Structural basis of Toxoplasma gondii MIC2-associated protein interaction with MIC2

Abstract

Toxoplasma gondii parasites must actively invade host cells to propagate. Secretory microneme proteins have been shown to be important for both gliding motility and active invasion. MIC2-M2AP is a protein complex that is essential for productive motility and rapid invasion by binding to host cell surface receptors. To investigate the architecture of the MIC2 and M2AP complex, we identified the minimal domains sufficient for interaction and solved the NMR solution structure of the globular domain of M2AP. We found that M2AP adopts a modified galectin fold similar to the C-terminal domain of another microneme protein, MIC1. NMR and immunoprecipitation analyses implicated hydrophobic residues on one face of the M2AP galectin fold in binding to the membrane proximal sixth thrombospondin type I repeat domain of MIC2. Our findings provide a second example of a galectin fold adapted for microneme protein-protein interactions and suggest a conserved strategy for the assembly and folding of diverse protein complexes.

Keywords: Cell Invasion; Galectin; M2AP; MIC2; Microneme; Nuclear Magnetic Resonance (NMR); Protein Complex; Toxoplasma gondii.

FIGURE 1.

Interaction of MIC2 domain deletion mutants with M2AP. Expression and IP of constructs in EtM1/TgM2KO/EtM2 parasites (A and B) or in CHO cells (C). The top blots show expression of the constructs as detected by immunoblotting (IB) with MsαMyc, and the bottom blots show IP with RbαM2AP and detection of MIC2 constructs (A–C) with MsαMyc. A, top, schematic showing the full-length (IMC), ΔI, and ΔM MIC2 with a Myc tag upstream of the TM domain, and mature M2AP. B, top, schematic showing the TSR domains deleted in each of the constructs. C, left, schematic of constructs containing individual TSRs co-expressed in CHO cells with mature M2AP (termed ΔproM2AP).

FIGURE 2.

The NMR-derived solution structure of the M2AP β-domain and chemical shift mapping of the M2AP-MIC2 interaction. A, the final family of 10 NMR structures from the final ARIA iteration of solution structure calculation of M2AP. B, schematic representation of the solution structure of M2AP showing the juxtaposition of the β strand in β sandwich. A representative structure has been chosen from the low energy family and displayed in the same orientation as in A. C, schematic representation of the superposition of the structures of M2AP β-domain (orange) and TgMIC1-CT (green) bound to the second EGF of TgMIC6 (cyan) (Protein Data Bank code 2K2S). D, solvent-accessible hydrophobic patch shown in blue on surface representation of M2AP. Residues delineating the surface are indicated. E, overlay of two-dimensional ¹H,¹⁵N HSQC spectra of ¹⁵N,¹³C-labeled M2AP in the presence (red) and absence (black) of 6-fold molar excess of unlabeled TSR5–6. Selected resonance assignments are indicated. F, molecular surface of M2AP color-coded is in red according to chemical shift perturbations upon interaction with TSR5–6. The structure is shown along the β2-β11-β4-β5-β6-β7 face (left) similar to the views in A and B. The β1-β12-β3-β8-β9-β10 face is also shown (right) in a similar orientation as C and D with the hydrophobic patch delineated and shaded in blue.

FIGURE 3.

Positively charged residues on the β2-β11-β4-β5-β6-β7 face of M2AP are not required for interaction with MIC2 TSR6. A, expression of M2AP mutants and TSR6 in CHO cells (lysate lanes) and immunoprecipitation with RbαM2AP followed by detection of M2AP mutants and TSR6 with MsαMyc (IP lanes). B, quantification of expression versus interaction of M2AP mutants with TSR6. Interaction:expression ratios for mutant M2APs were normalized to that of WT M2AP set to 100%. Data are compiled from three independent experiments. Error bars represent S.D. n.s., not significant.

FIGURE 4.

Several residues in the hydrophobic patch of mature M2AP are required for interaction with TSR6 and TSR5–6 in CHO cells. A and C, expression of M2AP mutants and TSR5–6 or TSR6 (Lysate lanes) in CHO cells and IP with RbαM2AP. B and D, quantification of expression versus interaction of M2AP mutants with TSR6 or TSR5–6. Interaction:expression ratios for mutant M2APs were normalized to that of WT M2AP set to 100%. Data were compiled from at least three independent experiments. *** p value < 0.001; ** p value < 0.006. Error bars represent S.D. n.s., not significant. E, upfield methyl region from the ¹H NMR spectra for the four M2AP mutants overlaid with that of wild type M2AP. The WT and W134A major peaks are truncated.

FIGURE 5.

Expression of M2AP hydrophobic patch mutants in Δm2ap parasites confirms their role in tight binding to MIC2. A, immunofluorescence staining of M2AP and MIC2 to assess trafficking to the micronemes. Overnight replicated RH wildtype or Δm2ap parasites were fixed and stained with rabbit anti-M2AP and mouse mAb 6D10 anti-MIC2. Arrows indicate retention in a post-Golgi compartment. Bar, 5 μm. B, immunofluorescence staining of VP1 and MIC2 to assess retention in a post-Golgi compartment(s). Parasites were stained as for A except for using affinity purified rabbit anti-VP1. Arrows indicate retention in a post-Golgi compartment. C, expression of M2AP mutants and TSR6 in Δm2ap parasites (Lysate lanes) and immunoprecipitation with RbαM2AP (IP lanes). MW, molecular weight. D, quantification of expression versus interaction of M2AP mutants with TSR6. Interaction:expression ratios for mutant M2APs were normalized to that of WT M2AP set to 100%. Data are compiled from three independent experiments. Error bars represent S.D. n.s., not significant.