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Structural basis of recognition of interferon-α receptor by tyrosine kinase 2

Nature Structural & Molecular Biology volume 21, pages 443–448 (2014)Cite this article

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Abstract

Tyrosine kinase 2 (TYK2) is a member of the Janus kinase (JAK) family of nonreceptor tyrosine kinases, which are essential for proper signaling in immune responses and development. Here we present a 2.0-Å-resolution crystal structure of a receptor-binding fragment of human TYK2, encompassing the FERM and SH2 domains, in complex with a so-called 'box2'-containing intracellular peptide motif from the interferon-α receptor chain 1 (IFNAR1). The TYK2-IFNAR1 interface reveals an unexpected receptor-binding mode that mimics a SH2 domain–phosphopeptide interaction, with a glutamate replacing the canonical phosphotyrosine residue. This structure provides the first view, to our knowledge, of a JAK in complex with its cognate receptor and defines the molecular logic through which JAKs have evolved to interact with divergent receptor sequences.

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Figure 1: The structure of the TYK2 FERM-SH2 receptor-binding module in complex with IFNAR1.
Figure 2: IFNAR1 interacts with TYK2 in three segments.
Figure 3: The TYK2–IFNAR1 complex resembles a SH2-phosphopeptide interaction.
Figure 4: IFNAR1 interface contacts stabilize TYK2 interaction.
Figure 5: TYK2-IFNAR1-interface residues are conserved in other cytokine receptors and JAKs.

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Acknowledgements

We thank X. Ma, M. Ultsch, J. Payandeh, L. Wu and the staff at the Stanford Synchrotron Radiation Lightsource (SSRL) for their technical advice and assistance. We also thank the Structural Biology Expression Group, Microchemistry and Proteomics Laboratory and DNA Sequencing facilities at Genentech for their technical support. Portions of this research were carried out at the SSRL, a Directorate of SLAC National Accelerator Laboratory and an Office of Science User Facility operated for the US Department of Energy (DOE) Office of Science by Stanford University. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the US National Institutes of Health (NIH), National Institute of General Medical Sciences (NIGMS; including P41GM103393). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.

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  1. Department of Structural Biology, Genentech, South San Francisco, California, USA

    Heidi J A Wallweber, Christine Tam, Yvonne Franke, Melissa A Starovasnik & Patrick J Lupardus

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Contributions

H.J.A.W. performed protein purification, crystallization and biochemical experiments. C.T. and Y.F. performed design and cloning of TYK2 constructs. H.J.A.W. and M.A.S. participated in data analysis and manuscript preparation. P.J.L. solved and analyzed the structure, prepared figures and wrote the manuscript.

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Correspondence to Patrick J Lupardus.

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Integrated supplementary information

Supplementary Figure 1 Biochemical analysis of the TYK2–IFNAR1 complex

Comparison of size exclusion chromatograms of (a) the TYK2 FERM–SH2 to the (b) TYK2 FERM–SH2+IFNAR1 single chain fusion. Recombinant TYK2 FERM–SH2 (residues 23–566) or TYK2 FERM–SH2 (residues 23–583) fused to IFNAR1 (residues 465–512) were expressed in insect cells, purified by Ni-affinity and analyzed by size exclusion chromatography on a Superdex 200 size exclusion column. Labeled fractions were then analyzed by SDS-PAGE. The solution behavior and monodispersity of TYK2 FERM–SH2 without IFNAR1 was compromised when compared to the correctly sized monodisperse peak seen when IFNAR1 chain was present. (c) Size exclusion chromatogram showing co-elution of TYK2 FERM–SH2 (residues 23–583) and IFNAR1 peptide (residues 465–512) after protease-mediated cleavage of the linker sequence. Marked fractions were analyzed by SDS-PAGE, and “L” denotes load sample. Protein standards are overlaid on the chromatogram and shown as a gray dashed line. (d) LC/MS analysis of TYK2 non-covalently associated with IFNAR1. A peak fraction from the purification of the TEV protease-cleaved TYK2/IFNAR1 complex shown in (c) was analyzed by HPLC followed by MS-TOF to determine intact mass of the protein contents of that fraction. Two peaks were observed, the first corresponding to IFNAR1 peptide (top panel) and the second corresponding to the TYK2 FERM/SH2 (bottom panel). The sequence of the TEV-cleavable TYK2/IFNAR1 construct analyzed is shown underneath the chromatogram in (c).

Supplementary Figure 2 Structural analysis and crystal packing of the TYK2–IFNAR1 complex

(a) Stereo view cartoon diagram of the TYK2 FERM–SH2 in complex with IFNAR1. TYK2 is colored as a rainbow, with blue starting at the N-terminus to red at the C-terminus. IFNAR1 is colored yellow. (b) Stereo view of electron density (2Fobs-Fcalc contoured at 1σ) around the SH2 domain and IFNAR1 peptide. The backbone is shown as a ribbon with sidechains shown as stick models, with TYK2 colored as in (a). (c) Analysis of crystal packing and contacts near the IFNAR1 peptide. Front view and side view of the TYK2–IFNAR1 complex showing nearest neighbors to IFNAR1 in the crystal lattice. IFNAR1 does not participate in any crystal contacts, indicating the peptide is bound in the native conformation.

Supplementary Figure 3 Secondary structure of the TYK2 FERM-SH2 domain, and comparison of TYK2 primary sequence to the other human JAK family members

Secondary structure elements are labeled using conventions established for the FAK FERM domain and SH2 domain of Lck. Secondary structural elements are displayed above the alignments, with helices displayed as cylinders, strands displayed as block arrows, and loops displayed as lines. Secondary structure is labeled and domain structure colors matched between the structure and the alignment. Residues contacting IFNAR1 are shaded in yellow.

Supplementary Figure 4 Details of the TYK2 FERM-SH2 interdomain linker structure and SH2 domain comparison

(a) Close up view showing the L1 linker connecting the F1 and F2 subdomains of the FERM domain along with the C-terminal L3 linker. (b) Close-up view showing the L2 linker that connects the FERM F3 subdomain to the SH2 domain. All linkers are shown in orange, with key residues shown as stick models. (c-e) Comparison of the TYK2 SH2 domain to the SHP2 N-SH2. Annotated view of the (c) TYK2 SH2 (blue) and (d) SHP2 N-SH2 (PDB ID 1AYB, purple) secondary structure elements, to illustrate similarities and differences between the TYK2 SH2 and a canonical phosphopeptide-binding SH2 domain. (e) Overlay of the SHP2 N-SH2 domain onto the TYK2 SH2 domain (RMSD 2.0Å over 89 residues). Alignments generated using SUPERPOSE.

Supplementary Figure 5 Differential scanning fluorimetry analysis of single-chain TYK2–IFNAR1 wild-type and mutant complexes

Curves are plotted and overlaid for each individual experiment, with apparent melting temperature (Tm), standard deviation (SD), P-value (calculated by unpaired, two-tailed students t-test) comparing the mutants against wild type, and number of samples (n) are printed below each graph. At the bottom, representative fitted curves are plotted and overlaid for each of the proteins to illustrate the experimental curve differences between the wild type and IFNAR1 mutants.

Supplementary Figure 6 Pull-down analysis of His-tagged TYK2 (a) and GST-tagged IFNAR1 (b) wild type and mutants. Full gel images are shown here for the experiments in Figure 4b and c.

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Wallweber, H., Tam, C., Franke, Y. et al. Structural basis of recognition of interferon-α receptor by tyrosine kinase 2. Nat Struct Mol Biol 21, 443–448 (2014). https://doi.org/10.1038/nsmb.2807

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