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Convergent evolution of a parasite-encoded complement control protein-scaffold to mimic binding of mammalian TGF-β to its receptors, TβRI and TβRII

2022; Elsevier BV; Volume: 298; Issue: 6 Linguagem: Inglês

10.1016/j.jbc.2022.101994

ISSN

1083-351X

Autores

Ananya Mukundan, Chang‐Hyeock Byeon, Cynthia S. Hinck, Kyle T. Cunningham, Tiffany Campion, Danielle J. Smyth, Rick M. Maizels, Andrew P. Hinck,

Tópico(s)

Coagulation, Bradykinin, Polyphosphates, and Angioedema

Resumo

The mouse intestinal helminth Heligmosomoides polygyrus modulates host immune responses by secreting a transforming growth factor (TGF)-β mimic (TGM), to expand the population of Foxp3+ Tregs. TGM comprises five complement control protein (CCP)-like domains, designated D1-D5. Though lacking homology to TGF-β, TGM binds directly to the TGF-β receptors TβRI and TβRII and stimulates the differentiation of naïve T-cells into Tregs. However, the molecular determinants of binding are unclear. Here, we used surface plasmon resonance, isothermal calorimetry, NMR spectroscopy, and mutagenesis to investigate how TGM binds the TGF-β receptors. We demonstrate that binding is modular, with D1-D2 binding to TβRI and D3 binding to TβRII. D1-D2 and D3 were further shown to compete with TGF-β(TβRII)2 and TGF-β for binding to TβRI and TβRII, respectively. The solution structure of TGM-D3 revealed that TGM adopts a CCP-like fold but is also modified to allow the C-terminal strand to diverge, leading to an expansion of the domain and opening potential interaction surfaces. TGM-D3 also incorporates a long structurally ordered hypervariable loop, adding further potential interaction sites. Through NMR shift perturbations and binding studies of TGM-D3 and TβRII variants, TGM-D3 was shown to occupy the same site of TβRII as bound by TGF-β using both a novel interaction surface and the hypervariable loop. These results, together with the identification of other secreted CCP-like proteins with immunomodulatory activity in H. polygyrus, suggest that TGM is part of a larger family of evolutionarily plastic parasite effector molecules that mediate novel interactions with their host. The mouse intestinal helminth Heligmosomoides polygyrus modulates host immune responses by secreting a transforming growth factor (TGF)-β mimic (TGM), to expand the population of Foxp3+ Tregs. TGM comprises five complement control protein (CCP)-like domains, designated D1-D5. Though lacking homology to TGF-β, TGM binds directly to the TGF-β receptors TβRI and TβRII and stimulates the differentiation of naïve T-cells into Tregs. However, the molecular determinants of binding are unclear. Here, we used surface plasmon resonance, isothermal calorimetry, NMR spectroscopy, and mutagenesis to investigate how TGM binds the TGF-β receptors. We demonstrate that binding is modular, with D1-D2 binding to TβRI and D3 binding to TβRII. D1-D2 and D3 were further shown to compete with TGF-β(TβRII)2 and TGF-β for binding to TβRI and TβRII, respectively. The solution structure of TGM-D3 revealed that TGM adopts a CCP-like fold but is also modified to allow the C-terminal strand to diverge, leading to an expansion of the domain and opening potential interaction surfaces. TGM-D3 also incorporates a long structurally ordered hypervariable loop, adding further potential interaction sites. Through NMR shift perturbations and binding studies of TGM-D3 and TβRII variants, TGM-D3 was shown to occupy the same site of TβRII as bound by TGF-β using both a novel interaction surface and the hypervariable loop. These results, together with the identification of other secreted CCP-like proteins with immunomodulatory activity in H. polygyrus, suggest that TGM is part of a larger family of evolutionarily plastic parasite effector molecules that mediate novel interactions with their host. Helminth parasites are major human and animal health burdens in tropical regions of the world, with up to two billion infected humans worldwide (1Hotez P.J. 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Yin M. et al.Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease.Nature. 1992; 359: 693-699Crossref PubMed Scopus (2586) Google Scholar). The dysregulation of the TGF-β pathway has been implicated in the pathogenesis of several human diseases, including inflammatory bowel disease (25Ihara S. Hirata Y. Koike K. TGF-beta in inflammatory bowel disease: a key regulator of immune cells, epithelium, and the intestinal microbiota.J. Gastroenterol. 2017; 52: 777-787Crossref PubMed Scopus (117) Google Scholar), renal and cardiac fibrosis (26Kim K.K. Sheppard D. Chapman H.A. TGF-beta1 signaling and tissue fibrosis.Cold Spring Harb. Perspect. Biol. 2018; 10a022293Crossref Scopus (229) Google Scholar, 27Hu H.H. Chen D.Q. Wang Y.N. Feng Y.L. Cao G. Vaziri N.D. et al.New insights into TGF-beta/Smad signaling in tissue fibrosis.Chem. Biol. 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Berenguer-Llergo A. Badia-Ramentol J. Iglesias M. et al.TGFbeta drives immune evasion in genetically reconstituted colon cancer metastasis.Nature. 2018; 554: 538-543Crossref PubMed Scopus (860) Google Scholar). Thus, TGF-β is a key therapeutic target in its own right (32Akhurst R.J. Targeting TGF-beta signaling for therapeutic gain.Cold Spring Harb. Perspect. Biol. 2017; 9a022301Crossref PubMed Scopus (108) Google Scholar, 33Derynck R. Turley S.J. Akhurst R.J. TGFbeta biology in cancer progression and immunotherapy.Nat. Rev. Clin. Oncol. 2021; 18: 9-34Crossref PubMed Scopus (146) Google Scholar). TGM, in contrast to the single-domain structure of TGF-β, is composed of five modular domains, designated D1 – D5, all with distant sequence homology to proteins of the complement control protein (CCP) family (15Johnston C.J.C. Smyth D.J. Kodali R.B. White M.P.J. Harcus Y. Filbey K.J. et al.A structurally distinct TGF-beta mimic from an intestinal helminth parasite potently induces regulatory T cells.Nat. Commun. 2017; 8: 1741Crossref PubMed Scopus (87) Google Scholar). CCP domains are approximately 60 to 65 amino acids in length with multiple short β-strands tethered together by two highly conserved disulfide bonds in a CysI-CysIII and CysII-CysIV topology (34Kirkitadze M.D. Barlow P.N. Structure and flexibility of the multiple domain proteins that regulate complement activation.Immunol. Rev. 2001; 180: 146-161Crossref PubMed Scopus (173) Google Scholar). They are usually found in arrays and are present in numerous proteins, including the family of proteins that regulate complement, such as decay accelerating factor, factor H, and complement C3b/C4b receptor 1 (CR1) (34Kirkitadze M.D. Barlow P.N. Structure and flexibility of the multiple domain proteins that regulate complement activation.Immunol. Rev. 2001; 180: 146-161Crossref PubMed Scopus (173) Google Scholar). In H. polygyrus, more than 30 CCP-containing proteins have been identified (35Smyth D.J. Harcus Y. White M.P.J. Gregory W.F. Nahler J. Stephens I. et al.TGF-beta mimic proteins form an extended gene family in the murine parasite Heligmosomoides polygyrus.Int. J. Parasitol. 2018; 48: 379-385Crossref PubMed Scopus (25) Google Scholar, 36Hewitson J.P. Ivens A.C. Harcus Y. Filbey K.J. McSorley H.J. Murray J. et al.Secretion of protective antigens by tissue-stage nematode larvae revealed by proteomic analysis and vaccination-induced sterile immunity.PLoS Pathog. 2013; 9e1003492Crossref PubMed Scopus (43) Google Scholar), including in addition to TGM and nine TGM homologs (35Smyth D.J. Harcus Y. White M.P.J. Gregory W.F. Nahler J. Stephens I. et al.TGF-beta mimic proteins form an extended gene family in the murine parasite Heligmosomoides polygyrus.Int. J. Parasitol. 2018; 48: 379-385Crossref PubMed Scopus (25) Google Scholar), H. polygyrus Alarmin Release Inhibitor (HpARI) and H. polygyrus Binds Alarmin Receptor and Inhibits (HpBARI), which suppress innate and adaptive type II immune responses, by binding IL-33 and its receptor ST2, respectively (37Osbourn M. Soares D.C. Vacca F. Cohen E.S. Scott I.C. Gregory W.F. et al.HpARI protein secreted by a helminth parasite suppresses interleukin-33.Immunity. 2017; 47: 739-751.e735Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 38Chauche C. Vacca F. Chia S.L. Richards J. Gregory W.F. Ogunkanbi A. et al.A truncated form of HpARI stabilizes IL-33, amplifying responses to the cytokine.Front. Immunol. 2020; 11: 1363Crossref PubMed Scopus (8) Google Scholar, 39Vacca F. Chauche C. Jamwal A. Hinchy E.C. Heieis G. Webster H. et al.A helminth-derived suppressor of ST2 blocks allergic responses.Elife. 2020; 9e54017Crossref PubMed Scopus (21) Google Scholar). Similar to TGM, HpARI and HpBARI contain multiple CCP domains (three and two, respectively) and contain large insertions not present in canonical CCP domains (15Johnston C.J.C. Smyth D.J. Kodali R.B. White M.P.J. Harcus Y. Filbey K.J. et al.A structurally distinct TGF-beta mimic from an intestinal helminth parasite potently induces regulatory T cells.Nat. Commun. 2017; 8: 1741Crossref PubMed Scopus (87) Google Scholar, 37Osbourn M. Soares D.C. Vacca F. Cohen E.S. Scott I.C. Gregory W.F. et al.HpARI protein secreted by a helminth parasite suppresses interleukin-33.Immunity. 2017; 47: 739-751.e735Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 38Chauche C. Vacca F. Chia S.L. Richards J. Gregory W.F. Ogunkanbi A. et al.A truncated form of HpARI stabilizes IL-33, amplifying responses to the cytokine.Front. Immunol. 2020; 11: 1363Crossref PubMed Scopus (8) Google Scholar). Here, we characterized the individual domains of TGM and investigated the nature of the TGM:TβRI and TGM:TβRII binding interactions, using surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and NMR. Binding of TGM to the TGF-β receptors was found to be modular in nature, with D1-D2 and D3 binding TβRI and TβRII, respectively. TGM was additionally shown to bind to similar structural motifs on TβRI and TβRII as TGF-β, indicating that TGM truly mimics TGF-β, despite its lack of structural similarity. The solution structure of TGM-D3 was determined and showed that TGM-D3 assumes the overall fold of a CCP domain with two key differences: (1) a loop and a short helix replace two β-strands and (2) a long (23-amino acid) structurally ordered insertion within the hypervariable loop (HVL). These modifications lead to a significant lateral expansion of the domain and create potential interaction surfaces on opposite faces of the protein. Through NMR binding studies, as well as binding studies of TGM-D3 and TβRII variants, TGM-D3 is shown to engage TβRII through one of its two potential interaction surfaces, as well as through the HVL. These new structural data illuminate how H. polygyrus has adapted its own CCP domain–containing proteins for the purpose of protein mimicry and host immunomodulation. Previous in vitro TGF-β bioassays demonstrated that only TGM domains 1 to 3 were required for induction of CD4+ CD25+ Foxp3+ Tregs from naïve murine T cells or activation of a TGF-β reporter in a mouse embryonic fibroblast cell line (35Smyth D.J. Harcus Y. White M.P.J. Gregory W.F. Nahler J. Stephens I. et al.TGF-beta mimic proteins form an extended gene family in the murine parasite Heligmosomoides polygyrus.Int. J. Parasitol. 2018; 48: 379-385Crossref PubMed Scopus (25) Google Scholar). Proteins lacking domains 4 and 5 (TGM-D123) retained ability to induce TGF-β signaling, albeit with reduced potency in T-cell assays, while removal of any or all of domains 1 to 3 completely abolished activity. TGM was furthermore shown to require both TβRI and TβRII to elicit TGF-β signaling (15Johnston C.J.C. Smyth D.J. Kodali R.B. White M.P.J. Harcus Y. Filbey K.J. et al.A structurally distinct TGF-beta mimic from an intestinal helminth parasite potently induces regulatory T cells.Nat. Commun. 2017; 8: 1741Crossref PubMed Scopus (87) Google Scholar), as TGM activity was inhibited by both SB431542, a TβRI kinase inhibitor (40Inman G.J. Nicolas F.J. Callahan J.F. Harling J.D. Gaster L.M. Reith A.D. et al.SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7.Mol. Pharmacol. 2002; 62: 65-74Crossref PubMed Scopus (1300) Google Scholar), and ITD-1, which stimulates ubiquitin-dependent degradation of TβRII (41Willems E. Cabral-Teixeira J. Schade D. Cai W. Reeves P. Bushway P.J. et al.Small molecule-mediated TGF-beta type II receptor degradation promotes cardiomyogenesis in embryonic stem cells.Cell Stem Cell. 2012; 11: 242-252Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Previous SPR measurements demonstrated that TGM binds TβRII with micromolar affinity, similar but weaker than TGF-β1 and -β3, but unlike TGF-β1 and TGF-β3, which only bind TβRI with low nanomolar affinity once bound to TβRII, TGM binds TβRI with low nanomolar affinity in the absence of TβRII (14Grainger J.R. Smith K.A. Hewitson J.P. McSorley H.J. Harcus Y. Filbey K.J. et al.Helminth secretions induce de novo T cell Foxp3 expression and regulatory function through the TGF-beta pathway.J. Exp. Med. 2010; 207: 2331-2341Crossref PubMed Scopus (357) Google Scholar). It is unknown which domains of TGM bind to TβRI and TβRII or if TβRI and TβRII directly contact one another, as in the TGF-β receptor complex. To investigate this, the individual domains TGM-D1, TGM-D2, and TGM-D3, along with full-length TGM (TGM-FL), were expressed and purified for SPR binding studies with the TGF-β receptors. The injection of these domains over biotinylated avi-tagged TβRI captured on a streptavidin-coated sensor chip yielded robust concentration-dependent responses when TGM-D2 or TGM-FL was injected, but not when TGM-D1 or TGM-D3 was injected (Fig. 1, A–D). The KD values derived by globally fitting the TGM-D2 and TGM-FL sensorgrams to a (1:1) kinetic model were 310 nM and 13 nM, respectively (Table 1). Thus, TGM-D2 is evidently the main binding partner for TβRI, but nonetheless lacks the full binding capacity of TGM. The same series of injections, performed over biotinylated avi-tagged TβRII captured on a streptavidin-coated sensor chip, yielded robust responses when TGM-D3 or TGM-FL was injected, but not when TGM-D1 or TGM-D2 was injected (Fig. 1,F–I). The KD values derived from the TGM-FL and TGM-D3 sensorgrams were 610 nM and 910 nM, respectively (Table 1). Thus, TGM-D3 accounts for most of the binding affinity of TGM-FL for TβRII.Table 1TGM:TβRI and TGM:TβRII binding as assessed by SPRSurfaceAnalyteFitted parametersaFitted parameters were derived from kinetic analysis of a single injection series.kon (M−1 s−1)koff (s−1)KD (nM)Rmax (RU)TβRITGM-D1NDbNot determined due to weak signal.NDbNot determined due to weak signal.NDbNot determined due to weak signal.NDaFitted parameters were derived from kinetic analysis of a single injection series.TβRITGM-D2(3.0 ± 0.1) × 105(9.1 ± 0.1) × 10−2310 ± 1089.6 ± 0.7TβRITGM-D3NDbNot determined due to weak signal.NDbNot determined due to weak signal.NDbNot determined due to weak signal.NDaFitted parameters were derived from kinetic analysis of a single injection series.TβRITGM-D12(6.7 ± 0.1) × 104(1.6 ± 0.1) × 10−324 ± 1429 ± 1TβRIcMeasured on a lower density chip compared to that used for TβRI:TGM-D2 and TβRI:TGM-D12.TGM-FL(5.9 ± 0.1) × 104(7.8 ± 0.2) × 10−413 ± 1193 ± 1TβRIITGM D1NDbNot determined due to weak signal.NDbNot determined due to weak signal.NDbNot determined due to weak signal.NDaFitted parameters were derived from kinetic analysis of a single injection series.TβRIITGM D2NDbNot determined due to weak signal.NDbNot determined due to weak signal.NDbNot determined due to weak signal.NDaFitted parameters were derived from kinetic analysis of a single injection series.TβRIITGM D1D2NDbNot determined due to weak signal.NDbNot determined due to weak signal.NDbNot determined due to weak signal.NDaFitted parameters were derived from kinetic analysis of a single injection series.TβRIITGM D3(6 ± 1) × 1050.6 ± 0.1910 ± 2033.0 ± 0.4TβRIITGM FL(2 ± 6) × 107(1 ± 4) × 10−1610 ± 10215 ± 2a Fitted parameters were derived from kinetic analysis of a single injection series.b Not determined due to weak signal.c Measured on a lower density chip compared to that used for TβRI:TGM-D2 and TβRI:TGM-D12. Open table in a new tab TGM-D3's full and TGM-D2's partial recapitulation of TGM binding affinity for TβRII and TβRI, respectively, suggested that TGM-D1 might contribute to binding of TβRI. Thus, we assessed binding of a construct containing both TGM-D1 and TGM-D2, designated TGM-D12, to TβRI and TβRII using SPR. This didomain construct bound robustly to TβRI, but did not bind at all to TβRII (Fig. 1, E and J). The KD derived from kinetic analysis of the TGM-D12:TβRI sensorgrams was 24 nM, which is within a factor of two of that of TGM-FL (Table 1). Thus, TGM-D1 also contributes to the binding to TβRI. ITC experiments, which in contrast to SPR are carried out entirely in solution and do not require any tagging, were also performed to assess binding of the individual domains of TGM to TβRI and TβRII. In accord with the SPR results, titration of TGM-D2, TGM-D12, and TGM-FL into TβRI and TGM-D3 and TGM-FL into TβRII yielded readily measurable binding isotherms with large negative enthalpies (Figs. 2, A–E and S1, A–E). In contrast, titration of TGM-D1 and TGM-D3 over a similar range of concentrations into TβRI and TGM-D1 and TGM-D2 into TβRII did not (Fig. S1, F–M). In further accord with the SPR results, the fitted KD values for binding of TGM-D12 to TβRI and TGM-D3 to TβRII were comparable to those of TGM-FL and were generally consistent with those measured by SPR (Table S2). In contrast, and as expected based on the SPR results, the KD for binding of TGM-D2 to TβRI was significantly increased (ca. 50-fold) relative to TGM-FL (Table S2). ITC, in addition to providing KD values, also provides values for the stoichiometry, and as shown, TGM-FL binds both TβRI and TβRII with near 1:1 stoichiometry (Table S2). The near 1:1 stoichiometry is also observed for the TGM subdomains shown to bind TβRI and TβRII, TGM-D12 and TGM-D3, respectively, but for TGM-D2 binding to TβRI, the stoichiometry was closer to 0.5. The differing stoichiometry for binding of TGM-D2 and TGM-D12 to TβRI is likely due the weaker affinity of the former interaction, which makes accurate data fitting difficult. Thus, as discussed in a following section, an alternative method was used and this established 1:1 stoichiometry for the TGM-D2 to TβRI interaction. To assess potential shared binding sites on TβRI and TβRII, ITC competition experiments were performed in which KDs and enthalpies for TβRI and TβRII binding to their partners were measured under noncompetitive and competitive conditions. In the case of TβRI, titration of the TGF-β3(TβRII)2 complex into TβRI yielded a fitted KD of 61 nM (Fig. 2F and Table S3), which is similar to the KD of 25 nM when TGM-D12 was titrated into TβRI (Fig. 2B and Table S2). However, unlike TGM-D12:TβRI binding which had a large negative enthalpy, −19 kcal mol−1 (Table S2), binding of TGF-β3(TβRII)2 to TβRI had a much smaller negative enthalpy, −4.2 kcal mol−1, even at an increased temperature (Table S3). In light of similar KDs, but significantly different enthalpies, the competition experiment with TβRI was performed by titrating TGM-D12 into the cell loaded with the TGF-β3(TβRII)2(TβRI)2 ternary complex (Fig. 2G). This yielded no heat, indicating that TGM-D12 and TGF-β3(TβRII)2 compete for binding to TβRI. TGF-β3, and TGF-β homodimers in general, is well known to be practically insoluble in the unbound form, except under either very acidic (pH 4.0) or basic (pH 11.0) conditions (42Pellaud J. Schote U. Arvinte T. Seelig J. Conformation and self-association of human recombinant transforming growth factor-beta3 in aqueous solutions.J. Biol. Chem. 1999; 274: 7699-7704Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Therefore, competition experiments in which TβRII is tit

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