Motility of the apicomplexan malaria parasite Plasmodium falciparum is enabled by a multiprotein glideosome complex, whose core is the class XIV myosin motor, PfMyoA, and a divergent Plasmodium actin (PfAct1). Parasite motility is necessary for host-cell invasion and virulence, but studying its molecular basis has been hampered by unavailability of sufficient amounts of PfMyoA. Here, we expressed milligram quantities of functional full-length PfMyoA with the baculovirus/Sf9 cell expression system, which required a UCS (UNC-45/CRO1/She4p) family myosin chaperone from Plasmodium spp. In addition to the known light chain myosin tail interacting protein (MTIP), we identified an essential light chain (PfELC) that co-purified with PfMyoA isolated from parasite lysates. The speed at which PfMyoA moved actin was fastest with both light chains bound, consistent with the light chain–binding domain acting as a lever arm to amplify nucleotide-dependent motions in the motor domain. Surprisingly, PfELC binding to the heavy chain required that MTIP also be bound to the heavy chain, unlike MTIP that bound the heavy chain independently of PfELC. Neither the presence of calcium nor deletion of the MTIP N-terminal extension changed the speed of actin movement. Of note, PfMyoA moved filaments formed from Sf9 cell–expressed PfAct1 at the same speed as skeletal muscle actin. Duty ratio estimates suggested that as few as nine motors can power actin movement at maximal speed, a feature that may be necessitated by the dynamic nature of Plasmodium actin filaments in the parasite. In summary, we have reconstituted the essential core of the glideosome, enabling drug targeting of both of its core components to inhibit parasite invasion.

Motility of the apicomplexan parasite Plasmodium falciparum, the causative agent of malaria, is enabled by the glideosome, a multi-protein complex containing the class XIV myosin motor, PfMyoA. Parasite motility is necessary for invasion into host cells and for virulence. Here we show that milligram quantities of functional PfMyoA can be expressed using the baculovirus/Sf9 cell expression system, provided that a UCS (UNC-45/CRO1/She4p) family myosin co-chaperone from Plasmodium spp. is co-expressed with the heavy chain. The homologous chaperone from the apicomplexan Toxoplasma gondii does not functionally substitute. We expressed a functional full-length PfMyoA with bound myosin tail interacting protein (MTIP), the only known light chain of PfMyoA. We then identified an additional "essential" light chain (PfELC) that co-purified with PfMyoA isolated from parasite lysates. PfMyoA expressed with both light chains moved actin at ~3.8 µm/sec, more than twice that of PfMyoA-MTIP (~1.7 µm/sec), consistent with the light chain binding domain acting as a lever arm to amplify nucleotide-dependent motions in the motor domain. Surprisingly, PfMyoA moved skeletal actin or expressed P. falciparum actin at the same speed. Duty ratio estimates suggest that PfMyoA may be able to move actin at maximal speed with as few as 6 motors. Under unloaded conditions, neither phosphorylation of Ser19 of the heavy chain, phosphorylation of several Ser residues in the N-terminal extension of MTIP, or calcium affected the speed of actin motion. These studies provide the essential framework for targeting the glideosome as a potential drug target to inhibit invasion by the malaria parasite.