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. 2020 Oct 13;9:e60581. doi: 10.7554/eLife.60581

Figure 1. Crystal structures of PfMyoA in the Post-rigor (PR) and the Pre-Powerstroke (PPS) states.

(a) (Left) The PfMyoA motor is located in the intermembrane space of the parasite. PfMyoA (blue) binds two light chains, PfELC and myosin tail interacting protein (MTIP). MTIP connects the motor to the glideosome-associated proteins (GAP) complex, which is anchored in the inner membrane complex (IMC). PfMyoA cyclically interacts with PfAct1 filaments, which are bound to adhesins from the parasite plasma membrane (PPM); these adhesins also bind receptors from the host cell plasma membrane (HPM). The displacement of PfAct1 filaments by PfMyoA drives parasite gliding motility. (Right) The crystal structure of the motor domain of PfMyoA has been solved (Robert-Paganin et al., 2019), but the lever arm structure was not known. (b,c) Overall structures of the full-length PfMyoA motor in the PPS and PR states, displayed so that their N-terminal subdomains adopt a similar orientation. As expected, the orientation of the converter and lever arm differs in these two states. (d) The lever arm has been built in these two states of the motor, revealing the structure of the two bound light chains, PfELC and MTIP, displayed here in a similar orientation. The kink in the lever arm helix at the end of the converter (pliant region in orange; last helix of the converter in deep olive green) induces different converter/PfELC interfaces in the PPS (interface A, left) compared to the PR state (interface B, right). To illustrate that the two interfaces are different, two reporter residues are displayed as spheres, A722 from the converter and V83 from PfELC. These residues are the part of interface A but not the part of interface B. (e) and (f) represents the recovery stroke (left) and the powerstroke (right) for PfMyoA and scallop myosin 2 (ScMyo2), respectively. Structures of ScMyo2 used: PR (PDB code 1S5G); PPS (PDB code 1QVI).

Figure 1.

Figure 1—figure supplement 1. The atypical and tunable mechanical cycle of PfMyoA.

Figure 1—figure supplement 1.

(a) Motor cycle of myosin motors. The nucleotide free state, strongly bound to actin, is called rigor. Binding of ATP in the rigor state detaches the head from actin. Upon detachment, the motor first populates the post-rigor state (PR) with ATP bound. Isomerization toward the pre-powerstroke state (PPS) allows ATP hydrolysis. This state binds weakly to actin. The sequential release of the products of hydrolysis drives the lever arm swing and thus the powerstroke, which generates force. Detachment of the motor upon ATP binding starts a new cycle. (b) PfMyoA properties are tuned by a phosphorylation in the N-terminal extension of the motor domain (SEP19). When the motor is phosphorylated, it moves actin with a high velocity spending a short fraction of the cycle strongly bound to actin. When the motor is dephosphorylated, it produces more force at the expense of speed. (c) and (d) show the mechanism of force production in scallop myosin 2 (ScMyo2) and PfMyoA, respectively. In ScMyo2, the mobility of the SH1-helix is a key element for the mechanism driving the powerstroke of most myosins and requires the presence of a conserved glycine (G695), the so-called fulcrum, at the basis of the SH1-helix (Kinose et al., 1996; Kad et al., 2007; Preller et al., 2011). In PfMyoA, the SH1-helix is immobile, due to the presence of a serine at the fulcrum (S691). The lack of mobility of the fulcrum is compensated by a phosphorylatable N-terminal extension (SEP19) and sequence adaptations in the connector.

Figure 1—figure supplement 2. The crystal structure of the PfMyoA-PR state displays a kink at the pliant region.

Figure 1—figure supplement 2.

(a) Overall structure of PfMyoA•ΔNter-PR. (b) PfMyoA•ΔNter-PR and PfMyoA•FL-PR superimpose perfectly (rmsd 0.330 Å), indicating that the deletion does not change or alter the protein fold. (c) The PR state of PfMyoA•FL displays a kink in the lever arm at the pliant region (orange). The converter thus becomes far from the α5* and α5*’ helices, which are destabilized (not modeled since no density is indicated in the electron density map). (d) Crystal packing shows that the kinked lever arm is involved in a large surface of the crystal packing. (e), (f), (g) Small-angle X-ray scattering experiment investigating the conformation of PfMyoA in solution. (e) When the motor is bound to ADP and Pi analogs, the theoretical curve computed from the PfMyoA•FL-PPS structure (chain A) fits well to the SAXS experimental curve (χ2 = 3.32). (f) When the motor is bound to MgADP, the experimental curve fits poorly to the theoretical curve from the kinked PfMyoA•FL-PR crystal structure (χ2 = 302), (g) The SAXS experimental curve fits better to the theoretical curve obtained from a model of the PR state in which no kink occurs at the end of the converter (open conformation) (χ2 = 19.2). (h) A fit of the theoretical curve in the PR condition with the software Oligomer (Ryan et al., 2020). The fit has been performed with the closed PfMyoA•FL-PR structure obtained from the crystal with a kink at the pliant region (interface A) and a computed open PfMyoA•FL-PR structure (interface B from the PfMyoA•FL-PPS structure) (see Figure 1). Calculations predict 98% of the sample in the open conformation (χ2 = 8.69).

Figure 1—figure supplement 3. Electron density in the PfMyoA structures.

Figure 1—figure supplement 3.

For all structures, the lever arm and the connectors are displayed, and the 2Fo-Fc map is shown, contoured at 1.0 σ. (a) PfMyoA•FL-PR at 2.5 Å resolution. (b) PfMyoA•FL-PPS at 3.9 Å resolution. (c) PfMyoA•ΔNter-PR at 3.3 Å resolution.

Figure 1—figure supplement 4. The orientation of the lever arm of PfMyoA in PPS differs in the structures of the MD and of the FL.

Figure 1—figure supplement 4.

(a) The orientation of the converter in the post-rigor state (PR) of PfMyoA is identical in the structures of the full-length (FL) and of the motor domain (MD) constructs (PDB code 6I7D chain A). (b) In the pre-powerstroke state (PPS), the lever arm is 30° more primed in the FL structure compared to that found in the MD structure (PDB code 6I7E). (c) Superimposition on the N-terminal subdomain of PfMyoA.FL-PPS and PfMyoA•MD-PPS. On the left, overall view showing that the conformation of the motor domain is similar for the two structures. On the right, zoom on the transducer and on the connectors of the active site (P-loop, Switch-1 and Switch-2) show that the conformation of these elements, which is characteristic of the state, is highly similar. On the following panels, all the structures are superimposed on the SH2-helix. (d) The positions of both the relay-helix and the converter vary between the FL and the MD constructs. Since the SH1-helix was reported to be immobile in PfMyoA (Frénal et al., 2017), its position only slightly differs between the two constructs. (e) The priming of PfMyoA•FL-PPS is identical to the priming of TgMyoA•MD-PPS (PDB code 6DUE). The communication described between the SH1- and the relay helices (polar bond between S691 and Q494) (Frénal et al., 2017) is similar as well as the orientation of the lever arm. (f) Comparison of the position of the connectors between the PPS and the rigor-like states in PfMyoA (Left), and ScMyo2 (Right). The priming described in the PfMyoA•FL-PPS structure indicates that the SH1-helix remains mostly immobile: this connector is only rotated 10° during the powerstroke. This rotation is of smaller amplitude compared to that of conventional myosins such as ScMyo2 (~30°).