Vg9Vd2 T cells respond in a TCR-dependent fashion to both microbial and host-derived pyrophosphate compounds (phosphoantigens, or P-Ag). Butyrophilin-3A1 (BTN3A1), a protein structurally related to the B7 family of costimulatory molecules, is necessary but insufficient for this process. We performed radiation hybrid screens to uncover direct TCR ligands and cofactors that potentiate BTN3A1's P-Ag sensing function. These experiments identified butyrophilin-2A1 (BTN2A1) as essential to Vg9Vd2 T cell recognition. BTN2A1 synergised with BTN3A1 in sensitizing P-Ag-exposed cells for Vg9Vd2 TCR-mediated responses. Surface plasmon resonance experiments established Vg9Vd2 TCRs used germline-encoded Vg9 regions to directly bind the BTN2A1 CFG-IgV domain surface. Notably, somatically recombined CDR3 loops implicated in P-Ag recognition were uninvolved. Immunoprecipitations demonstrated close cell-surface BTN2A1-BTN3A1 association independent of P-Ag stimulation. Thus, BTN2A1 is a BTN3A1-linked co-factor critical to Vg9Vd2 TCR recognition. Furthermore, these results suggest a composite-ligand model of P-Ag sensing wherein the Vg9Vd2 TCR directly interacts with both BTN2A1 and an additional ligand recognized in a CDR3-dependent manner.

SummaryMembranes of Gram-negative bacteria, mitochondria and chloroplasts receive and fold b-barrel transmembrane proteins through the action of polypeptide transport-associated (POTRA) domains. In Escherichia coli, folding substrates are inserted into the outer membrane by the essential protein YaeT, a prototypic Omp85 protein. Here, the articulation between tandem POTRA domains in solution is defined by nuclear magnetic resonance (NMR) spectroscopy, indicating an unprecedented juxtaposition. The novel solution orientations of all five POTRA domains are revealed by small-angle X-ray scattering of the entire 46 kDa periplasmic region. NMR titration studies show that strands from YaeT's canonical folding substrate, PhoE, bind non-specifically along alternating sides of its mixed b sheets, thus providing an ideal platform for helping to fold nascent outer-membrane proteins. Together, this provides the first structural model of how multiple POTRA domains recruit substrates from the periplasmic solution into the outer membrane.

The Mla pathway is believed to be involved in maintaining the asymmetrical Gramnegative outer membrane via retrograde phospholipid transport. The pathway is composed of 3 components: the outer membrane MlaA-OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here we solve the crystal structure of MlaC in its phospholipid free closed apo conformation, revealing a pivoting βsheet mechanism which functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC we provide evidence that the inner membrane MlaFEDB machinery exports phospholipids to MlaC in the periplasm. Furthermore we confirm that the phospholipid export process occurs through the MlaD component of the MlaFEDB complex and that this process is independent of ATP. Our data provides evidence of an apparatus for lipid export away from the inner membrane and suggests that the Mla pathway may have a role in anterograde phospholipid transport.

Graphical Abstract Highlights d BTNL3 binds directly and specifically to Vg4 + TCRs via its IgV domain d The superantigen-like binding mode focuses on germlineencoded TCR regions d In contrast, gd TCR binding to a clonally restricted antigen is CDR3-mediated d Mutagenesis indicates parallels with BTN3A1-mediated activation of Vg9Vd2 T cells SUMMARY Butyrophilin (BTN) and butyrophilin-like (BTNL/Btnl) heteromers are major regulators of human and mouse gd T cell subsets, but considerable contention surrounds whether they represent direct gd T cell receptor (TCR) ligands. We demonstrate that the BTNL3 IgV domain binds directly and specifically to a human Vg4 + TCR, ''LES'' with an affinity ($15-25 mM) comparable to many ab TCR-peptide major histocompatibility complex interactions. Mutations in germline-encoded Vg4 CDR2 and HV4 loops, but not in somatically recombined CDR3 loops, drastically diminished binding and T cell responsiveness to BTNL3-BTNL8-expressing cells. Conversely, CDR3g and CDR3d loops mediated LES TCR bindingto endothelial protein C receptor, a clonally restricted autoantigen, with minimal CDR1, CDR2, or HV4 contributions. Thus, the gd TCR can employ two discrete binding modalities: a non-clonotypic, superantigenlike interaction mediating subset-specific regulation by BTNL/BTN molecules and CDR3-dependent, antibody-like interactions mediating adaptive gd T cell biology. How these findings might broadly apply to gd T cell regulation is also examined.