Reconstitution of the core of the malaria parasite glideosome with recombinant Plasmodium class XIV myosin A and Plasmodium actin

Abstract

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.

Structural role of essential light chains in the apicomplexan glideosome

“…Therefore, we propose that the binding of essential light chains and their respective myosin binding sites mutually induces folding of both components and thereby stiffens the myosin lever arm. In turn, the myosins are capable of undergoing a larger step size and thus increase their speed, which is in agreement with the previous measurements of both T. gondii and P. falciparum myosin A motors [14,16,17].…”

“…However, precipitation occurred again and we speculated that the presence of MTIP bound to PfMyoA is a prerequisite for binding of PfELC. In agreement with this hypothesis, previous reports have shown that PfELC can be co-purified with full-length MyoA only in the presence of MTIP from insect cells [17]. To determine the affinity of PfELC via isothermal titration calorimetry we first formed a complex between MTIP and the PfMyoA neck region peptide (PfMyoA-C, residues 775-816; S2 Fig and see Fig 3B) and then titrated in PfELC.…”

“…Both T. gondii ELCs (TgELC1 and TgELC2) as well as P. falciparum PfELC have been previously shown to bind to the C-terminus of MyoA [13,14,17]. Whereas for PfELC, two binding sites of the PfMyoA C-terminus (PfMyoA residues 786-803 and 801-818; Fig 3A) were identified [14], only one binding site was experimentally confirmed for TgELCs (TgMyoA 775-795; Fig 3A) [15, 16].…”

“…Previous reports have shown that the presence of P. falciparum and T. gondii essential light chains increases the speed of the myosin A motor twofold [14,16,17]. To understand the functional role of ELCs on a molecular level, we characterized TgELC2 in a free and bound state in complex with TgMyoA-C ELC (see Fig 3B).…”

“…MLC1 (in P. falciparum : myosin A tail-interacting protein, MTIP) binds at the very C-terminus of MyoA, while ELC is expected to interact with the C-terminus of MyoA upstream of MLC1 [15]. Two ELC homologs recognizing the same MyoA region, termed TgELC1 and TgELC2, were identified in T. gondii [16], whereas only one PfELC homolog is known in P. falciparum [14, 17]. Myosin together with its light chains and the glideosome associated protein 45 (GAP45) has been shown to form a pre-complex in the earlier stages of intracellular parasite development [7], which subsequently assembles with the remaining glideosome members (GAP40 and GAP50).…”