Molecular basis for receptor tyrosine kinase A-loop tyrosine transphosphorylation

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.

Fig. 1 |. The FGFR2K^R678G Crouzon syndrome substitution accelerates phosphorylation of A-loop tyrosines without elevating intrinsic kinase activity.

a, Turnover rates (min⁻¹) of unphosphorylated FGFR2K^WT and nine variants harboring distinct pathogenic mutations. Note that the turnover rate of the R678G mutant is indistinguishable from the wild type. Data are mean ± s.d. (n = 3). Statistical analysis was performed via a two-tailed unpaired Student’s t test. b–d, Top, kinetics of overall tyrosine transphosphorylation in FGFR2K^WT, FGFR2K^E565A and FGFR2K^R678G assayed by native gel electrophoresis. Middle, LC–MS spectra showing transphosphorylation on A-loop tandem tyrosines (Y656 and Y657) in samples corresponding to those analyzed above. Bottom, quantitation of LC–MS relative ion intensities. Kinase assays were done independently twice with similar results. e, Phosphorylation on Y656 precedes that of Y657. MS/MS spectra of 0P (top), 1P (middle) and 2P (bottom) FGFR2K^WT A-loop tryptic peptides. Note the increase by 80 Da in the mass of the y4 ion (but not the y3 ion) in the mono-phosphorylated peptide as compared to the non-phosphorylated peptide, demonstrating phosphorylation on Y656 (but not Y657). f, MALDI–TOF mass spectrometry analysis of the effects of substituting A-loop tandem tyrosines (YY) with phenylalanines (FF) on the substrate phosphorylation activities of FGFR2K^WT, FGFR2K^E565A and FGFR2K^R678G, respectively, in each case shown relative to unphosphorylated FGFR2K^WT as measured at 0.5 min. Data are mean ± s.d. (n = 3). g, Immunoblot analyses of whole extracts of untreated or FGF1-treated L6 myoblasts stably expressing wild-type FGFR2c or its R-to-G variant probed with an anti-p-FGFR (Y656/Y657), an anti-FGFR2 or an anti-β-tubulin antibody. Experiments were performed in biological triplicates with similar results. Full-length gels are shown in Supplementary Fig. 15a. h, Left, kinetics of overall tyrosine transphosphorylation in FGFR2K^R678A and FGFR2K^R678E assayed by native gel electrophoresis. Middle and right, LC–MS analysis of transphosphorylation on A-loop tandem tyrosines of samples at 0.5 min (middle) and corresponding quantitation (right). Experiments were done independently twice with similar results.

Fig. 2 |. FGFR3^R669E promotes formation of an A-loop-transphosphorylating asymmetric complex.

a, Middle, overall view of the crystal structure of the FGFR3K^R669E asymmetric complex shown as a cartoon superimposed on a semitransparent surface. Enzyme- and substrate-acting kinases are in green and blue, respectively. Bound AMP-PCP molecules are shown as sticks. Left and right, surface regions mediating asymmetric complex formation are highlighted in magenta (enzyme) and yellow (substrate), respectively. b, Close-up view of contacts at the enzyme’s catalytic site. c,d, Expanded views of the dimer interface distal to the active site, with hydrogen bonds and hydrophobic interactions shown in c and d, respectively. e, Reversion of the engineered glutamic acid at position 669 to an arginine residue introduces an electrostatic clash with K659 of the enzyme kinase. f, Sequence alignment of the kinase domains of FGFR1–FGFR4. Residues of enzyme and substrate kinases that mediate the FGFR3K^R669E A-loop-transphosphorylating asymmetric complex interface are highlighted in blue and green, respectively. Tandem tyrosine phosphorylation sites in the A-loop are in red.

Fig. 3 |. The crystallographically deduced A-loop-transphosphorylation asymmetric complex forms in solution.

a, Overlays of leucine/valine regions of ¹H/¹³C methyl HMQC spectra for FGFR2K^R678E (middle) and FGFR2K^R678G (right) mutants acquired at either high (1.5 mM for R678E; 2.0 mM for FGFR2K^R678G) or low (0.1 mM) concentrations. Peaks sustaining >20% intensity loss are boxed. Left, corresponding spectrum of FGFR2K^WT at 1.2 mM is shown for comparison. HMQC experiments were performed independently twice with similar results. b, Dilution-dependent reappearance of peaks corresponding to L675 (top) and V709 (bottom) for FGFR2K^R678E. c, CPMG dispersion curves for I707 in FGFR2K^R678E (top) and FGFR2K^R678G (bottom) at the protein concentrations shown. Curves plotted in blue and black represent data collected at 800 MHz and 600 MHz, respectively. Note that the 0.1 mM FGFR2K^R678E dataset in blue was collected at 900 MHz. d, Plots of k_ex × R_ex derived from CPMG relaxation dispersion experiments for FGFR2K^R678E and FGFR2K^R678G as a function of protein concentration. Plots were globally fitted using multiple residues to estimate dimerization K_d values (boxed above). Error bars for k_ex × R_ex and K_d values reflect errors from non-linear least squares fits. e, Correlation plots of R_ex values for FGFR2K^R678E (left) and FGFR2K^K659E (right) determined at 1.3 mM and 0.4 mM (FGFR2K^R678E) and 1.2 mM and 0.4 mM (FGFR2K^K659E), respectively. A slope of 1.0 is indicated by the dashed line. For d and e, n = 1 using independent samples; two technical replicates were acquired for select CPMG frequencies. The center value is the optimal fit to the data using equation (2). For e, the solid line is a linear correlation with the best fit slope to the data reported and a y-intercept of 0.

Fig. 4 |. Functional validation of the crystallographically deduced A-loop-transphosphorylating mechanism in vitro and in vivo.

a, Expanded view of the FGFR3K^R669E asymmetric complex interface highlighting the key contribution of (i) the salt bridge between R571 (enzyme) and D668 (substrate), and (ii) the hydrogen bond between R568 (enzyme) and D668 (substrate) backbone (in each case shown as dashed lines). b,c, Introduction of a R568E/R571E double substitution in the enzyme kinase (b) or a D668R single substitution in the substrate kinase (c) are predicted to inhibit A-loop-transphosphorylating asymmetric complex formation by eliminating both salt-bridge and hydrogen-bonding interactions and by introducing electrostatic clashes. d, Equivalent residues in FGFR1–FGFR4 that mediate salt bridges and hydrogen bonds at the asymmetric complex interface and their corresponding substitution to residues with opposite charge, engineered to abolish dimerization. e, Kinetic analyses by native gel electrophoresis (top), immunoblotting (middle) and time-resolved LC–MS of A-loop-tyrosine phosphorylation (bottom) in wild-type FGFR2Ks and its variants harboring mutations predicted to disrupt the asymmetric complex. Kinase assays were done independently twice with similar results. f, Immunoblot analyses of whole lysates of buffer-treated or FGF1-stimulated L6 myoblasts overexpressing either full-length wild-type FGFR2 or corresponding variants harboring dimer-breaking substitutions. Blots were probed with anti-p-FGFR (Y656/Y657), anti-FGFR2 and anti-β-tubulin antibodies. Experiments were performed in biological triplicates with similar results. Full-length gels are shown in Supplementary Fig. 15b,c.

Fig. 5 |. In vitro and in vivo complementation assays reinforce the existence of an asymmetric A-loop-tyrosine transphosphorylation complex.

a, Cartoon representation of heterodimerization of FGFR3^D668R and FGFR3^R568E/R571E in which FGFR3^D668R assumes the role of enzyme, while FGFR3^R568E/R571E acts as substrate. Locations of mutated residues (E568 and E571, red; R668, blue) are highlighted. b, Kinetic analysis of phosphorylation of A-loop tyrosines in reactions containing equimolar amounts of FGFR2K^D677R and FGFR2K^R577E/R580E by native gel electrophoresis (top), immunoblotting with an anti-p-FGFR antibody (middle) and time-resolved LC–MS (bottom). Kinase assays were done independently twice with similar results. c, Lysates from buffer-treated or FGF1-treated L6 myoblasts stably expressing either wild-type FGFR2c, FGFR2c^R577E/R580E or FGFR2c^D677R alone, or co-expressing FGFR2c^R577E/R580E and FGFR2c^D677R, in each case analyzed by immunoblotting using antibodies specific for selected target proteins and their phosphorylated forms. An anti-β-tubulin antibody was used as a loading control. Experiments were performed in biological triplicates with similar results. Full-length gels are shown in Supplementary Fig. 15d,e.

Fig. 6 |. Asymmetric complex formation induces reciprocal allosteric changes in enzyme and substrate kinases.

a,c, Overlays of ¹H/¹³C HMQC (leucine/valine region) spectra of 0.4 mM isotopically labeled FGFR2K^WT (a; blue) or FGFR2K^K659E (c; red) either alone or together with 0.8 mM unlabeled substrate kinase (that is, FGFR2K^R577E/R678E). Peaks sustaining >20% loss of intensity are boxed. Experiments were performed independently twice with similar results. b,d, R_ex values (with range depicted by a boxed colored bar) derived from CPMG relaxation dispersion experiments for FGFR2K^WT (b) or FGFR2K^K659E (d) mixed with unlabeled FGFR2K^R577E/R678E mapped onto the enzyme-acting kinase in the asymmetric complex crystal structure. e, Changes in R_ex values of selected residues in FGFR2K^WT or FGFR2K^K659E enzyme kinase induced upon addition of substrate (that is, FGFR2K^R577E/R678E). f, Reductions in CPMG-derived R_ex values in FGFR2K^K659E enzyme kinase when A-loop tyrosines (annotated YY) of FGFR2K^R577E/R678E substrate kinase are substituted to YF, FY and FF. e,f, n = 1 using independent samples for each set of CPMG measurements acquired at two magnetic field strengths; error bars reflect the fitted error to equation (2). a,c,e,f, Isotopically enriched kinases contained in mixtures are indicated by asterisks. g–j, Induced-fit model for A-loop-tyrosine transphosphorylation. g, Asymmetric complex formation of FGFR kinases (enzyme and substrate in green and blue, respectively) is thermodynamically inhibited by a charge repulsion between K659 in the enzyme-acting kinase and R669 in the incoming substrate-acting kinase (both residues highlighted in pink). h, Energetic gains in extracellular FGF-induced FGFR dimerization offset these repulsive forces, facilitating formation of a C lobe–C lobe-mediated asymmetric kinase dimer. HS, heparan sulfate. i, Asymmetric complex formation imparts upon the substrate A-loop a more phosphorylatable conformation (indicated as a change in color to yellow). j, This encourages the A-loop of the enzyme to adopt the active state (depicted by a change in color to red), resulting in the formation of an A-loop-tyrosine transphosphorylation complex as revealed by the crystal structure. k,l, Immunoblot analyses of L6 myoblast cell lines overexpressing either full-length mouse wild-type VEGFR2 (k) or human wild-type insulin receptor (IR) (l), together with variants harboring either a R1080G substitution (k) or a G1211R substitution (l) (in each case corresponding to FGFR2 R678) plus dimer-disrupting substitutions R929E/R932E and D1079R (k) or R1116E/R1119E and D1210R substitutions (l) (in each case corresponding to FGFR2 R577E/R580E and D677R). Cells were stimulated with either VEGF (k) or insulin (l) at the concentrations shown. Whole-cell lysates were analyzed by immunoblotting using antibodies specific for p-VEGFR2, VEGFR2 (k) or antibodies specific for phosphorylated human insulin receptor (p-hIR) or human insulin receptor (hIR) (l). k,l, An antibody to β-tubulin was used as a loading control. Experiments were performed in biological triplicates with similar results. Full-length gels are shown in Supplementary Fig. 15f,g.