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

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

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

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

Crystal structures of <i>Plasmodium</i> actin I and II.

<p>(<b>A</b>) <i>P. berghei</i> actin II (<i>Pb</i>ActII; yellow) superimposed on α-actin (1eqy <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004091#ppat.1004091-McLaughlin1" target="_blank">[39]</a>; cyan). (<b>B</b>) <i>P. falciparum</i> actin I (<i>Pf</i>ActI; red) superimposed on α-actin. In both (<b>A</b>) and (<b>B</b>), ATP, subdomains 1–4, and several regions discussed in the text are indicated. Both N and C termini reside in subdomain 1; the N terminus is visible at the front, and the C-terminal helix is on the back side. Note that the C-terminal helix is not visible in actin I. The C-terminal part and the nearby hydrophobic cluster with Trp357 are shown in the zoomed view on the right and the region involved in intra-filament contacts in subdomain 3 in the box at the lower left corner. The blue and pink dots in (<b>B</b>) indicate the approximate positions of the structural elements shown in detail in (<b>C</b>) and (<b>D</b>), respectively. (<b>C</b>) Lys 207 and Glu188 are at an intimate distance in actin I. A similar salt bridge is present between the corresponding residues in latrunculin-bound α-actin, but the hydrogen-bonding distance is longer without the drug. (<b>D</b>) The proline-rich loop with Gly115 in actin I superimposed on that of α-actin. Note the bending of the loop in actin I, due to the more flexible glycine residue.</p

ATP binding sites of actin I and II.

<p>(<b>A</b>) The Asn17 side chain in actin I is part of a cluster formed by the Asn17 Nδ and main chain N atoms as well as Nζ of Lys19. Together, they could form an oxyanion hole for stabilizing a negative charge on one of the β-phosphate oxygen atoms in a reaction intermediate. (<b>B</b>) The active-site water structure in actin I is conserved, and W39 is in an almost inline position for a nucleophilic attack to the ATP γ-phosphate. (<b>C</b>) The catalytic water in actin II has moved further away from the ATP γ-phosphate, is mobile, and is likely a double conformation of the water bound directly to His161. (<b>D</b>) Phosphate release rates of the wild-type <i>Plasmodium</i> actins in the calcium- or magnesium-bound states compared to α-actin, the actin I–α-actin chimera and the actin I mutants F54Y and G115A. Error bars represent standard deviation (n = 3).</p

Filament structure of <i>Plasmodium</i> actin I compared to α-actin.

<p>(<b>A</b>) The cryo-EM structure of actin I filament at 25 Å resolution (left) in comparison with rabbit skeletal muscle α-actin filtered to a comparable resolution (right; EM database entry EMD-5168 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004091#ppat.1004091-Fujii1" target="_blank">[28]</a>). (<b>B</b>) Symmetry refinement of actin I confirms that the change in cross-over distance is caused mainly by a change in helical rotation when compared with actin II and canonical rabbit α-actin. (<b>C</b>) Fourier Shell correlation of actin I half data sets used for 3D reconstruction. The resolution can be estimated at 25 Å based on the 0.5 criterion.</p

Electron micrographs of <i>Plasmodium</i> actin filaments.

<p>(<b>A</b>) In the absence of stabilizing agents, actin I forms only short structures lacking helical symmetry. (<b>B,C</b>) Actin II readily forms filaments varying from hundreds of nm to 1–2 µm in length. (<b>D,E</b>) In the presence of JAS, both parasite actins form long helical filaments. (<b>F</b>) Length distributions of two <i>Plasmodium</i> actin isoforms and three actin I mutants. Note the logarithmic scale of the Y axis.</p