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 a-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 b4-a4 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.

Structural Basis of Toxoplasma gondii MIC2-associated Protein Interaction with MIC2

“…4B). Similar results were obtained with constructs expressing M2AP mutants and 15 N HSQC spectra of 15 N, 13 C-labeled M2AP in the presence (red) and absence (black) of 6-fold molar excess of unlabeled TSR5-6. Selected resonance assignments are indicated.…”

“…The protein was then expressed in SHuffle Escherichia coli strain (New England Biolabs). For 15 N, 13 C labeling of wild type M2AP, minimal media containing 0.07% 15 NH 4 Cl and 0.2% [ 13 C 6 ]glucose was used; otherwise Luria-Bertani media was used. For protein production, cultures were grown at 37°C before induction with 1 mM isopropyl -Dthiogalactopyranoside at 18°C for 12 h. The protein was purified using nickel-nitrilotriacetic acid-agarose (Promega).…”

“…The distance restraint was provided by 13 C, 1 H, 15 N nuclear Overhauser effect spectroscopy (NOESY)-heteronuclear multiple quantum coherence (HSQC) experiments (mixing time, 100 ms at 800 MHz).…”

“…Crystal structures of the MIC2 I-domain (13) and the I-domain with the first TSR (14) suggested that the adhesive function of the I-domain is regulated by conformational changes, potentially induced by tension created during gliding motility. Low resolution data from small angle x-ray scattering implied that MIC2-M2AP adopts a dimeric structure with MIC2 appearing as an elongated rod and M2AP appearing as a "foot" at the C-terminal end of the M-domain (14).…”

Shape change in the receptor for gliding motility in Plasmodium sporozoites

“…Sporozoites are important targets of pre-erythrocytic malaria vaccines. However, we know little about the structure and arrangement of the two most important vaccine targets on sporozoite surfaces, the circumsporozoite (CS) protein (1-3) and thrombospondin repeat anonymous protein (TRAP) (4,5). CS is a constitutive sporozoite surface protein and has a glycophosphatidylinositol anchor.…”