Raw data file for Fig 5A and 5B.

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.

肌动蛋白（actin）是一类在真核生物中可形成丝状体的高度保守蛋白质，其不仅参与细胞质内的关键生命过程，还具备细胞核内的功能。疟原虫（Plasmodium spp.）拥有两种肌动蛋白同工型，二者彼此之间以及与经典肌动蛋白（canonical actin）在结构与丝状体形成特性上均存在差异。肌动蛋白I（actin I）在运动过程中发挥关键作用，其相关特性已得到较为充分的表征。肌动蛋白II（actin II）的结构与功能目前尚不完全明确，但突变分析已揭示其在雄性配子发生以及卵囊阶段具备两项关键功能。本研究针对疟原虫肌动蛋白II开展了表达分析、高分辨率丝状体结构解析以及生化特性表征，证实该蛋白在雄性配子体与合子中均有表达，并发现其在这两个阶段均以丝状体样结构定位于细胞核内。与肌动蛋白I不同，肌动蛋白II在体外可高效形成长丝状体；无论是否添加jasplakinolide，其近原子分辨率结构均极为相似。与其他肌动蛋白相比，肌动蛋白II在开放度与扭转角、活性位点、D环以及插头区域存在细微但显著的差异，这些差异有助于丝状体的稳定性。本研究通过突变分析探究了肌动蛋白II的功能，结果表明长而稳定的丝状体对于雄性配子发生是必需的；而其在卵囊阶段的第二项功能则需要通过组氨酸73的甲基化进行精准调控。肌动蛋白II通过经典的成核-延伸机制进行聚合，其稳态临界浓度约为0.1 μM，与肌动蛋白I以及经典肌动蛋白一致。与肌动蛋白I类似，二聚体是肌动蛋白II在平衡状态下的稳定存在形式。