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Molecular basis for receptor tyrosine kinase A-loop tyrosine transphosphorylation

Nature Chemical Biology volume 16, pages 267–277 (2020)Cite this article

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Abstract

A long-standing mystery shrouds the mechanism by which catalytically repressed receptor tyrosine kinase domains accomplish transphosphorylation of activation loop (A-loop) tyrosines. Here we show that this reaction proceeds via an asymmetric complex that is thermodynamically disadvantaged because of an electrostatic repulsion between enzyme and substrate kinases. Under physiological conditions, the energetic gain resulting from ligand-induced dimerization of extracellular domains overcomes this opposing clash, stabilizing the A-loop-transphosphorylating dimer. A unique pathogenic fibroblast growth factor receptor gain-of-function mutation promotes formation of the complex responsible for phosphorylation of A-loop tyrosines by eliminating this repulsive force. We show that asymmetric complex formation induces a more phosphorylatable A-loop conformation in the substrate kinase, which in turn promotes the active state of the enzyme kinase. This explains how quantitative differences in the stability of ligand-induced extracellular dimerization promotes formation of the intracellular A-loop-transphosphorylating asymmetric complex to varying extents, thereby modulating intracellular kinase activity and signaling intensity.

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Fig. 1: The FGFR2KR678G Crouzon syndrome substitution accelerates phosphorylation of A-loop tyrosines without elevating intrinsic kinase activity.
Fig. 2: FGFR3R669E promotes formation of an A-loop-transphosphorylating asymmetric complex.
Fig. 3: The crystallographically deduced A-loop-transphosphorylation asymmetric complex forms in solution.
Fig. 4: Functional validation of the crystallographically deduced A-loop-transphosphorylating mechanism in vitro and in vivo.
Fig. 5: In vitro and in vivo complementation assays reinforce the existence of an asymmetric A-loop-tyrosine transphosphorylation complex.
Fig. 6: Asymmetric complex formation induces reciprocal allosteric changes in enzyme and substrate kinases.

Data availability

Atomic coordinates and structure factors of the FGFR3R669E asymmetric complex have been deposited in the Protein Data Bank under accession 6PNX. Raw mass spectrometry files and Mascot generic format files have been deposited in the MassIVE database under accession MSV000084018. All other data generated or analyzed during this study are included in this published article and its associated Supplementary Information.

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Acknowledgements

The authors are indebted to N. Cowan for critically reading and editing the manuscript. This work was supported by National Institute of Dental and Craniofacial Research (NIDCR) grant R01 DE13686 (to M.M.), National Institute of General Medical Sciences (NIGMS) grant R01 GM117118 (to N.J.T.), NIGMS grant R35 GM127040 (to Y.Z.), National Institute of Neurological Disorders and Stroke (NINDS) grant P30 NS050276 and Shared Instrumentation Grant RR027990 (to T.A.N.), China Scholarship Council (CSC) and China Association for Science and Technology (CAST) (to L.C.), National Cancer Institute (NCI) predoctoral grant F99CA212474 (to W.M.M.) and the Natural Science Foundation of China (NSFC) grant 81930108 (to G.L.). An NMR cryoprobe at New York University was supported by an NIH S10 grant (OD016343). Data collection at the New York Structural Biology Center was made possible by a grant from NYSTAR. Computing resources were provided by New York University-ITS. We dedicate this work to the memory of J.M., who died suddenly before submission.

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Author notes

  1. These authors contributed equally: Lingfeng Chen, William M. Marsiglia, Huaibin Chen.

  2. Deceased: Jinghong Ma

Authors and Affiliations

  1. School of Chemical Engineering, Nanjing University of Science and Technology, Nanjing, China

    Lingfeng Chen & Guang Liang

  2. Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY, USA

    Lingfeng Chen, Huaibin Chen, Lili Fu, Jinghong Ma & Moosa Mohammadi

  3. Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China

    Lingfeng Chen, Lili Fu, Guang Liang & Xiaokun Li

  4. Department of Chemistry, New York University, New York, NY, USA

    William M. Marsiglia, Joseph Katigbak, Yingkai Zhang & Nathaniel J. Traaseth

  5. Department of Cell Biology and Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY, USA

    Hediye Erdjument-Bromage & Thomas A. Neubert

  6. Department of Cell and Molecular Biology, University of Rhode Island, Kingston, RI, USA

    David J. Kemble & Gongqin Sun

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Contributions

H.C. purified and crystallized FGFR3KR669E, and contributed to the initial analysis of the crystal structure. L.C. expressed and purified all structure-based FGFRK proteins, established stable cell lines, generated cell-based and kinase assay data (Fig. 1, 4 and 6 and Supplementary Figs. 2, 8, 9 and 14–16), prepared the structural figures and participated in the design of experiments and in editing and revising the manuscript. W.M.M. expressed and purified all FGFR2K samples for NMR studies, acquired and interpreted the NMR data (Figs. 3 and 6, and Supplementary Figs. 5, 7 and 10–12) and participated in editing and revising the manuscript. G.S. and D.J.K. provided the catalytic turnover rates data (Fig. 1a and Supplementary Fig. 1). T.A.N. and H.E.-B. generated and interpreted LC–MS data. G.L. and X.L. contributed to manuscript revision. J.M. and L.F. engineered bacterial and lentiviral expression constructs. J.K. and Y.Z. provided the molecular dynamics simulation data (Supplementary Fig. 6). N.J.T. directed the NMR studies, interpreted NMR datasets, and participated in writing the manuscript. M.M. conceived and directed the project, solved, refined, analyzed and interpreted the crystal structure of the FGFR3KR669E asymmetric complex and wrote the manuscript.

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Correspondence to Nathaniel J. Traaseth or Moosa Mohammadi.

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Chen, L., Marsiglia, W.M., Chen, H. et al. Molecular basis for receptor tyrosine kinase A-loop tyrosine transphosphorylation. Nat Chem Biol 16, 267–277 (2020). https://doi.org/10.1038/s41589-019-0455-7

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