Structural Characterization of Apical Membrane Antigen 1 (AMA1) from Toxoplasma gondii

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 Å 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.Toxoplasma gondii, the etiological agent of toxoplasmosis, is a prevalent global pathogen capable of establishing acute and chronic infections in nearly all warm blooded animals (1, 2). Although largely asymptomatic in healthy individuals, T. gondii infections can be lethal to a developing fetus and immunocompromised cancer and AIDS patients (3-6). Toxoplasmosis can also result in severe ocular infections in both children and adults, and encysted forms of the parasite have recently been implicated in neuropsychiatric disorders, such as schizophrenia (7-9).The success of T. gondii stems from its ability to persist in the environment, utilize several modes of transmission (10), and, importantly, to infect a broad range of host cells (1). A dominant feature that endows T. gondii and, in fact, all Apicomplexan parasites, including Plasmodium, Babesia, Cryptosporidium, and Neospora, with the ability to efficiently invade host cells is a multiprotein complex assembled at the moving junction (MJ) 4 (2, 11). The MJ is an electron-dense, ringlike structure formed between the plasma membranes of the apical tip of the motile parasite and the target host cell (12). During invasion, T. gondii is rapidly engulfed within a parasitophorous vacuole (PV) as the MJ traverses in a posterior direction along the length of the parasite (13, 14). As it migrates, the MJ serves as a molecular sieve, selectively filtering host proteins from the PV (12, 15), thereby protec...

Dissecting the interface between apicomplexan parasite and host cell: Insights from a divergent AMA–RON2 pair

“…Minimal structural rearrangement of TgAMA4 is required to accommodate TgRON2 L1 D3, consistent with both the lack of an extended DII loop and the lack of a deep groove, as observed for other AMAs (Fig. 3D) (14,25). As initially predicted from our evolutionary analyses, the structural diversity of TgAMA4 and TgRON2 L1 D3 gives rise to a markedly divergent binary complex within the AMA-RON2 families; TgRON2 L1 D3 extends from an N-terminal helix, through a kinked cystine loop that forms a knobin-hole interaction with a surface pocket in TgAMA4 (Fig.…”

“…The TgAMA1 and TgAMA2 head groups are connected to the TMD through linkers of <10 residues, resulting in close proximity to the parasite surface (Fig. 5, Top) (25). TgAMA3 presents a considerably longer, 93-residue linker rich in Pro, Glu, and Val residues, which we modeled in a semiextended, kinked conformation to be ∼175 Å from the TMD (Fig.…”

“…2B) (14,25,26). The similarity between TgAMA4 and other AMAs, however, is restricted to the DI and DII cores (rmsd with TgAMA1 of 2.4 Å over 265 Cα, representing approximately half of the Cαs in the TgAMA4 structure).…”

The moving junction of apicomplexan parasites: a key structure for invasion

“…Once secreted at the surface of the parasite, AMA1 is a type I integral membrane protein, with a short cytosolic region and the bulk of the protein forming the ectodomain. Recent structural studies on Plasmodium and Toxoplasma AMA1 (Pizarro et al, 2005;Crawford et al, 2010) have confirmed that they are structurally conserved: the ectodomain is composed of three architectural domains and contains a conserved hydrophobic trough surrounded by polymorphic loops. However, the understanding of the precise role of AMA1 in invasion and of the mode of action of anti-AMA1 invasion-inhibitory antibodies has remained incomplete.…”

Expression, Purification, and Biological Characterization of Babesia microti Apical Membrane Antigen 1

“…The precise biological role of AMA1 is not known, but evidence based on its subcellular localization and data from genetic knockout and transspecies expression studies suggest that it facilitates invasion of red blood cells (RBCs) and further growth of intraerythrocytic Plasmodium parasites (14,15). Crystallization of complete and truncated forms of the extracellular region from Plasmodium vivax, P. falciparum, and Toxoplasma gondii (16)(17)(18) revealed that it contains three domains. Domains I and II are homologous to the PAN (plasminogen, apple, and nematode) domains, which facilitate protein-protein and protein-carbohydrate interactions among the members of a class of adhesion molecules (19).…”