Systems-Based Analysis of the Sarcocystis neurona Genome Identifies Pathways That Contribute to a Heteroxenous Life Cycle

Blazejewski, Tomasz; Nursimulu, Nirvana; Pszenny, Viviana; Dangoudoubiyam, Sriveny; Namasivayam, Sivaranjani; Chiasson, Melissa A.; Chessman, Kyle; Tonkin, Michelle; Swapna, Lakshmipuram S.; Hung, Stacy S.; Bridgers, Joshua; Ricklefs, Stacy M.; Boulanger, Martin J.; Dubey, Jitender P.; Porcella, Stephen F.; Kissinger, Jessica C.; Howe, Daniel K.; Grigg, Michael E.; Parkinson, John

Molecular modeling reveals that SnAMA1a is capable of intimately coordinating SnRON2. To examine if S. neurona AMA1 homologs can bind S. neurona RON2 homologs, we generated structural models of SnAMA1a and SnAMA1b, which show the highest sequence identity (49 and 44%, respectively) with TgAMA1. Both models possess a PAN-like domain architecture for DI and DII (SnAMA1a, Fig. 4A ) consistent with homologs from other apicomplexans (35, 36) . A key feature of DII is an extended loop that packs into the groove of DI and regulates RON2 binding in related AMA1 proteins (37) . In the SnAMA1a model, a cysteine pair localized within the DII loop is a novel feature of AMA1s and may serve as a hinge to regulate loop displacement and, consequently, RON2 binding (Fig. 4A ). Furthermore, the SnAMA1a DII loop appears to be loosely anchored within the DI groove via a Val-Val pair surrounding a central Leu (Fig. 4A ). This is in contrast to TgAMA1 (Fig. 4C) , where a Trp-Trp pair surround a central Tyr, and the SnAMA1b model (Fig. 4B) , where a Trp-Leu pair surround a central Tyr (Fig. 4B) . These models suggest that AMA1 paralogs in S. neurona employ divergent strategies that control DII loop dynamics and govern access to the ligand-binding groove. Focusing on the interaction with RON2, removal of the apical segment of the DII loop from the model of SnAMA1a (mimicking the mature binding surface) led to a pronounced groove similar to the RON2 binding surface observed in TgAMA1 (Fig. 4D) . Indeed, an energy-minimized docked model revealed that SnRON2 domain 3 was accommodated in a U-shaped conformation (SnRON2D3; Fig. 4E ) with an overall topology conserved with respect to the TgAMA1 costructure with a synthetic TgRON2D3 peptide (Fig. 4D) (37) . Key features of the TgAMA1-TgRON2sp interface appear to be conserved at the SnAMA1-SnRON2D3 interface, including a RON2 proline residue that occupies an AMA1 pocket exposed by displacement of the DII loop ( Fig. 4D and E, yellow arrow) and a reliance on hydrophobic interactions to engage AMA1.