The p47 immunity-related GTPase (IRG) Irgb6 plays a pioneering role in host defense against Toxoplasma gondii infection. Irgb6 is recruited to the parasitophorous vacuole membrane (PVM) formed by T. gondii and disrupts it. Despite the importance of this process, the molecular mechanisms accounting for PVM recognition by Irgb6 remain elusive because of lack of structural information on Irgb6. Here we report the crystal structures of mouse Irgb6 in the GTP-bound and nucleotide-free forms. Irgb6 exhibits a similar overall architecture to other IRGs in which GTP binding induces conformational changes in both the dimerization interface and the membrane-binding interface. The membrane-binding interface of Irgb6 assumes a unique conformation, composed of N- and C-terminal helical regions forming a phospholipid binding site. In silico docking of phospholipids further revealed membrane-binding residues that were validated through mutagenesis and cell-based assays. Collectively, these data demonstrate a novel structural basis for Irgb6 to recognize T. gondii PVM in a manner distinct from other IRGs.

The p47 immunity-related GTPase (IRG) Irgb6 plays a pioneering role in host defense against Toxoplasma gondii infection. It is recruited to the parasitophorous vacuole membrane (PVM) formed by T. gondii and disrupts it. Despite the importance of this process, the molecular mechanisms accounting for PVM recognition by Irgb6 remain elusive due to lack of structural information on Irgb6. Here we report the crystal structures of mouse Irgb6 in the GTP-bound and nucleotide-free forms. Irgb6 exhibits a similar overall architecture to other IRGs in which GTP-binding induces conformational changes in both the dimerization interface and the membrane-binding interface. The membrane-binding interface of Irgb6 assumes a unique conformation, composed of N- and C-terminal helical regions forming a phospholipid binding site. In silico docking of phospholipids further revealed membrane binding residues that were validated through mutagenesis and cell-based assays. Collectively, these data demonstrate a novel structural basis for Irgb6 to recognize T. gondii PVM in a manner distinct from other IRGs.

The mechanism of T cell triggering upon engagement with a peptide-MHC (pMHC) complex remains a challenging problem. In order to observe structural and dynamics changes in the T cell receptor (TCR) upon pMHC binding, we carried out coarse grained molecular dynamics simulations of TCR-only and TCR-pMHC systems starting from a recently solved cryo-EM structure of the TCR and CD3 co-receptors. The simulations were performed in biological membranes for an aggregated length of 2 ms. We observed that, while unengaged TCRs adopted conformations that bent and restricted the dynamics of the CD3 co-receptors, the pMHC-bound TCRs adopted elongated conformations that allowed CD3 co-receptors to diffuse more freely. In this way, the TCR-pMHC pair acted as a "drawbridge", licensing the dynamics of the CD3 co-receptors, resulting in signal transmission across the plasma membrane.

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Upon infection with Toxoplasma gondii, host cells produce immune-related GTPases (IRGs) to kill the parasite. T. gondii counters this response by releasing ROP18 kinase, which inactivates IRG GTPases and inhibits their recruitment to the T. gondii parasitophorous vacuole (PV). However, the molecular mechanisms of this process are entirely unknown. Here we report the atomic structures of Irgb6 with a phosphomimetic mutation by ROP18. The mutant has lower GTPase activity and is not recruited to the PV membrane (PVM). The crystal structure shows the mutant exhibit a distinct conformation from the physiological nucleotide-free form, thus preventing GTPase cycling. This change allosterically modifies the conformation of the membrane-binding interface, preventing physiological PVM-binding. Docking simulation of PI5P also supports the impaired binding of the mutant to PVM. We thus demonstrate the structural basis for T. gondii escape from host cell-autonomous defense, and provide a structural model for regulating enzymatic activity by phosphorylation.