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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.

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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.

References

  1. Hunter, T. Signaling–2000 and beyond. Cell 100, 113–127 (2000).

    CAS  PubMed  Google Scholar 

  2. Lemmon, M. A. & Schlessinger, J. Cell signaling by receptor tyrosine kinases. Cell 141, 1117–1134 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Blume-Jensen, P. & Hunter, T. Oncogenic kinase signalling. Nature 411, 355–365 (2001).

    CAS  PubMed  Google Scholar 

  4. Hubbard, S. R. Autoinhibitory mechanisms in receptor tyrosine kinases. Front Biosci. 7, d330–d340 (2002).

    CAS  PubMed  Google Scholar 

  5. Hubbard, S. R. Juxtamembrane autoinhibition in receptor tyrosine kinases. Nat. Rev. Mol. Cell Biol. 5, 464–471 (2004).

    CAS  PubMed  Google Scholar 

  6. Schlessinger, J. Signal transduction. Autoinhibition control. Science 300, 750–752 (2003).

    CAS  PubMed  Google Scholar 

  7. Wybenga-Groot, L. E. et al. Structural basis for autoinhibition of the Ephb2 receptor tyrosine kinase by the unphosphorylated juxtamembrane region. Cell 106, 745–757 (2001).

    CAS  PubMed  Google Scholar 

  8. Hubbard, S. R. & Miller, W. T. Receptor tyrosine kinases: mechanisms of activation and signaling. Curr. Opin. Cell Biol. 19, 117–123 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Zhang, X., Gureasko, J., Shen, K., Cole, P. A. & Kuriyan, J. An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor. Cell 125, 1137–1149 (2006).

    CAS  PubMed  Google Scholar 

  10. Kovacs, E., Zorn, J. A., Huang, Y., Barros, T. & Kuriyan, J. A structural perspective on the regulation of the epidermal growth factor receptor. Annu. Rev. Biochem. 84, 739–764 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Jura, N. et al. Catalytic control in the EGF receptor and its connection to general kinase regulatory mechanisms. Mol. Cell 42, 9–22 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Nolen, B., Taylor, S. & Ghosh, G. Regulation of protein kinases; controlling activity through activation segment conformation. Mol. Cell 15, 661–675 (2004).

    CAS  PubMed  Google Scholar 

  13. Pellicena, P. & Kuriyan, J. Protein–protein interactions in the allosteric regulation of protein kinases. Curr. Opin. Struct. Biol. 16, 702–709 (2006).

    CAS  PubMed  Google Scholar 

  14. Boggon, T. J. & Eck, M. J. Structure and regulation of Src family kinases. Oncogene 23, 7918–7927 (2004).

    CAS  PubMed  Google Scholar 

  15. Binns, K. L., Taylor, P. P., Sicheri, F., Pawson, T. & Holland, S. J. Phosphorylation of tyrosine residues in the kinase domain and juxtamembrane region regulates the biological and catalytic activities of Eph receptors. Mol. Cell. Biol. 20, 4791–4805 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Furdui, C. M., Lew, E. D., Schlessinger, J. & Anderson, K. S. Autophosphorylation of FGFR1 kinase is mediated by a sequential and precisely ordered reaction. Mol. Cell 21, 711–717 (2006).

    CAS  PubMed  Google Scholar 

  17. Wu, J. et al. Small-molecule inhibition and activation-loop trans-phosphorylation of the IGF1 receptor. EMBO J. 27, 1985–1994 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Kan, S. H. et al. Genomic screening of fibroblast growth-factor receptor 2 reveals a wide spectrum of mutations in patients with syndromic craniosynostosis. Am. J. Hum. Genet. 70, 472–486 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. McDonell, L. M., Kernohan, K. D., Boycott, K. M. & Sawyer, S. L. Receptor tyrosine kinase mutations in developmental syndromes and cancer: two sides of the same coin. Hum. Mol. Genet. 24, R60–R66 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Wilkie, A. O. Bad bones, absent smell, selfish testes: the pleiotropic consequences of human FGF receptor mutations. Cytokine Growth Factor Rev. 16, 187–203 (2005).

    CAS  PubMed  Google Scholar 

  21. Chen, H. et al. A molecular brake in the kinase hinge region regulates the activity of receptor tyrosine kinases. Mol. Cell 27, 717–730 (2007).

    PubMed  PubMed Central  Google Scholar 

  22. Chen, H. et al. Elucidation of a four-site allosteric network in fibroblast growth factor receptor tyrosine kinases. Elife 6, e21137 (2017).

    PubMed  PubMed Central  Google Scholar 

  23. Rosen, M. K. et al. Selective methyl group protonation of perdeuterated proteins. J. Mol. Biol. 263, 627–636 (1996).

    CAS  PubMed  Google Scholar 

  24. Pervushin, K., Riek, R., Wider, G. & Wuthrich, K. Attenuated T2 relaxation by mutual cancellation of dipole–dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc. Natl Acad. Sci. USA 94, 12366–12371 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Mittermaier, A. & Meneses, E. Analyzing protein–ligand interactions by dynamic NMR spectroscopy. Methods Mol. Biol. 1008, 243–266 (2013).

    CAS  PubMed  Google Scholar 

  26. Mittermaier, A. K. & Kay, L. E. Observing biological dynamics at atomic resolution using NMR. Trends Biochem. Sci. 34, 601–611 (2009).

    CAS  PubMed  Google Scholar 

  27. Chen, H. et al. Cracking the molecular origin of intrinsic tyrosine kinase activity through analysis of pathogenic gain-of-function mutations. Cell Rep. 4, 376–384 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Huang, Z. et al. Structural mimicry of A-loop tyrosine phosphorylation by a pathogenic FGF receptor 3 mutation. Structure 21, 1889–1896 (2013).

    CAS  PubMed  Google Scholar 

  29. Korzhnev, D. M., Kloiber, K., Kanelis, V., Tugarinov, V. & Kay, L. E. Probing slow dynamics in high molecular weight proteins by methyl-TROSY NMR spectroscopy: application to a 723-residue enzyme. J. Am. Chem. Soc. 126, 3964–3973 (2004).

    CAS  PubMed  Google Scholar 

  30. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D. Biol. Crystallogr. 67, 235–242 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D. Biol. Crystallogr. 66, 213–221 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D. Biol. Crystallogr. 60, 2126–2132 (2004).

    PubMed  Google Scholar 

  33. Songyang, Z. et al. Catalytic specificity of protein-tyrosine kinases is critical for selective signalling. Nature 373, 536–539 (1995).

    CAS  PubMed  Google Scholar 

  34. Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2, 1896–1906 (2007).

    CAS  PubMed  Google Scholar 

  35. Byron, S. A. et al. The N550K/H mutations in FGFR2 confer differential resistance to PD173074, dovitinib, and ponatinib ATP-competitive inhibitors. Neoplasia 15, 975–988 (2013).

    PubMed  PubMed Central  Google Scholar 

  36. Maier, J. A. et al. ff14SB: improving the accuracy of protein side chain and backbone parameters from ff99SB. J. Chem. Theory Comput. 11, 3696–3713 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Meagher, K. L., Redman, L. T. & Carlson, H. A. Development of polyphosphate parameters for use with the AMBER force field. J. Comput. Chem. 24, 1016–1025 (2003).

    CAS  PubMed  Google Scholar 

  38. Allner, O., Nilsson, L. & Villa, A. Magnesium ion–water coordination and exchange in biomolecular simulations. J. Chem. Theory Comput. 8, 1493–1502 (2012).

    CAS  PubMed  Google Scholar 

  39. Webb, B. & Sali, A. Protein structure modeling with MODELLER. Curr. Protoc. Bioinformatics 54, 5.6.1–5.6.37 (2017).

    Google Scholar 

  40. Bae, J. H. et al. Asymmetric receptor contact is required for tyrosine autophosphorylation of fibroblast growth factor receptor in living cells. Proc. Natl Acad. Sci. USA 107, 2866–2871 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Salomon-Ferrer, R., Gotz, A. W., Poole, D., Le Grand, S. & Walker, R. C. Routine microsecond molecular dynamics simulations with AMBER on GPUs. 2. explicit solvent particle mesh ewald. J. Chem. Theory Comput. 9, 3878–3888 (2013).

    CAS  PubMed  Google Scholar 

  42. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    CAS  PubMed  Google Scholar 

  43. Carver, J. P. & Richards, R. E. General 2-site solution for chemical exchange produced dependence of T2 upon Carr–Purcell pulse separation. J. Magn. Reson. 6, 89–105 (1972).

    CAS  Google Scholar 

  44. Luz, Z. & Meiboom, S. Nuclear magnetic resonance study of the protolysis of trimethylammonium ion in aqueous solution-order of the reaction with respect to solvent. J. Chem. Phys. 39, 366–370 (1963).

    CAS  Google Scholar 

Download references

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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