Ancient MAPK ERK7 is regulated by an unusual inhibitory scaffold required for Toxoplasma apical complex biogenesis

Abstract

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.

Keywords: cilium; intrinsically disordered protein; kinase; scaffold.

Fig. 1.

AC9 is required to complete the parasite lytic cycle. (A) AC9^AID-3×HA (green) localizes to the apical cap (magenta) and is lost when parasites are treated with IAA. (Scale bars, 5 μm.) (B) Quantification of plaque number comparing growth of parental, AC9^AID, and WT-complemented AC9^AID parasites grown with and without IAA. (C) AC9^AID parasites naturally egress from host cells in −IAA but are found as floating vacuoles when grown in IAA. (Scale bars, 20 μm.) (D) Quantification of egress of the indicated strains induced by a calcium ionophore and grown in ±IAA. (E) Quantification of invasion of the indicated strains grown in ±IAA. (F) Western blot of soluble secreted proteins from AC9^AID and AC9^AID/IAA. Microneme secretion was tracked with anti-MIC2 and the constitutively secreted dense granule protein GRA39 was used as a control. All error bars are SD.

Fig. 2.

Loss of AC9 disrupts the parasite apical complex. (A) AC9^AID and AC9^AID/IAA parasites were stained with ROP2 (green) and RON11 (magenta). (B) AC9^AID and AC9^AID/IAA parasites were stained with antibodies to the HA tag (magenta) and the conoid marker SAS6L (green). Arrowheads indicate the position of the maternal apical complex. (Scale bars, 5 μm.) (D–F) TEM images of the apical complex from negatively stained detergent-extracted (C) AC9^AID parasites, (D) AC9^AID/IAA parasites, and AC9^AID WT-complemented parasites grown in (E) −IAA and (F) +IAA.

Fig. 3.

AC9 tightly binds ERK7. (A) ERK7-Ty (green) colocalizes with AC9-HA (magenta) at the apical cap. (B) Proximity ligation (magenta) of ERK7 and AC9 reveal bright foci at the parasite apical cap. Parasites are counterstained with anti–β-tubulin (note that this antibody does not stain the apical complex, likely due to antigen accessibility). (Scale bars, 5 μm.) (C) Sequence logo for AC9_419–452 highlights invariant C-terminal residues. (D) Binding of AC9_419–452 to ERK7_1–358 was measured by fluorescence polarization and the K_D was calculated from global fit of three replicate experiments of three technical replicates each.

Fig. 4.

AC9 is a scaffold that drives ERK7 apical localization. (A and B) ERK7-3×myc (green) localization is lost upon degradation of (A) AC9^AID-3×HA with growth in IAA (magenta), which is rescued in the (B) AC9 WT complement. White arrowheads indicate the apical cap. (C) K_I of WT AC9_401–452 and 3×Glu AC9_401–452 was determined by competition with fluorescein-labeled AC9_419–452; 95% CI: WT, 13.4 to 17.7 nM; 3×Glu, 2.4 to 3.6 μM. (D) ERK7-3×myc (green) localization was compared in 3×Glu-complemented (magenta) parasites as in A and B. (E) Growth of 3×Glu-complemented (green) AC9^AID parasites expressing mCherry-tubulin (magenta) with or without IAA. Orange arrowheads indicate the expected location of conoid foci. (F) Quantification of plaque number comparing growth of AC9^AID, WT-complemented AC9^AID, and 3xGlu-complemented AC9^AID parasites grown with and without IAA. (Scale bars, 5 μm.) All error bars are SD.

Fig. 5.

AC9 binds ERK7 in an inhibitory conformation. (A) Overview of AC9–ERK7 interaction. ERK7 is blue. A 1.5σ 2F_O − F_C electron density map (black) is shown around AC9 (yellow). (B–G) Contacts between AC9 (yellow) and ERK7 (blue) are compared with ERK2 (gray) at (B and C) the MAPK-docking domain, (D and E) activation loop/substrate binding site, and (F and G) kinase active site. (H) K_I of AC9 mutants was determined by competition with fluorescein-labeled wild-type AC9. Wild-type competition curve (K_I 15 nM) is shown for comparison; 95% CI: AC9_W438A, 1.3 to 2.0 μM; AC9_R421A/K423A (RK/AA), 55 to 74 nM. ERK2 images are from PDB ID codes 4H3Q (C) and 6OPG (E and G). All error bars are SD.

Fig. 6.

AC9 is an inhibitory regulator of ERK7 kinase activity. (A) Quantification of phosphorylation of MBP by the indicated kinases in the presence and absence of 10 μM AC9_401–452, normalized to activity without AC9. (B) Binding of kinase-interacting-motif from rat MEK2 to TgERK7 was measured by fluorescence polarization; 95% CI: 10.5 to 14.5 μM. (C) Quantification of TgERK7 phosphorylation of MBP or a chimeric ELK1/MEK2_4–16 substrate in the presence of the indicated AC9_401–452 concentration (n = 3 biological replicates). (D) Model for AC9 regulation of ERK7 specificity. AC9 occupies the ERK7 active site, preventing the binding of nonspecific substrates. However, the AC9 docking-site interaction is suboptimal and can be competed off by true ERK7 substrates, which are released after phosphorylation, allowing AC9 to rebind. All error bars are SD.