Co-sedimentation of <i>Pf</i>-Frm1 with <i>Plasmodium</i> and pig actins.

<p><b>A</b>, <i>Pf</i>-Frm1 induces actin polymerization in low-salt conditions. <i>Pf</i>-Act1 and <i>Pb</i>-Act2 were incubated alone and in the presence of <i>Pf</i>-Frm1-FH1FH2, <i>Pf</i>-Frm1-FH2, and <i>Pf</i>-Frm1-FH2Δlasso in G-buffer, followed by ultracentrifugation at 435000 g. The supernatants and pellets were separated, and equal amounts were applied to the SDS gel, which was stained with Coomassie Brilliant Blue. The supernatants and pellets are labeled at the bottom of the gel as S and P, respectively. The presence or absence of the different <i>Pf</i>-Frm versions is indicated with + and − signs above. The first eight lanes contain <i>Pf</i>-Act1, the next eight <i>Pb</i>-Act2, and the last six only the formins in G-buffer. <b>B</b>, All three <i>Pf</i>-Frm versions associate with <i>Plasmodium</i> actin filaments. The actins were allowed to polymerize in the presence or absence of the formins in F-buffer and separated on SDS-PAGE, as in panel A. The first eight lanes contain <i>Pf</i>-Act1, the next eight <i>Pb</i>-Act2, and the last six only the formins in F-buffer. The arrowhead shows the small amount of <i>Pf</i>-Frm1-FH2Δlasso left in the soluble fraction in the presence of <i>Pb</i>-Act2 in F-buffer. <b>C</b>, All three formins also co-sediment with pig muscle actin. The experiment was performed as in panel B, and the presence or absence of the formins is indicated above the figure. The molecular weight standards of 70, 55, and 40 kDa are indicated. The same standards were used in all gels shown. The arrowheads indicate the positions of actin, <i>Pf</i>-Frm1-FH1FH2, <i>Pf</i>-Frm1-FH2, and <i>Pf</i>-Frm1-FH2Δlasso in the gel, from left to right.</p

Solution structures of the <i>Pf</i>-Frm1 domains.

<p><b>A</b>, Raw synchrotron SAXS data for <i>Pf</i>-Frm1-FH1FH2 (red), <i>Pf</i>-Frm1-FH2 (blue), and <i>Pf</i>-Frm1-FH2Δlasso (green). <b>B</b>, Distance distribution functions derived from the SAXS data; coloring as in panel A. <b>C</b>, SANS data for <i>Pf</i>-Frm1-FH1FH2. A radius of gyration (R_g) of 5.7 nm and a maximum particle dimension (D_max) of 18 nm can be estimated from the data. <b>D</b>, Averaged <i>ab initio</i> dummy atom models for <i>Pf</i>-Frm1-FH1FH2 (red) and <i>Pf</i>-Frm1-FH2 (blue) created by DAMMIN. The extensions at the extremities most likely correspond to the FH1 domain. <b>E</b>, Averaged <i>ab initio</i> models for <i>Pf</i>-Frm1-FH2 created by DAMMIN (blue) and GASBOR (cyan). The two figures are related by a 90° rotation about the X-axis. Both methods produce very similar models that fit the data. <b>F</b>, Averaged <i>ab initio</i> model for <i>Pf</i>-Frm1-FH2Δlasso (green) created by DAMMIN, superimposed on the structure of <i>Pf</i>-Frm1-FH1FH2 (red). Note that <i>Pf</i>-Frm1-FH2Δlasso is highly elongated, lacking a compact domain in the middle. <b>G</b>, SRCD spectra for the <i>Pf</i>-Frm1 variants. Coloring as in panel A. <i>Pf</i>-Frm1-FH2 has the highest relative helical content; see text for details on spectral deconvolution.</p

Purification and characterization of the recombinant proteins.

<p><b>A</b>, SDS-PAGE analysis showing the purity of the proteins used in this study. The gel was stained using Coomassie Brilliant Blue. The samples are 1. <i>Pf</i>-Frm1-FH1FH2, 2. <i>Pf</i>-Frm1-FH2, 3. <i>Pf</i>-Frm1-FH2Δlasso, 4. pig muscle actin, 5. <i>Pf</i>-Act1, 6. <i>Pb</i>-Act2, and 7. <i>Pf</i>-Pfn. The molecular weights of the standard proteins in kDa are indicated on the right. The differences in the molecular weights of the <i>Plasmodium</i> and pig actins are accounted for by the 6×His-tags present in the recombinant <i>Plasmodium</i> actins. <b>B</b>, Size-exclusion chromatograms showing a single peak for <i>Pf</i>-Frm1-FH1FH2 (red) and <i>Pf</i>-Frm1-FH2 (blue) and two peaks for <i>Pf</i>-Frm1-FH2Δlasso (green). The molecular weights of the peaks of <i>Pf</i>-Frm1-FH1FH2 and <i>Pf</i>-Frm1-FH2 correspond to the size of dimers, and the two peaks of <i>Pf</i>-Frm1-FH2Δlasso to a dimer and a monomer, as determined by MALS (vertical lines with colors corresponding to those of the chromatograms).</p

Effect of <i>Pf</i>-Frm1 on actin polymerization kinetics.

<p>The effect of <i>Pf</i>-Frm1 on the kinetics of actin polymerization was tested by measuring the change in fluorescence upon incorporation of 5% pyrene-actin into growing actin polymers. The actin concentration used in all experiments was 5 µM. The initial rates (ΔF/s) were calculated as the slope of the linear part (120 s) of the fluorescence curves. In order to facilitate comparison, the samples containing only actin were set to the value 1. <b>A–B</b>, Different concentrations of <i>Pf</i>-Frm1-FH1FH2. <b>C–D</b>, Different concentrations of <i>Pf</i>-Frm1-FH2. <b>E–F</b>, Different concentrations of <i>Pf</i>-Frm1-FH2Δlasso.</p

Effect of <i>Pf</i>-Pfn on actin polymerization kinetics in the presence of <i>Pf</i>-Frm1.

<p>The effect of <i>Pf</i>-Pfn added at a 1∶1 or 2∶1 molar ratio to actin on the kinetics of actin polymerization in the presence of the different <i>Pf</i>-Frm1 domains was tested by measuring the change in fluorescence upon incorporation of 5% pyrene-actin into growing actin polymers. The actin concentration used in all experiments was 5 µM. <b>A</b>, <i>Pf</i>-Pfn together with 10 nM <i>Pf</i>-Frm1-FH1FH2. <b>B</b>, <i>Pf</i>-Pfn together with 10 nM <i>Pf</i>-Frm1-FH2. <b>C</b>, <i>Pf</i>-Pfn together with 10 nM <i>Pf</i>-Frm1-FH2Δlasso.</p

Model for the dimerization of <i>Plasmodium</i> formin 1.

<p><b>A</b>, Sequence alignment between the <i>Pf</i>-Frm1 FH2 domain and the best template identified by Phyre for homology modeling (mouse mDia1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033586#pone.0033586-Nezami1" target="_blank">[43]</a>). <i>Pf</i>-Frm1-FH2 includes both the lasso and linker domains, while <i>Pf</i>-Frm1-FH2Δlasso has only the linker segment. <b>B</b>, Comparison of the dimer homology model of <i>Pf</i>-Frm1 (shown as cartoons; the dimerization in the model is built identical to that seen in the mDia1 template structure 3O4X <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033586#pone.0033586-Nezami1" target="_blank">[43]</a>) with the SAXS model built by using the core FH2 domains as rigid bodies (green) and building the linker, lasso, and FH1 segments by chain-like assemblies of dummy residues (cyan). The SAXS model was made with BUNCH, applying P2 symmetry, but no distance restraints. The Chi-value against the raw SAXS data is 1.4, reflecting a very good fit to the measurement; the fit is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033586#pone.0033586.s001" target="_blank">Figure S1B</a>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033586#pone.0033586.s001" target="_blank">Figure S1</a> also contains further rigid body refinement analysis of the homology model. <b>C</b>, A close-up view into the model of the lasso segment (green) in a <i>Pf</i>-Frm1 dimer; the FH2 domain monomers are indicated in blue and red. The orange segment represents the flexible linker region.</p

Properties of the three formin versions derived from SEC/MALS and SAXS.

<p>The given volume is that of the respective averaged <i>ab initio</i> dummy bead model. The Chi-value describes the fit between the experimental scattering data and the model; with values close to unity reflecting a good fit.</p>*<p>MW = molecular weight.</p>**<p>R_g = radius of gyration.</p>***<p>D_max = maximum particle dimension.</p

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.</p

Data collection and refinement statistics.

Data collection and refinement statistics.</p

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 uM ATP was 1.4x106 M-1 s-1). (Lower) The panel shows the ensemble average of 598 binding events. Each event was synchronized to its start [arrow] and end point [arrow] defined by the change in system stiffness, determined from the amplitude of a 200 Hz sinusoidal forcing function applied by the optical tweezers. The synchronized data were averaged. The average displacement at the start of the event (~4 nm) is smaller than that at the end of the event (~5.5 nm). This observation implies the power stroke is generated in two phases, ~4 nm followed by a further motion of ~1.5 nm.</p