Structure of the micronemal protein 2 A/I domain from Toxoplasma gondii

Abstract

Toxoplasma gondii is a widespread zoonotic pathogen capable of causing serious disease in humans and animals. As an obligate intracellular parasite, T. gondii relies on the orchestrated secretion of proteins from its apical complex organelles including the multimodular, transmembrane micronemal protein 2 (MIC2) that couples recognition of the host cell with cytoskeletal reorganization of the parasite to drive invasion. To probe the basis by which the von Willebrand Factor A (vWA)-Integrin like module of TgMIC2 engages the host cell, we solved the crystal structure of a truncated form of TgMIC2A/I (TgMIC2A/Ic) phased by iodide SIRAS and refined to a resolution of 2.05 Å. The TgMIC2A/Ic core is organized into a central twisted beta sheet flanked by α-helices consistent with a canonical vWA fold. A restricted basic patch serves as the putative heparin binding site, but no heparin binding was detected in native gel shift assays. Furthermore, no metal was observed in the metal ion dependent adhesion site (MIDAS). Structural overlays with homologous A/I domains reveal a divergent organization of the MIDAS β4-α4 loop in TgMIC2A/Ic, which is stabilized through the burial of Phe195 into a deep pocket formed by Gly185. Intriguingly, Gly185 appears to be unique among A/I domains to TgMIC2A/I suggesting that the divergent loop conformation may also be unique to TgMIC2A/I. Although lacking the C-terminal extension, the TgMIC2A/Ic structure reported here is the first of an A/I domain from an apicomplexan parasite and provides valuable insight into defining the molecular recognition of host cells by these widespread pathogens.

Figure 1

Biological assembly and overall structure of TgMIC2A/Ic. A: Single unit of the heterohexameric organization of TgMIC2, with the A/I domain (green sphere) positioned at the apical end of the full transmembrane protein. B: Sequence alignment of TgMIC2A/I with PfTRAP, vWFA1, Mac-1, and Integrin α2β1. MIDAS residues are indicated by purple triangles, C-terminal cysteines in orange boxes, divergent C-terminal region in black box and Gly185 and Phe195 as yellow and orange triangles, respectively. Secondary structure and residue numbering is indicated for TgMIC2A/I. C-terminii of TgMIC2A/Ic and TgMIC2A/If constructs demarcated by green and blue arrows, respectively. C: Size exclusion trace and associated SDS-PAGE gel of TgMIC2A/Ic (green) and TgMIC2A/If (blue) showing elution of TgMIC2A/Ic as dimer and TgMIC2A/Ic as a monomer with the carbonic anhydrase standard (29 kDa), shown as a dotted line. D: Stereo image of TgMIC2A/Ic colored from N (blue) to C (red) terminus, highlighting the conserved vWA fold and associated topology diagram with disulfide bond represented by a dotted line. E: Electrostatic surface of TgMIC2A/Ic reveals a hydrophobic patch at the dimer interface (left panel), which, in the structurally homologous integrin α2β1 (yellow, PDB ID 1AOX) and vWA (green, PDB ID 1AUQ), is concealed by the C-terminal extension (right panel).

Figure 2

Structure–function analysis of TgMIC2A/I. (A) Left panel—electrostatic surface representation of TgMIC2A/Ic highlighting the spatially separated regions of potential functional importance: putative heparin-binding basic patch (green arrow) and the MIDAS (yellow arrow). Right panel—heparin binding native gel shift assay in the presence (+) and absence (−) of heparin for a known heparin binding protein and TgMIC2A/If. No heparin binding was observed with TgMIC2AI/f. (B) Left panel—Overlay of TgMIC2A/Ic (dark gray) and Mac-1 (light grey, PDB ID 1BHO) highlighting the reorganized alpha 4 and 5 helices in TgMIC2A/Ic (green) compared to the analogous regions in Mac-1 (orange). TgMIC2A/Ic Phe195 on α4, anchors the α4 helix by docking into a deep pocket made possible by Gly185. Ultimately, this reorganized loop results in Asp188 of the MIDAS motif being shifted 4.4 Å from the analogous Asp of Mac-1 effectively eliminating the ability to coordinate a metal.