EUROISMAR 2019

TGF-β stimulates the proliferation and differentiation of regulatory T-cells (T_regs) to promote peripheral immune tolerance. The mouse parasite Heligmosomoides polygyrus manipulates the host immune system through the induction of Fox3p+ T_regs using the five-domain complement control protein (CCP) protein, TGM. Though lacking any homology to the TGF-βs, TGM binds directly to the TGF-β receptors, TβRI and TβRII, to induce immunosuppressive signaling. To determine how the five domains of TGM, TGM-D1-D5, bind and assemble TβRI and TβRII into a signaling complex, TGM-D1, -D2, and -D3 were produced in E. coli and refolded. Through NMR, ITC, and SPR, TGM-D2 and TGM-D3 were shown to be responsible for binding TβRI and TβRII, respectively, though the binding of TβRI by TGM-D2 was further potentiated by TGM-D1. Through NMR, we further showed that TβRII uses the same exposed β-strand to bind TGM-D3 as it does to bind TGF-βs, consistent with TGM-D3 and TGF-β competing for TβRII. Through SPR, we showed that TGM-D1D2 and TGF-β:TβRII compete for binding to TβRI, suggesting that TβRI uses its pre-helix extension to bind TGF-β:TβRII and TGM-D1D2. TGM-D3 was shown by NMR structural analysis to adopt a canonical CCP fold, but differed in that it forms a continuous half β-barrel rather than two unconnected antiparallel β-strands. Through assignment of TGM-D3 bound to TβRII, we showed that the majority of resonances perturbed by TβRII reside within a cleft on the outer surface of the barrel. This cleft is formed by two loop regions which are extended in TGM compared to canonical CCP domains, suggesting that these loop insertions represent important adaptations for binding the TGF-β receptors. The information derived from these studies is important not only for guiding efforts to determine the structure of TGM with TβRI and TβRII, but will also aid in the development of TGM as an immunosuppressive therapeutic.