Abstract
Apical membrane antigen 1 (AMA1) is an essential component of the moving junction complex used by Apicomplexan parasites to invade host cells. We report the 2.0 A resolution x-ray crystal structure of the full ectodomain (domains I, II, and III) of AMA1 from the pervasive protozoan parasite Toxoplasma gondii. The structure of T. gondii AMA1 (TgAMA1) is the most complete of any AMA1 structure to date, with more than 97.5% of the ectodomain unambiguously modeled. Comparative sequence analysis reveals discrete segments of divergence in TgAMA1 that map to areas of established functional importance in AMA1 from Plasmodium vivax (PvAMA1) and Plasmodium falciparum (PfAMA1). Inspection of the TgAMA1 structure reveals a network of apical surface loops, reorganized in both size and chemistry relative to PvAMA1/PfAMA1, that appear to serve as structural filters restricting access to a central hydrophobic groove. The terminal portion of this groove is formed by an extended loop from DII that is 14 residues shorter in TgAMA1. A pair of tryptophan residues (Trp(353) and Trp(354)) anchor the DII loop in the hydrophobic groove and frame a conserved tyrosine (Tyr(230)), forming a contiguous surface that may be critical for moving junction assembly. The minimalist DIII structure folds into a cystine knot that probably stabilizes and orients the bulk of the ectodmain without providing excess surface area to which invasion-inhibitory antibodies can be generated. The detailed structural characterization of TgAMA1 provides valuable insight into the mechanism of host cell invasion by T. gondii.
Highlights
Adults, and encysted forms of the parasite have recently been implicated in neuropsychiatric disorders, such as schizophrenia [7,8,9]
Despite an ambiguous orientation of TgRON2 in the membrane, recent studies have demonstrated a clear interaction between TgRON2 and the micronemal protein Apical membrane antigen 1 (AMA1) [17,18,19,20,21], a core component of the moving junction (MJ) complex conserved across the phylum
Increased evolutionary divergence is observed with respect to AMA1s from P. falciparum, P. vivax, and B. babesi, with DIII, in particular, displaying less than 10% sequence identity
Summary
Bioinformatics—Boundaries for DI, DII, and DIII were defined based on the paradigm established for PvAMA1 [43]. Expression, and Purification—A clone encoding the fully processed ectoplasmic domain of TgAMA1 was generated in a modified pAcGP67b vector (Pharmingen) incorporating a C-terminal hexahistidine tag and thrombin cleavage site. Crystallization and Data Collection—Crystals of TgAMA1 were initially identified in the Index Screen (Hampton Research) and subsequently refined to a final condition of 20% polyethylene glycol 3350, 100 mM HEPES, pH 7.5, and 50 mM NaCl. Small crystals were observed after 2 days and grew to a final size of 0.5 ϫ 0.1 ϫ 0.1 mm within 6 days. Initial phases were obtained by molecular replacement using MOLREP [57] with the individual DI and DII domains of PfAMA1 (Protein Data Bank code 2Q8A) pruned with CHAINSAW [58] to better reflect the TgAMA1 sequence.
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