Structure-based mapping of the TβRI and TβRII receptor binding sites of the parasitic TGF-β mimic, Hp-TGM

Abstract

TGF-β is a secreted signaling protein involved in many physiological processes: organ development, production and maintenance of the extracellular matrix, as well as regulation of the adaptive immune system. As a cytokine, TGF-β stimulates the differentiation of CD4⁺ T-cells into regulatory T-cells (T_regs) that act to promote peripheral immune tolerance. The murine parasite Heligmosomoides polygyrus takes advantage of this pathway to induce inducing Foxp3⁺ T_regs in a similar manner using a TGF-β mimic (TGM), comprised of five tandem complement control protein (CCP) domains, designated D1-D5. Despite having no structural homology to TGF-β or to TGF-β family proteins, TGM binds directly to the TGF-β type I and type II receptors, TβRI and TβRII. To further investigate, NMR titration, and SPR and ITC binding experiments were performed, showing that TGM-D2, with the aid of D1, binds TβRI and TGM-D3 binds TβRII. Competition ITC experiments showed that TGM-D3 competes with TGF-β for binding to TβRII, consistent with TGM-D3-induced NMR chemical shift perturbations of TβRII which aligned with the solvent inaccessible areas of TβRII upon binding TGF-β. Thus, TGM-D3 binds to the same edged β-strand of TβRII that is used to bind TGF-β. Competition ITC experiments demonstrated that TGM-D1D2 and TGF-β3:TβRII compete for binding to TβRI, while TGM-D2-induced NMR chemical shift perturbation of TβRI showed that TGM-D2 binds to the same pre-helix extension of TβRI as does the TGF-β/TβRII binary complex. The solution structure of TGM-D3 revealed that while it has the overall structure of a CCP domain, TGM-D3 has an insertion in the hypervariable loop uncommon to CCP domains. These findings suggest that parasitic TGM, despite its lack of structural similarity to TGF-β, evolved to take advantage of the binding regions of the mammalian TGF-β type I and type II receptors. The structure of this TGM domain, along with the predicted structure of other H. polygyrus secreted proteins reported in the literature, suggest that TGM is part of a larger family of evolutionarily-adapted immunomodulatory CCP-containing proteins.