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Structural basis of the proinflammatory signaling complex mediated by TSLP

Nature Structural & Molecular Biology volume 21, pages 375–382 (2014)Cite this article

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Abstract

Thymic stromal lymphopoietin (TSLP), a cytokine produced by epithelial cells at barrier surfaces, is pivotal for the development of widespread chronic inflammatory disorders such as asthma and atopic dermatitis. The structure of the mouse TSLP-mediated signaling complex reveals how TSLP establishes extensive interfaces with its cognate receptor (TSLPR) and the shared interleukin 7 receptor α-chain (IL-7Rα) to evoke membrane-proximal receptor-receptor contacts poised for intracellular signaling. Binding of TSLP to TSLPR is a mechanistic prerequisite for recruitment of IL-7Rα to the high-affinity ternary complex, which we propose is coupled to a structural switch in TSLP at the crossroads of the cytokine-receptor interfaces. Functional interrogation of TSLP-receptor interfaces points to putative interaction hotspots that could be exploited for antagonist design. Finally, we derive the structural rationale for the functional duality of IL-7Rα and establish a consensus for the geometry of ternary complexes mediated by interleukin 2 (IL-2)–family cytokines.

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Figure 1: Structure of the TSLP–TSLPR–IL-7Rα complex.
Figure 2: Molecular dynamics simulation to characterize unbound TSLP.
Figure 3: Degeneracy of IL-7Rα in TSLP and IL-7 signaling complexes.
Figure 4: ITC and SPR binding studies of the assembly of the ternary TSLP–TSLPR–IL-7Rα complex.
Figure 5: Cellular binding and activity assays with TSLP, TSLPR and IL-7Rα mutants.

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Acknowledgements

We thank the staff of beamlines P13 (PetraIII, Deutsches Elektronen-Synchrotron) and Proxima 2A (SOLEIL) for their technical support and beamtime allocation; R. Loris for access to a MicroCal iTC-200 instrument; A. Garcia-Pino and S. De Gieter for assistance during ITC measurements; B. Vekemans and G. Vandriessche for X-ray fluorescence and mass spectrometry measurements, respectively, to confirm the presence of calcium ions and PEG 4000 in crystal form 1A. Supercomputing resources (Stevin Supercomputer Infrastructure) and services were provided by Ghent University, the Hercules Foundation (Belgium) and the Flemish government, department Economie, Wetenschap & Innovatie (EWI). K.V. is supported as postdoctoral fellow by Research Foundation Flanders, Belgium (FWO). Y.B. and L.V. are supported as predoctoral fellows of the Agentschap voor Innovatie door Wetenschap en Technologie (IWT). This work was supported by grants from the FWO (no. G0C2214N to S.N.S. and F.P.), the Hercules Foundation (no. AUGE-11-029 to S.N.S.) and Ghent University (Methusalem grant to J.T.).

Author information

Authors and Affiliations

  1. Department of Biochemistry & Microbiology, Unit for Structural Biology, Laboratory for Protein Biochemistry and Biomolecular Engineering, Ghent University, Ghent, Belgium

    Kenneth Verstraete, Loes van Schie, Yehudi Bloch & Savvas N Savvides

  2. Department of Medical Protein Research, Vlaams Interuniversitair Instituut voor Biotechnologie and Ghent University, Ghent, Belgium

    Laurens Vyncke, Jan Tavernier & Frank Peelman

  3. Center for Molecular Modeling, Ghent University, Ghent, Belgium

    Ewald Pauwels

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  1. Kenneth Verstraete
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  2. Loes van Schie
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Contributions

K.V. designed the strategy for recombinant protein production and crystallization. K.V. and L.v.S. created expression constructs, performed protein expression and purification and performed crystallization experiments. K.V. determined and analyzed crystallographic structures with contributions from L.v.S. and S.N.S. K.V. and L.v.S. performed ITC and SPR binding studies. Y.B. performed MALS experiments. K.V., F.P. and S.N.S. designed site-directed mutants and L.V. carried out site-directed mutagenesis. F.P. designed, performed and optimized cellular binding and activity assays with contributions from J.T. E.P. carried out molecular dynamics simulations. K.V. and S.N.S. wrote the manuscript with contributions from all authors. S.N.S. conceived and supervised the project.

Corresponding authors

Correspondence to Kenneth Verstraete or Savvas N Savvides.

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Integrated supplementary information

Supplementary Figure 1 Isolation and crystallization of glycan-minimized ternary TSLP–TSLPR– IL-7Rα complexes.

(a) SEC elution profiles of binary TSLPN123Q–TSLPRN122Q complex expressed in HEK293S MGAT1–/– (blue profile) and ternary TSLPN123Q–TSLPRN122Q–IL-7Rα complex (grey profile). Elution volumes of protein standards are indicated above the chromatograms. The Coomassie-stained SDS-PAGE gel shows the binary complex before and after EndoH-treatment, and the resulting ternary complex used to obtain crystal forms 1A and 1B. The molecular masses calculated from the amino acid sequences (i.e. without glycosylation) for recombinant mouse TSLP, TSLPR and IL-7Rα are 15 kDa, 23 kDa and 27 kDa respectively. The TSLPN123Q–TSLPRN53Q–IL-7Rα complex (crystal form 2) was assembled and purified via an identical approach. (b) Cartoon representation of the three structures for the ternary TSLP:TSLPR:IL-7Rα complex determined in this study. Disulfide-bridges are shown as yellow spheres. Potential N-linked glycosylation sites are indicated as blue spheres. The single GlcNAc-residue on TSLPRAsn53 modeled in crystal form 1A is shown as a magenta mesh. (c) Structural superposition of the IL-7RαD2–TSLPRD2 assembly observed in crystal form 2 against the corresponding assembly observed in crystal form 1B.

Supplementary Figure 2 Structure-based sequence comparison between TSLP and IL-7 and evolutionary conserved features in TSLP.

(a) Mature sequences for hTSLP (NP_149024.1), mTSLP (NP_067342.1), mIL-7 (NP_032397.1) and hIL-7 (NP_000871.1) were aligned based on a structural superposition of mTSLP and hIL-7 (pdb 3DI2, chain A) and are annotated according to secondary structure. The 56 residues from which the Cα atoms were included in the structural alignment are highlighted by grey shaded boxes. Dashed lines indicate missing segments in the structural models. Conserved residues are shown in white on a black background. Semi-conserved residues are in black bold and are boxed. Residues in mTSLP and hIL-7 colored blue interact with IL-7Rα, residues of mTSLP colored orange interact with TSLPR. The basic cassette located in the CD-loop of hTSLP is highlighted in red. (b) Structural superposition of mTSLP (blue) and hIL-7 (yellow). mTSLP residues Trp132 and Tyr34, and the corresponding residues in hIL-7, are shown as sticks. (c) View of the evolutionarily conserved hydrophobic core of mTSLP and coordination of the buried water molecule (red sphere), and the internal void volume in the core of TSLP (black mesh). (d) Sequence alignments for sequences of mature TSLP from different mammalian species. mTSLP residues interacting with TSLPR (orange) and IL-7Rα (blue) are indicated. Black lines indicate the conserved disulfide pattern of TSLP. The stretch of basic residues in the CD-loop, present in a subset of TSLP sequences, is highlighted in red. TSLP sequences were derived from the NCBI-database: M. musculus: NP_067342.1, H. sapiens: NP_149024.1, R. norvegicus: XP_001067649.1, M. mulatta: XP_001100503.1, F. catus: XP_003981290.1, O. garnettii: XP_003788993.1, E. caballus: NP_001157535.1, A. melanoleuca: XP_002919224.1

Supplementary Figure 3 Cross-species sequence alignments for the TSLPR ectodomain.

The secondary structure elements correspond to mTSLPR. Dashed lines indicate that the corresponding region was missing in the structural model. Strands are labeled A1-G1 for TSLPRD1 and A2-G2 for TSLPRD2. Conserved residues are shown as white letters on a black background. Semi-conserved residues are shown in black bold and are boxed. mTSLPR residues colored blue interact with mTSLP. Black lines indicate the disulfide pattern of mTSLPR. The atypical PSXW(S/T) motif in TSLPRD2 is indicated by a red box. The predicted transmembrane (TM)-region is shaded blue. Sequences were derived from the NCBI-database: M. musculus: NP_057924.3, H. sapiens: NP_071431.2, R. norvegicus: NP_604460.2, S. scrofa: XP_003361089.1, S. simum: XP_004440836.1, O. aries: XP_004022563.1, O. rosmarus: XP_004400052.1, C. lupus: XP_005641109.1.

Supplementary Figure 4 Cross-species sequence alignments for the IL-7Rα ectodomain.

The secondary structure elements correspond to mIL-7Rα. Dashed lines indicate that the corresponding region was missing in the structural model. Strands are labeled A1-G1 for IL-7RαD1 and A2-G2 for IL-7RαD2. Conserved residues are shown as white letters on a black background. Semi-conserved residues are black bold and are boxed. mIL-7Rα residues colored blue interact with mTSLP, hIL-7Rα residues that interact with hIL-7 are colored orange. Positions that interact with both TSLP and IL-7 are indicated by red dots. Black lines indicate the disulfide bridge network in IL-7Rα. The WSXWS motif in IL-7RαD2 is indicated by a red box. The predicted transmembrane (TM)-region is shaded blue. Sequences were derived from the NCBI-database: M. musculus: NP_032398.3, H. sapiens: NP_002176.2, F. catus: XP_003981510.1, L. africana: XP_003408018.1, C. lupus: XP_855408.1, B. taurus: NP_001192816.1, O. aries: XP_004017102.1, M. domestica: XP_001373020.1 and G. gallus: NP_001073575.1.

Supplementary Figure 5 Biophysical studies to dissect the assembly of TSLP complexes and cellular activity assays to probe observed receptor-receptor contacts at site III.

(a) Coomassie-stained SDS-PAGE gel illustrating the purity of the samples for glycosylated TSLP, TSLPR, IL-7Rα and binary TSLP–TSLPR complex used in the binding studies. (b) ITC thermogram for the titration of 4 mM mIL-7Rα with 46 μM mTSLP. (c-d) Recovered ITC-samples from the titration of TSLP with TSLPR (c) and the titration of IL-7Rα with TSLP–TSLPR (d) were analyzed by MALS. (e) Single-cycle kinetics SPR data for the binding of TSLPR to immobilized TSLP. (f) Cellular activity assays employing TSLPR and IL-7Rα mutants to probe receptor-receptor contacts in site III. TSLP-induced STAT5 activity was measured using a luciferace-based reporter system in HEK293T cells expressing wild type and/or mutant forms of either TSLPR or IL-7Rα upon incubation with wild type TSLP. The determined half-maximum effective concentration (EC50) values for STAT5-activation assays were determined as follows: Control experiment with wild type TSLPR and IL-7Rα (EC50= 0.27 nM); IL-7RαH185A (EC50= 0.31 nM); TSLPRQ162A (EC50= 0.28 nM), TSLPRR180S (EC50= 0.38 nM), and TSLPRD177S (EC50= 0.39 nM). Each experiment was carried out in triplicate and error bars were calculated as s.e.m.

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Verstraete, K., van Schie, L., Vyncke, L. et al. Structural basis of the proinflammatory signaling complex mediated by TSLP. Nat Struct Mol Biol 21, 375–382 (2014). https://doi.org/10.1038/nsmb.2794

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