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ABSTRACT The Toxoplasma inner membrane complex (IMC) is a specialized organelle that is crucial for the parasite to establish an intracellular lifestyle and ultimately cause disease. The IMC is composed of both membrane and cytoskeletal components, further delineated into the apical cap, body, and basal subcompartments. The apical cap cytoskeleton was recently demonstrated to govern the stability of the apical complex, which controls parasite motility, invasion, and egress. While this role was determined by individually assessing the apical cap proteins AC9, AC10, and the mitogen-activated protein kinase ERK7, how the three proteins collaborate to stabilize the apical complex is unknown. In this study, we use a combination of deletion analyses and yeast two-hybrid experiments to establish that these proteins form an essential complex in the apical cap. We show that AC10 is a foundational component of the AC9:AC10:ERK7 complex and demonstrate that the interactions among them are critical to maintaining the apical complex. Importantly, we identify multiple independent regions of pairwise interaction between each of the three proteins, suggesting that the AC9:AC10:ERK7 complex is organized by multivalent interactions. Together, these data support a model in which multiple interacting domains enable the oligomerization of the AC9:AC10:ERK7 complex and its assembly into the cytoskeletal IMC, which serves as a structural scaffold that concentrates ERK7 kinase activity in the apical cap. IMPORTANCE The phylum Apicomplexa consists of obligate, intracellular parasites, including the causative agents of toxoplasmosis, malaria, and cryptosporidiosis. Hallmarks of these parasites are the IMC and the apical complex, both of which are unique structures that are conserved throughout the phylum and required for parasite survival. The apical cap portion of the IMC has previously been shown to stabilize the apical complex. Here, we expand on those studies to determine the precise protein-protein interactions of the apical cap complex that confer this essential function. We describe the multivalent nature of these interactions and show that the resulting protein oligomers likely tether ERK7 in the apical cap. This study represents the first description of the architecture of the apical cap at a molecular level, expanding our understanding of the unique cell biology that drives Toxoplasma infections. 

Apicomplexan parasites use a specialized cilium structure called the apical complex to organize their secretory organelles and invasion machinery. The apical complex is integrally associated with both the parasite plasma membrane and an intermediate filament cytoskeleton called the inner-membrane complex (IMC). While the apical complex is essential to the parasitic lifestyle, little is known about the regulation of apical complex biogenesis. Here, we identify AC9 (apical cap protein 9), a largely intrinsically disordered component of the Toxoplasma gondii IMC, as essential for apical complex development, and therefore for host cell invasion and egress. Parasites lacking AC9 fail to successfully assemble the tubulin-rich core of their apical complex, called the conoid. We use proximity biotinylation to identify the AC9 interaction network, which includes the kinase extracellular signal-regulated kinase 7 (ERK7). Like AC9, ERK7 is required for apical complex biogenesis. We demonstrate that AC9 directly binds ERK7 through a conserved C-terminal motif and that this interaction is essential for ERK7 localization and function at the apical cap. The crystal structure of the ERK7–AC9 complex reveals that AC9 is not only a scaffold but also inhibits ERK7 through an unusual set of contacts that displaces nucleotide from the kinase active site. ERK7 is an ancient and autoactivating member of the mitogen-activated kinase (MAPK) family and its regulation is poorly understood in all organisms. We propose that AC9 dually regulates ERK7 by scaffolding and concentrating it at its site of action while maintaining it in an “off” state until the specific binding of a true substrate.