their human counterparts. Thus, they are attractive targets for drug and/or vaccine development. In addition, understanding cell motility at the molecular level enhances our understanding of fundamental cellular processes from development to disease, including many infectious diseases, cardiac and skeletal muscle disorders, and cancer. The specific objectives of the proposed project include (i) characterizing the structure of Plasmodium actin filaments and its complexes at high resolution, (ii) elucidating the mechanism of ATP hydrolysis and its link to actin polymerization, (iii) structural and functional characterization of all Plasmodium myosins, and (iv) structure determination of Plasmodium actin-myosin complexes. We use most modern structural biology techniques complemented by biochemical and biophysical methods to gain a holistic understading of the motor complex. Particularly innovative is our novel approach to generate crystallizable actin filament mimics.