Data collection and refinement statistics.

<p>Values in parentheses are for the highest resolution shell.</p>a<p>R_merge = ∑<i>_hkl</i> ∑<i>_i</i> |I<i>_hkl,i</i> - [I<i>_hkl</i>]|/∑<i>_hkl</i> ∑<i>_i</i> I<i>_hkl,i</i>, where [I<i>_hkl</i>] is the is the average of symmetry related observations of a unique reflection.</p>b<p>R_work = ∑|F_obs-F_calc|/∑F_obs, where F_obs and F_calc are the observed and the calculated structure factors, respectively.</p>c<p>R_free is R using 5% (apo) or 10% (complex) of reflections randomly chosen and omitted from refinement.</p

Specificity between SporoAMA1 and SporoRON2-D3 is achieved through interactions within both the cysteine loop and the connecting coil.

<p>A<b>.</b> Apical view of sporoAMA1 (green surface) bound to sporoRON2-D3 (gold cartoon) (left), showing conservation of the overall AMA1/RON2 binding paradigm with generic AMA1 (purple surface) – generic RON2 synthetic peptide (sp; green cartoon) (right; PDB 2Y8T). Note that the extreme C-terminal portion of the sporoRON2 peptide is disordered as a result of its relatively “early” exit from the stabilizing environment of the hydrophobic groove and therefore is not resolved in this structure. B. Cysteine loop interactions clearly differ between sporoAMA1-sporoRON2-D3 (left) and generic AMA1-generic RON2sp (right). Hydrogen bonds shown as dotted black lines. Colored as in (A). C. Additional specificity is gained through interactions with sporoAMA1 (green surface) or generic AMA1 (purple surface) and the RON2 coil that connects the N-term helix to the cysteine loop. Central groove residue (sporoAMA1 Ser252 or generic AMA1 Tyr230) and generic AMA1 groove residue Met233 colored dark grey. Beta-hairpin loop 3 and variable loop 4 colored light grey. Side chains of sporoRON2-D3 (gold cartoon) and generic RON2sp (green cartoon) involved in specificity shown as sticks.</p

The sporo- and generic versions of RON2-domain 3 interact only with their respective sporo- and generic AMA1 partners.

<p>A. Molar equivalents of GST, GST-gD3, or GST-sD3 were added to lysates of RHΔ<i>hxgprt</i> and after NP-40 solubilization the material that did not bind to the GST fusions (“flow-through”) or that was pelleted with the fusions (“pull-down”) was resolved by polyacrylamide gel electrophoresis and analyzed by immunoblotting with antibodies to generic AMA1 or SAG1 as a control for loading and nonspecific pelleting. Parentheses indicate parasite equivalents of the different fractions relative to input (1×). Size markers indicated in kDa. B. GST-pull-down experiments were performed as described in (A) except using RHΔ<i>hxgprt</i> that were transiently expressing sporoAMA1-HA and the sporoAMA1 was detected using antibodies to the HA-epitope tag.</p

Preincubation of parasites with GST-sporoRON2-D3 specifically impedes sporozoite but not tachyzoite invasion.

<p>Tachyzoites (A) and sporozoites (B) were pretreated with molar equivalents of GST, GST-gD3, GST-sD3, and a mixture of GST-gD3/GST-sD3, and then permitted to invade a monolayer of HFFs for 45 minutes, following temperature synchronization. The number of intracellular parasites was determined by differential staining before and after permeabilization. The percent of invaded (intracellular) parasites relative to the total number was determined by counting parasites in 10 randomly selected fields from each of three coverslips for each condition. The counting and analysis were done blinded. A single asterisk indicates p<0.05 for the difference relative to the GST control; double asterisks indicate p<0.01 relative to this control.</p

SporoAMA1 presents a highly guarded apical groove.

<p>A. Stacked three domain architecture of sporoAMA1 shown in the predicted organization to the <i>T. gondii</i> sporozoite plasma membrane with the three ectodomains indicated as DI in burgundy, DII in green and DIII in blue. Disulfides are shown as yellow sticks. Dotted line indicates extended Pro/Glu rich region between the conserved portion of DIII and the transmembrane domain (grey rectangle) that leads through to the C-terminal domain (grey oval). B. Apical view of apo sporoAMA1 structure, with core structure shown as grey surface and DI surface loops that guard the apical groove shown as burgundy cartoon and semi-transparent surfaces, and the DII loop as a green cartoon and semi-transparent surface. N-linked glycosylation on Asn230 shown as sticks. Numbers indicate surface loops that frame the apical groove. Inset: sporoAMA1 DII loop residues Phe376 and Trp377 (green) are pinned into the apical groove by Pro227 at the tip of loop 2 (burgundy).</p

SporoAMA1 DIII reorganization upon ligand binding provides possible insight into signal transduction mechanisms.

<p>SporoAMA1 shown in predicted organization to the <i>T. gondii</i> sporozoite plasma membrane, with DI and DII shown as a grey surface and DIII shown as blue cartoon with a semi-transparent blue surface, sporoRON2-D3 shown as a gold surface, and disulfides shown as yellow sticks. Dotted lines indicate extended Pro/Glu rich region between the conserved portion of DIII and the transmembrane domain (grey rectangle) that leads through to the C-terminal domain (grey oval/sphere). Left – Apo SporoAMA1. Right – SporoAMA1 bound to sporoRON2-D3.</p

<i>T. gondii</i> sporoRON2 and sporoAMA1 are conserved in other <i>Apicomplexans</i> and are distinct from generic RON2 and generic AMA1.

<p>A. The <i>Toxoplasma</i> sporoRON2 polypeptide sequences were aligned with their respective homologues in <i>Eimeria tenella, Neospora caninum, Plasmodium</i> spp. (<i>P. falciparum</i> and <i>P. vivax</i>), <i>Babesia spp.</i> (<i>B.bovis</i> and <i>B.microti</i>), and <i>Theileria spp.</i> (<i>T. annulata</i> and <i>T. parva</i>) using ClustalW (as part of MegAlign software (Lasergene)) and an anchored phylogenetic tree was generated using the standard algorithm. The clusters including generic and sporozoite-specific versions of each protein are so-labeled. Bootstrapping analysis was performed to determine confidence intervals (1000 trials). B<b>.</b> Alignment of domain 3 (D3) of the indicated RON2 homologues was performed in ClustalW. Residues identical to that of <i>T. gondii</i> sporoRON2 are indicated with shading on the upper panel, while residues identical to those of <i>T. gondii</i> generic RON2 are boxed on the lower panel. Numbers indicate amino acid position of <i>T. gondii</i> sporoRON2 from the starting Methionine. C. As for (A) except using the predicted sporoAMA1 polypeptide sequences.</p

SporoRON2 shows partial colocalization with ROP2/3/4 but little if any with RON4.

<p>Infected HFF monolayers were infected with M4 sporozoites for 2–3 hours, and then were methanol-fixed, and stained with rabbit anti-sporoRON2 (Rb-anti-sRON2) and either mouse (Mu) anti-RON4 (A), anti-ROP2/3/4 (B), or anti-sporoAMA1 (C). Images where two parasites were present in the same field are shown except for (B) where no such fields were found. The image shown in (B), however, is representative of the pattern consistently observed with these two antibodies. Scale bars represent 2 µm.</p

SporoAMA1 localizes apically in sporozoites.

<p>Extracellular sporozoites (A) or HFF monolayers infected with sporozoites for 2–3 hours (B–D), were formaldehyde-fixed, permeabilized with triton X-100, and stained with mouse anti-sporoAMA1 (Mu-anti-sAMA1) and rabbit (Rb) anti-MIC5 (B) or anti-MIC10 (A, C and D). Images shown in (C) and (D) are for adjacent parasites in the same field that were too far apart to capture in one image; both are shown to convey the reproducibility of the pattern observed. Scale bars represent 2 µm.</p

Hydrogen bond interactions observed in the sporoAMA1-sporoRON2-D3 (reported here) and generic AMA1-RON2sp (PDB 2Y8T; chains A and B, respectively) co-structures, aligned based on RON2 sequences with similar interactions bolded and the cysteine loop region residues are identified by italicized text.

<p>Hydrogen bond interactions observed in the sporoAMA1-sporoRON2-D3 (reported here) and generic AMA1-RON2sp (PDB 2Y8T; chains A and B, respectively) co-structures, aligned based on RON2 sequences with similar interactions bolded and the cysteine loop region residues are identified by italicized text.</p