Structure and function ofPlasmodiumactin II in the parasite mosquito stages

Abstract

AbstractActins are filament-forming, highly-conserved proteins in eukaryotes. They are involved in essential processes in the cytoplasm and have also nuclear functions. Malaria parasites (Plasmodiumspp.) 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 zygote. Here, we present expression analysis, high-resolution filament structures, and biochemical characterization ofPlasmodiumactin II. We show that it is expressed in male gametocytes and zygotes and associated with the nucleus in both stages in filament-like structures. Unlike actin I, actin II readily forms long filamentsin 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. Mutational analyses suggest that long and stable filaments are necessary for male gametogenesis, while the second function in the zygote stage also requires finetuned regulation by methylation of histidine 73. Actin II polymerizesviathe 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.Significance statementMalaria is a parasitic infection caused byPlasmodiumspp., which belong to the phylum Apicomplexa. In 2020, approximately 627000 people died from nearly 241 million malaria cases registered worldwide. In contrast to other apicomplexan parasites,Plasmodiumspp. encode two actin isoforms. While actin I is part of the glideosome complex, essential for locomotion, actin II is present in the mosquito stages, where it has functions in gametogenesis and in the zygote. The exact function of actin II is still unclear, and information at the molecular level is limited. We show here that actin II is associated with the nucleus in gametocytes and zygotes and performs specific functions requiring both long filaments and fine-tuning by methylation of histidine 73. We determined the structures of the filamentous form of actin II at near-atomic resolution and characterized its polymerization propertiesin vitro. Our study provides a molecular basis for the differences between actins of the malaria parasite and humans, as well as between the two parasite actin isoforms. Furthermore, thein vivostudies provide insights into the function of actin II in the parasite.