High-resolution structures of malaria parasite actomyosin and actin filaments

Figures

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  Fig 1. High-resolution reconstructions of filamentous Act1 (left) and MyoA-decorated Act1 (right). In the density maps, the central individual actin protomers and myosins are depicted in different colors. Jasplakinolide (JAS) and ADP are shown in red and yellow, respectively. The top middle part shows density maps around individual protein molecules and small molecule ligands as labeled. The bottom row shows density maps of selected structural motifs, as labeled, within the interfaces in the complexes. Additionally, density around phosphorylated Ser19 (S19-P) is shown.

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  Fig 2. A model of the light chains ELC and MTIP bound to MyoA. (A) An unsharpened map (gray) filtered with LAFTER shows the location of the MyoA motor (blue) and the light chains ELC (magenta) and MTIP (cyan). (B) The Act1:MyoA:ELC:MTIP model in the rigor state shown in the context of the membrane-delimited sub-pellicular compartment. An outline of GAC (dotted lines) in two randomly chosen orientations with approximate dimensions derived from the T. gondii GAC small-angle X-ray scattering (SAXS) low-resolution envelope [16]. (C) Results of single molecule optical tweezer assays are presented. (Upper) A displacement histogram for MyoA:ELC:MTIP at 2.5 μM ATP. Power stroke was determined as the shift in the Gaussian peak from 0 nm (gray line), which has an average ∼4.5 nm. (middle) An attached lifetime histogram for muscle actin-MyoA:ELC:MTIP events measured at 2.5 μM ATP. The detachment rate (grey line) was determined by fitting a single-exponential to the histogram. The event lifetime distribution (middle panel) fitted well to a mono-exponential decay (least squares fit gives a rate constant 4.1 s-1) consistent with detachment being controlled by ATP binding to the rigor complex (at 2.5…

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  Fig 3. Molecular interactions of MyoA with Act1. (A) MyoA is shown as cartoon and Act1 as surface representation. The two adjacent actin protomers are colored with distinct colors. The MyoA motifs contributing to the interface as well as MyoA subdomains, and the N-terminal helix and SH3 domain are highlighted and labeled. (B) The HCM-loop lies on one actin protomer, contacting actin SD1. The basic loop 2 (Lys632 and Lys634) interacts with the acidic N-terminus of Act1. (C) The HTH motif in the lower 50K domain contributes most specific hydrogen bonds in the interface, interacting with the D-loop (SD2) and the C-terminal patch of Act1 between SD1 and SD3 in close vicinity of the D-loop in the adjacent actin subunit. (D) Loop 3 and the HTH motif visualized approximately 190° rotated with respect to panel A. Loop 3 (576–577) interacts with the adjacent actin monomer on SD1. The activation loop in the lower 50K domain protrudes towards the next SD1, with Lys534 interacting with the actin N-terminus.

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  Fig 4. Internal cavities in filamentous and monomeric actin structures. The internal cavities were calculated using the CASTp server [74] and visualized as transparent surfaces using Chimera [66]. (left) The internal cavity in the Act1 filament and (middle) the corresponding channel in monomeric Act1 (4cbu; [15]). (right) The corresponding cavity in muscle actin (6djm; [38]) is shorter due to change from Ile76 to Val77 in Act1.

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  Fig 5. Details of the nucleotide binding site in the Act1 filament. (left) Density map around the nucleotide-binding site, including ADP (sticks), Mg2+ (green sphere) with coordinating water molecules and putative water molecules (red spheres) in the internal cavity. (right) Schematic presentation of ADP interactions in the filamentous Act1 active site.