Structure and function of Plasmodium actin II in the parasite mosquito stages.

Actins are filament-forming, highly-conserved proteins in eukaryotes. They are involved in essential processes in the cytoplasm and also have nuclear functions. Malaria parasites (Plasmodium spp.) have two actin isoforms that differ from each other and from canonical actins in structure and filament-forming properties. Actin I has an essential role in motility and is fairly well characterized. The structure and function of actin II are not as well understood, but mutational analyses have revealed two essential functions in male gametogenesis and in the oocyst. Here, we present expression analysis, high-resolution filament structures, and biochemical characterization of Plasmodium actin II. We confirm expression in male gametocytes and zygotes and show that actin II is associated with the nucleus in both stages in filament-like structures. Unlike actin I, actin II readily forms long filaments in vitro, and near-atomic structures in the presence or absence of jasplakinolide reveal very similar structures. Small but significant differences compared to other actins in the openness and twist, the active site, the D-loop, and the plug region contribute to filament stability. The function of actin II was investigated through mutational analysis, suggesting that long and stable filaments are necessary for male gametogenesis, while a second function in the oocyst stage also requires fine-tuned regulation by methylation of histidine 73. Actin II polymerizes via the classical nucleation-elongation mechanism and has a critical concentration of ~0.1 μM at the steady-state, like actin I and canonical actins. Similarly to actin I, dimers are a stable form of actin II at equilibrium

Data collection and refinement statistics.

<p>*Values in parentheses are for the highest-resolution shell.</p>#<p>R_meas is the redundancy-independent <i>R</i> factor <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004091#ppat.1004091-Diederichs1" target="_blank">[94]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004091#ppat.1004091-Weiss1" target="_blank">[95]</a>.</p>†<p>CC_1/2 is defined as the correlation coefficient between two random half data sets <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004091#ppat.1004091-Karplus1" target="_blank">[96]</a>.</p

Native PAGE analysis of the <i>Plasmodium</i> actins and α-actin.

<p>(<b>A</b>) <i>Plasmodium</i> actins I (lanes 1–4 and 9) and II (lanes 5–8 and 10) form small oligomers upon storage and after exchange of ATP to ADP. Both parasite actins were studied by native PAGE immediately and 48 h after purification in both ATP and ADP forms. Treatment of ADP-exchanged <i>Plasmodium</i> actins with a high concentration of reducing agent (10 mM TCEP) has no effect on the behavior of either of the actins. Exchange of ATP to ADP in α-actin (lanes 11–14) does not result in changes in the oligomeric state. Nt denotes the nucleotide. TCEP - and + denote either the normal 1 mM or an excessive 10 mM concentration, respectively. The approximate position of the different oligomers, corresponding to lane 2, are given on the left. Note that actin I and II run slightly differently on the gel. (<b>B–F</b>) The relative mobility <i>vs.</i> log MW (circles with the oligomeric state indicated on the side) and relative intensities of bands (bars) extracted from gel images of Coomassie-stained native PAGE gels containing ATP or ADP <i>Plasmodium</i> actin I (<b>B–D</b>) and ADP actin II (<b>E and F</b>) immediately or 48 h after purification. The dark grey bars denote the relative intensity of the bands compared to the most intense band and the light grey bars the relative intensity of the bands compared to the sum of all band intensities.</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

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

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

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

<i>Plasmodium</i> actin I–α-actin chimera forms long filaments.

<p>(<b>A</b>) Electron cryo-micrograph of the chimera filaments. (<b>B</b>) Negatively stained chimera filaments. (<b>C</b>) The crystal structure of the chimera (blue) resembles that of wild-type actin I (red). The zoomed views show the differences in the D-loop around Tyr54 (above) and the C-terminal helix and the hydrophobic residues nearby, which are in the canonical orientation in the chimera, unlike in actin I (below).</p

Cryo-EM image analysis of actin I and II.

<p>(<b>A</b>) Electron cryo-micrographs of actin I and II (left and right, respectively). Side-by-side average power spectrum of actin I and II. (<b>B</b>) Representative class averages from k-means clustering. Center: Histograms of measured cross-over distances reveal a larger half pitch for actin I. (<b>C</b>) Eigen images 1–2 from actin I and II k-means clustering reveal a constant pitch of the one-start helix, whereas Eigen images 3–4 confirm the difference in cross-over distance of actin I and II.</p