Ligand-Receptor and Receptor-Receptor Interactions Act in Concert to Activate Signaling in the Drosophila Toll Pathway

Abstract

In Drosophila, the signaling pathway mediated by the Toll receptor is critical for the establishment of embryonic dorso-ventral pattern and for innate immune responses to bacterial and fungal pathogens. Toll is activated by high affinity binding of the cytokine Spätzle, a dimeric ligand of the cystine knot family. In vertebrates, a related family of Toll-like receptors play a critical role in innate immune responses. Despite the importance of this family of receptors, little is known about the biochemical events that lead to receptor activation and signaling. Here, we show that Spätzle binds to the N-terminal region of Toll and, using biophysical methods, that the binding is complex. The two binding events that cause formation of the cross-linked complex are non-equivalent: the first Toll ectodomain binds Spätzle with an affinity 3-fold higher than the second molecule suggesting that pathway activation involves negative cooperativity. We further show that the Toll ectodomains are able to form low affinity dimers in solution and that juxtamembrane sequences of Toll are critical for the activation or derepression of the pathway. These results, taken together, suggest a mechanism of signal transduction that requires both ligand-receptor and receptor-receptor interactions. In Drosophila, the signaling pathway mediated by the Toll receptor is critical for the establishment of embryonic dorso-ventral pattern and for innate immune responses to bacterial and fungal pathogens. Toll is activated by high affinity binding of the cytokine Spätzle, a dimeric ligand of the cystine knot family. In vertebrates, a related family of Toll-like receptors play a critical role in innate immune responses. Despite the importance of this family of receptors, little is known about the biochemical events that lead to receptor activation and signaling. Here, we show that Spätzle binds to the N-terminal region of Toll and, using biophysical methods, that the binding is complex. The two binding events that cause formation of the cross-linked complex are non-equivalent: the first Toll ectodomain binds Spätzle with an affinity 3-fold higher than the second molecule suggesting that pathway activation involves negative cooperativity. We further show that the Toll ectodomains are able to form low affinity dimers in solution and that juxtamembrane sequences of Toll are critical for the activation or derepression of the pathway. These results, taken together, suggest a mechanism of signal transduction that requires both ligand-receptor and receptor-receptor interactions. What Happens during Toll Activation? Journal of Biological ChemistryVol. 280Issue 24PreviewIn Drosophila, the Toll receptor mediates cellular signal transduction pathways involved in both innate immune responses and in establishing embryonic dorso-ventral patterning. In order to become active, the Toll receptor must both dimerize and bind to its dimeric ligand, Spätzle. However, the exact sequence of the events leading to this activation and their biochemical nature is unknown. Full-Text PDF Open Access The Toll family of type I transmembrane receptors mediate cellular signal transduction pathways that are central to innate immune responses in both vertebrates and invertebrates (1Janeway C.A. Medzhitov R. Annu. Rev. Immunol. 2002; 20: 197-216Crossref PubMed Scopus (6190) Google Scholar). These receptors are responsible for detecting microbial molecular patterns, for example bacterial lipoproteins and lipopolysaccharides, and activating a cellular defense response. The overall structure of Toll receptors is evolutionarily conserved, with characteristic ectodomains consisting mainly of sequences of leucine rich repeats (LRRs) 1The abbreviations used are: LRR, leucine-rich repeat; TLR, Toll-like receptor; IL, interleukin; TIR, Toll/IL-1 receptor domain; C106, dimer of C-terminal 106 amino acids of Spätzle; EGF, epidermal growth factor; FGF, fibroblast growth factor; NGF, nerve growth factor; ITC, isothermal titration calorimetry; NFκB, nuclear factor κB; HEK, human embryonic kidney; Sulfo-GMBS, N-(γ-maleimidobutylody)sulfosuccinimide ester; Sulfo-KMUS (N-(κ-maleimidoundecanoyloxy)sulfosuccinimide ester; Ni-NTA, nickel-nitrilotriacetic acid; c(S), concentration as a function of sedimentation coefficient; c(M), concentration as a function of molecular mass. flanked by cysteine rich capping structures and an intracellular TIR (Toll/IL-1R) domain (2Rock F.L. Hardiman G. Timans J.C. Kastelein R.A. Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 588-593Crossref PubMed Scopus (1451) Google Scholar) (see Fig. 4B). Despite this structural conservation, different mechanisms have arisen for the detection of pathogen patterns in invertebrates and vertebrates (3Gangloff M. Gay N.J. Trends Biochem. Sci. 2004; 29: 294-300Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). In insects, for example, peptidoglycan is bound by a specific recognition protein and this complex then stimulates a cascade of serine proteases the terminal member of which processes an inactive proprotein form of the cytokine Spätzle to form an active protein ligand (4DeLotto Y. DeLotto R. Mech. Dev. 1998; 72: 141-148Crossref PubMed Scopus (152) Google Scholar, 5Weber A. Tauszig-Delamasure S. Hoffmann J. Lelièvre E. Gascan H. Ray K. Morse M. Imler J. Gay N. Nat. Immunol. 2003; 4: 794-800Crossref PubMed Scopus (340) Google Scholar, 6Bischoff V. Vignal C. Boneca I.G. Michel T. Hoffmann J.A. Royet J. Nat. Immunol. 2004; 5: 1175-1180Crossref PubMed Scopus (203) Google Scholar). By contrast, in mammals Toll-like receptors are thought to directly interact with microbial compounds (7Re F. Strominger J.L. J. Biol. Chem. 2002; 277: 23427-23432Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 8Miyake K. Trends Microbiol. 2004; 12: 186-192Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). Despite these differences, Toll receptors probably require ligand-induced dimerization or cross-linking to establish intracellular signal transduction, a feature shared with other type I transmembrane receptors (9Lemmon M.A. Schlessinger J. Trends Biochem. Sci. 1994; 19: 459-463Abstract Full Text PDF PubMed Scopus (433) Google Scholar). Such a mode of action was recently illustrated for the founding member of the family, Drosophila Toll (5Weber A. Tauszig-Delamasure S. Hoffmann J. Lelièvre E. Gascan H. Ray K. Morse M. Imler J. Gay N. Nat. Immunol. 2003; 4: 794-800Crossref PubMed Scopus (340) Google Scholar). In Drosophila, the Toll receptor is involved in dorso-ventral patterning of the embryo as well as innate immune responses (10Belvin M.P. Anderson K.V. Annu. Rev. Cell Dev. Biol. 1996; 12: 393-416Crossref PubMed Scopus (686) Google Scholar, 11Imler J.L. Hoffmann J.A. Trends Cell Biol. 2001; 11: 304-311Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). For example, female flies carrying loss-of-function mutations in Toll lay eggs that develop into embryos in which all cells adopt dorsal fates. In addition, these flies fail to express antimicrobial peptides in response to infection by Gram-positive bacteria and fungi and poorly resist these infections. Both processes require the same post-receptor components and the activating Spätzle ligand. However, differently regulated protease cascades are responsible for ligand generation in embryonic development and innate immune responses in adult flies (12Ligoxygakis P. Pelte N. Hoffmann J.A. Reichhart J.M. Science. 2002; 297: 114-116Crossref PubMed Scopus (279) Google Scholar). In the 1990s, genetic studies identified two classes of Toll gain-of-function alleles that cause a dominant ventralization of the embryo (13Schneider D.S. Hudson K.L. Lin T.Y. Anderson K.V. Gene Dev. 1991; 5: 797-807Crossref PubMed Scopus (261) Google Scholar). The first class (type I mutants) are point mutations in the extracellular domain of Toll involving specific cysteine residues in the juxtamembrane, cysteine-rich flank of the second block of LRRs. These cysteine residues are thought to be involved in disulfide bonds and disruption of these is believed to cause constitutive activation of the receptor. The second class of mutations (type II mutants) produces truncated ectodomains that are secreted from the embryo. The available evidence suggests that these mutations act by binding and then redistributing active Spätzle ligand in the perivitelline space of the embryo, causing inappropriate activation of receptors located at lateral and dorsal positions. The basic principle that signal transduction by Type 1 transmembrane receptors requires dimerization is long established (9Lemmon M.A. Schlessinger J. Trends Biochem. Sci. 1994; 19: 459-463Abstract Full Text PDF PubMed Scopus (433) Google Scholar). This has led to an expectation that active signaling complexes should be structurally symmetrical and that the sites of ligand interaction would be essentially equivalent. However, more recent structural and biophysical studies suggest that the events leading to ligand binding are complex and that some signaling assemblies are in fact asymmetrical. For example, cross-linking of the epidermal growth factor (EGF) receptor requires binding of two monomeric EGF molecules. After formation of ligand-receptor complexes, subsequent receptor dimerization may involve either second separate sites on ligand and receptor or a conformational rearrangement in the receptor allowing self-association (14Lemmon M.A. Bu Z. Ladbury J.E. Zhou M. Pinchasi D. Lax I. Engelman D.M. Schlessinger J. EMBO J. 1997; 16: 281-294Crossref PubMed Scopus (302) Google Scholar). The latter view has gained considerable support from structural analysis of EGF-receptor complexes (15Schlessinger J. Cell. 2002; 110: 669-672Abstract Full Text Full Text PDF PubMed Scopus (802) Google Scholar, 16Ogiso H. Ishitani R. Nureki O. Fukai S. Yamanaka M. Kim J.H. Saito K. Sakamoto A. Inoue M. Shirouzu M. Yokoyama S. Cell. 2002; 110: 775-787Abstract Full Text Full Text PDF PubMed Scopus (902) Google Scholar, 17Garrett T.P. McKern N.M. Lou M. Elleman T.C. Adams T.E. Lovrecz G.O. Zhu H.J. Walker F. Frenkel M.J. Hoyne P.A. Jorisson R.N. Nice E.C. Burgess A.W. Ward C.W. Cell. 2002; 110: 763-773Abstract Full Text Full Text PDF PubMed Scopus (625) Google Scholar). In another example, structures of the fibroblast growth factor (FGF) receptor in complex with FGF show that a molecule of heparin links together two heterodimers of FGF and FGF-R into a highly asymmetrical pentameric complex (18Harmer N.J. Chirgadze D. Hyun Kim K. Pellegrini L. Blundell T.L. Biophys. Chem. 2003; 100: 545-553Crossref PubMed Scopus (24) Google Scholar). Unlike the EGF and FGF pathways Drosophila Toll is activated by a dimeric ligand. The structure adopted by the active C-terminal fragment of Spätzle (C106) is a cystine knot, a fold held in common with neurotrophins such as NGF, and two monomers are held together as a covalent dimer by a single intermolecule disulfide bond (4DeLotto Y. DeLotto R. Mech. Dev. 1998; 72: 141-148Crossref PubMed Scopus (152) Google Scholar, 19Mizuguchi K. Parker J.S. Blundell T.L. Gay N.J. Trends Biochem. Sci. 1998; 23: 239-242Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Both in vivo and in vitro, C106 can form a heterotrimer with two molecules of the Toll receptor ectodomains (5Weber A. Tauszig-Delamasure S. Hoffmann J. Lelièvre E. Gascan H. Ray K. Morse M. Imler J. Gay N. Nat. Immunol. 2003; 4: 794-800Crossref PubMed Scopus (340) Google Scholar). Thus in contrast to EGF, Spätzle binding alone should be sufficient to cause receptor cross-linking. In this paper we report that Toll pathway activation probably requires receptor-receptor interactions as well as ligand induced cross-linking. Protein Production and Purification—Recombinant His-tagged Toll, Toll5B, and C106 proteins were produced in the baculovirus system and purified using Ni-NTA affinity chromatography as described in detail in (5Weber A. Tauszig-Delamasure S. Hoffmann J. Lelièvre E. Gascan H. Ray K. Morse M. Imler J. Gay N. Nat. Immunol. 2003; 4: 794-800Crossref PubMed Scopus (340) Google Scholar). In brief, constructs for Toll (residues 1–801), Toll5B (1–668), and full-length Spätzle proprotein were subcloned into pFastBac and sequenced and recombinant baculoviruses generated using the Bac-to-Bac system (Invitrogen). Viruses were amplified by infection of with and protein in the was by using For protein of cells were at of infection of or and the After were on a system with to binding After of protease His-tagged proteins were purified on and on or Spätzle C106 was generated from purified proprotein using as described (5Weber A. Tauszig-Delamasure S. Hoffmann J. Lelièvre E. Gascan H. Ray K. Morse M. Imler J. Gay N. Nat. Immunol. 2003; 4: 794-800Crossref PubMed Scopus (340) Google Scholar). isothermal titration a was were at by Toll or Toll5B at of or into the C106 fragment at concentration of In the C106 at a concentration of was into Toll or Toll5B at or were and at a of were into and as by the Protein were by amino analysis for Protein and with an protein concentration of 1 were into and Sulfo-KMUS (N-(κ-maleimidoundecanoyloxy)sulfosuccinimide were from to of as in Fig. and the to with The were at for and the was by of of 1 and were and the by were on an with a and an system for For the analysis of the complex and the Toll sedimentation were at or at The was were using The specific and were using T. T. S. S. J.C. in and Science. The of Scholar). were with an using and the system for of Toll were used and and were at of and The were using J. J. A. E. R.A. P. Biochem. 2004; PubMed Scopus Google Scholar). embryonic cells were in with and and in at a of were using and either of or Toll, the 1 residues to residues or residues to as well as of a the of the factor were into and were with Spätzle C106 or human at for and was with and 1 and and subsequent of and analysis was on a in for residues of Toll and of human were using using K. Blundell T.L. 1998; PubMed Scopus (355) Google Scholar). a Toll and used to the and the of binding full-length and a truncated Toll ectodomain and active, dimeric Spätzle ligand Toll ectodomain was into the Spätzle C106 protein This in the formation of a complex of two ectodomains binding 1 Spätzle dimer with a of By contrast, in the titration in which C106 was into Toll a complex with a and a higher of was This in affinity that the binding of a first Toll molecule the affinity for the second binding and that the two binding events are were and are in I. The in are the same in both that both the same binding This is by the of for which a factor of to the of the C106 ligand, has to be taken into Open in a were using Toll5B, a truncated ectodomain amino acids of sequence a of the second block and the C-terminal flank (see Fig. a known to be for signaling. with Toll5B the same as the full-length which that block II and the C-terminal flank to the binding of the ligand. and the of Both and of Toll and the of the different for complexes Toll and Spätzle, we first to the of a complex using cross-linking. were to a Toll C106 at and for at and the were then by The of to the in the of a is another cross-linking is used The is to to a protein complex consisting of two Toll monomers of and C106 dimer with the of a complex we to an in which the of C106 to a was as in We sedimentation on a of Toll with a of of the for the complex a with a sedimentation to a molecular of well with the of a complex. Toll a in has that some receptor ectodomains as dimers O. Science. PubMed Scopus Google and the complex binding of Toll to C106 be by such a we used to the of Toll ectodomain in from a analysis are two in sedimentation of and The analysis that the molecular of these are and The first is with a Toll and the second with a Toll The for the dimers probably a in the for the and which is taken into in the We then the of Toll using sedimentation These were at different of Toll and The results, as in Fig. were to a The of the is and the for was The molecular was and the is This suggests in contrast to C106 dimerization of Toll is of low affinity with a receptor for the Science. 2004; PubMed Scopus Google a structural of Spätzle is that binding of the ligand binding and that receptor dimerization C106 the N-terminal of a with have gained further into the interaction by the of an N-terminal of the Toll 4B). by analysis this fragment was to with full-length Toll ectodomain in the Ni-NTA in baculovirus cells was to N-terminal protein and that the fragment with the sequence to of the full-length Toll and that an this fragment protein C-terminal of block which is the first C-terminal cysteine-rich and block II with and C-terminal 4B). of the Ni-NTA the full-length Toll ectodomain and the fragment by a a at an to an molecular of analysis of this both full-length and truncated Toll 1 proteins in to Toll or or with of this are either that the is able to form a complex with the full-length protein or that the truncated with a molecular to that of of a of full-length Toll ectodomains a single at and the of the in both are at is to a to that of Toll the first more as such a is with the formation of a higher molecular that is Toll This suggests that the of the first as in in a high affinity receptor-receptor interaction by binding structural C-terminal of block receptor molecules interact and with low affinity (see Fig. of the and in the of Toll that the analysis of the fragment that the C-terminal residues of Toll are in we to other of the such as the juxtamembrane or transmembrane have an on signaling. these sequences are involved in ligand binding must be in for example by allowing receptor-receptor interactions to the transmembrane region of Drosophila Toll and that of the human Toll-like receptor a receptor were as illustrated in Fig. 1 of the Drosophila Toll ectodomain and transmembrane and the TIR and of the Drosophila Toll ectodomain the transmembrane and TIR in Fig. in both C106 is able to activation of an in cells and the is with that in Drosophila cells (5Weber A. Tauszig-Delamasure S. Hoffmann J. Lelièvre E. Gascan H. Ray K. Morse M. Imler J. Gay N. Nat. Immunol. 2003; 4: 794-800Crossref PubMed Scopus (340) Google Scholar). However, in the of is a of constitutive This suggests that is an role for the transmembrane in signaling and that are in the activation of the Drosophila Toll and This may be by the sequence in this region the two sequences are In this paper we biophysical of the interaction the ectodomain of Toll and the dimeric ligand Spätzle. show that two of binding in a complex Spätzle C106 is in and a complex Toll is in of the in these that the first and the second binding are and that the first has a higher affinity of binding than the This suggests negative the first binding for example cause structural in the symmetrical binding sites in the C106 a structural has recently of NGF, a dimeric cystine knot ligand structurally related to Spätzle K. Parker J.S. Blundell T.L. Gay N.J. Trends Biochem. Sci. 1998; 23: 239-242Abstract Full Text Full Text PDF PubMed Scopus (85) Google bound to a single molecule of the receptor Science. 2004; PubMed Scopus Google Scholar). In this the show that with two sites at the of the and that this first binding a of structural in the ligand, which the second in the The biophysical the for an titration of into a solution of an with that in Fig. and which a titration was suggest that in the complex and point to C106 and in receptor However, in contrast to the in the complex the of a second receptor may the of the class II of dominant alleles In C106 generated in of the is by Toll can be from the perivitelline of embryos D. C. Cell. Full Text PDF PubMed Scopus Google By contrast, in type II C106 a Toll receptor is to interact at the with full-length as this second interaction with a the of C106 to a The show that the binding of C106 to the truncated Type II ectodomain Toll5B is from that of the full-length the exact residues involved in ligand binding to be more this that the second block of and the cysteine-rich flank are involved in ligand This is with the that Toll type II dominant alleles bind to Spätzle C106 the fact that are truncated ectodomains (13Schneider D.S. Hudson K.L. Lin T.Y. Anderson K.V. Gene Dev. 1991; 5: 797-807Crossref PubMed Scopus (261) Google Scholar) (5Weber A. Tauszig-Delamasure S. Hoffmann J. Lelièvre E. Gascan H. Ray K. Morse M. Imler J. Gay N. Nat. Immunol. 2003; 4: 794-800Crossref PubMed Scopus (340) Google Scholar). In the of at in Fig. Toll N-terminal and the first and this suggests that the binding for Spätzle is this The extracellular of Toll are to adopt a structure to that in Morse M.A. Gay N.J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google and is to that the binding sites of are in a structure in that by cysteine rich Bazan J.F. G. K. M. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). is NGF, Spätzle has two binding sites to the in the Toll of the binding to the first block of suggests that the second the cysteine-rich and transmembrane play an role in receptor activation or some on their We evidence suggesting that block and other C-terminal are involved in receptor-receptor and that of the Toll in a these This suggests that in the of C106, the N-terminal ligand binding domain is to interaction and receptor signaling in the of ligand. We suggest that ligand binding this by conformational that Toll receptors into and signaling their This is to a on studies for the EGF receptor J. Cell Biol. PubMed Scopus Google Scholar). These that the EGF receptor a structural rearrangement ligand involving the of the that Toll transmembrane and intracellular on their are active C. Biol. Cell. PubMed Scopus Google Scholar, Y. T. Zhou S. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar, S. E. Hoffmann J.A. Imler J.L. P. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) and that is an of Toll ectodomains as in this the importance of the of and intracellular by and transmembrane This is by the of the active Type 1 dominant Toll receptors and second by the described which show that of the transmembrane domain the receptor. This that in the inactive these are in a that receptor self-association together with N-terminal (see The disulfide bonds of the C-terminal flank may a conformational on the juxtamembrane region that is in the Type 1 or ligand receptor In the related receptor for example, binding of factor domain the cysteine rich domain to from a structure to an S. P. Science. 2002; 297: PubMed Scopus Google which the conformational of these mechanism for Toll receptor activation has recently by Y. T. Zhou S. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar) further suggest that the cysteine generated in the Type 1 dominant is activated by formation of a covalent with another receptor. However, we were to show that of these dominant alleles in embryos than by was cross-linked in this J. is that of signaling function with the C-terminal flank is by the transmembrane domain of the receptor. the of sequences amino acids to the a of constitutive activation in receptors 4B). This to in the role of and transmembrane in the and activation of Drosophila and human Toll receptors signal together suggest a of activation for Drosophila Toll which is illustrated in Fig. This mechanism has in common with that for the EGF receptor (see However, in that the binding of EGF to two receptor molecules receptor we that ligand induced dimerization receptor-receptor which the signaling complex. structural studies of Toll ectodomains or complexes, as well as interactions on the be to this be to the of this for the activation of The at this is which is activated by from that activation of requires the secreted molecule and that binding of a conformational in the complex S. S. Y. T. N. Y. Y. K. S. Y. A. K. J. 2003; PubMed Scopus Google Scholar, T. A. D. N. J. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar, C.A. C. A. D.M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). We and E. Walker for and of this and Morse at for support and of the during the of this