Cytokine Spatzle binds to the Drosophila immunoreceptor Toll with a neurotrophin-like specificity and couples receptor activation

Abstract

Drosophila Toll functions in embryonic development and innate immunity and is activated by an endogenous ligand, Spätzle (Spz). The related Toll-like receptors in vertebrates also function in immunity but are activated directly by pathogen-associated molecules such as bacterial endotoxin. Here, we present the crystal structure at 2.35-Å resolution of dimeric Spz bound to a Toll ectodomain encompassing the first 13 leucine-rich repeats. The cystine knot of Spz binds the concave face of the Toll leucine-rich repeat solenoid in an area delineated by N-linked glycans and induces a conformational change. Mutagenesis studies confirm that the interface observed in the crystal structure is relevant for signaling. The asymmetric binding mode of Spz to Toll is similar to that of nerve growth factor (NGF) in complex with the p75 neurotrophin receptor but is distinct from that of microbial ligands bound to the Toll-like receptors. Overall, this study indicates an allosteric signaling mechanism for Toll in which ligand binding to the N terminus induces a conformational change that couples to homodimerization of juxtamembrane structures in the Toll ectodomain C terminus.

Keywords: Spz ligand; Toll receptor; crystallography; isothermal titration calorimetry; mass spectrometry.

Fig. 1.

Toll–Spz overall structure and ligand binding mode. (A) Schematic representation in two orientations (side view and concave view) with color-coded areas for Toll and Spz C106 dimer. The first N-acetylglucosamine residue of the glycan structures attached to Asn residues at positions 80, 140, 270, 346, and 391 is shown in light cyan spheres. (B) Space-filling models highlight the spatial restriction provided by the presence of the glycosylations. (C) Schematic representation of Spz C106 with the four-stranded antiparallel β-sheet labeled A–D and the two missing regions, the Trp loop, and the wing of each protomer indicated by dashed lines and corresponding missing residue numbers. The disulfide bonds of the cystine residues involved in the knot motif are shown along with the intermolecular bond involving Cys-98 of each chain. (D) Space-filling model of the Spz cystine-knot domain as observed in the complex structure, color-coded by polypeptide chain in Upper. In Lower, the same orientation highlights the footprint left by Toll across the dimeric surface of the ligand (interface colored in magenta for the proximal chain and pink for the distal one). (E) The asymmetry of the binding to Toll is represented by color-coding Toll residues that interact with Spz in either green or yellow depending on which chain that they contact.

Fig. 2.

Conformational changes induced by ligand binding and crystal packing. (A) Ribbon representation of Toll_N6 (residues 28–228 in cyan) superimposed on Toll_N13–VLR (magenta-gray) –Spz (green-yellow) reveals that conformational changes occur mainly in the Toll LRRNT region. The encapsulated close-up view shows a 4.6-Å shift of the Cα atom of D40 and a 3.6-Å shift for E61, which brings it in close proximity to Spz. (B) Ribbon representation of the Toll_N13–VLR–Spz complex superimposed on refolded Spz (black) (19) and a homology model of Spz based on NGF reveal steric hindrance by Toll LRRNT in the position of the wings. Arrows indicate potential repositioning of the wings.

Fig. 3.

Mutagenesis of Toll/Spz interface residues observed in the crystal structure. (A) Position in the interface of residues targeted for mutation: R14, K15, and D55 (boxed) and their symmetry-related counterpart on the other protomer. (B and C) Detailed views of the interactions between R14, K15, and D55 and the Toll ectodomain. (D) Spz mutants are defective in signaling. White bars indicate activity of 100 nM protein before protease cleavage and black bars after. Data are shown as relative luciferase induction and represented as mean ± SD (error bars).

Fig. 4.

Ligand-binding and dimerization abilities of Toll truncations. All constructs containing at least the N-terminal 399 residues are able to bind Spz C106 according to a range of biophysical techniques. White and gray triangles indicate VLR capping structures, black triangles native Toll N- and C-caps. The Spz construct is represented before and after TEV processing, which leaves an additional glycine residue at the N terminus of C106. The constructs Toll_N13–VLR and active Spz C106 have been used for crystallography.

Fig. 5.

Isothermal titration calorimetry of the Toll/Spz complex. Purified Spz C106 was titrated into the ITC measuring cell containing Toll_N13–VLR protein, which results in the formation of a complex of one truncated ectodomain binding a single Spz dimer with a dissociation constant between 30 and 50 nM, comparable with the affinity of full-length ectodomain. In the reciprocal titration in which Toll_N13–VLR was injected into Spz C106, a complex with the same stochiometry and dissociation constant was observed. A summary table of the thermodynamic parameters and a typical experiment is shown in Table S4.