Autophosphorylation activates c-Src kinase through global structural rearrangements

Abstract

The prototypical kinase c-Src plays an important role in numerous signal transduction pathways, where its activity is tightly regulated by two phosphorylation events. Phosphorylation at a specific tyrosine by C-terminal Src kinase inactivates c-Src, whereas autophosphorylation is essential for the c-Src activation process. However, the structural consequences of the autophosphorylation process still remain elusive. Here we investigate how the structural landscape of c-Src is shaped by nucleotide binding and phosphorylation of Tyr⁴¹⁶ using biochemical experiments, hydrogen/deuterium exchange MS, and atomistic molecular simulations. We show that the initial steps of kinase activation involve large rearrangements in domain orientation. The kinase domain is highly dynamic and has strong cross-talk with the regulatory domains, which are displaced by autophosphorylation. Although the regulatory domains become more flexible and detach from the kinase domain because of autophosphorylation, the kinase domain gains rigidity, leading to stabilization of the ATP binding site and a 4-fold increase in enzymatic activity. Our combined results provide a molecular framework of the central steps in c-Src kinase regulation process with possible implications for understanding general kinase activation mechanisms.

Keywords: ATP; Src; activation; biophysics; conformational change; enzyme mechanism; hydrogen/deuterium exchange; molecular dynamics; oncogene; phosphorylation; protein kinase.

Figure 1.

Tyr⁴¹⁶ phosphorylation increases c-Src kinase activity. A, structure of c-Src kinase (PDB code 1Y57). Coloring from the N to the C terminus: SH3 domain in yellow, SH2 domain in green, linker in gray, and kinase domain in blue. Important elements involved in kinase activation are depicted in red: Tyr⁴¹⁶ (spheres), Tyr⁵²⁷, A-loop, KER residues, and HRD motif. B, time-dependent substrate phosphorylation by c-Src full-length in its phosphorylated state (FLP) and the non-autophosphorylatable mutant FLU-Y416F. Note that this is the only experiment in which the Y416F mutant of c-Src was used. Inset, K_m (ATP) for the two different constructs.

Figure 2.

Bioanalytical characterization of different c-Src constructs. Shown are c-Src full-length in its unphosphorylated (orange) and phosphorylated state (red) and the kinase domain of c-Src in its unphosphorylated (dark cyan) and phosphorylated state (navy blue). A, aggregation of c-Src constructs at 42 °C was analyzed by absorbance at 350 nm. B, CD spectra of the different constructs. C, stability and unfolding cooperativity measured by CD spectroscopy. Thermal transitions were recorded at 207 nm. Inset, the temperature ranges spanning 10% to 90% of the protein unfolding signal. D, analysis of ANS binding to hydrophobic protein patches. Fluorescence spectra after excitation at 380 nm were recorded and buffer-corrected. E, tryptophan fluorescence emission quenching using acrylamide. Fluorescence signals at 328 nm were recorded after excitation at 295 nm. All measurements were performed at least in triplicate. a.u., arbitrary units.

Figure 3.

A, overview of an H/DX experiment. The protein is incubated in an excess of D₂O buffer, and backbone amide protons are exchanged in a time-dependent manner. After quenching the reaction, the protein is proteolytically digested. Peptides are chromatographically separated and introduced into a QTOF mass spectrometer, where these are additionally separated by ion mobility prior to m/z detection. By measuring time courses (in this study: 0 s (reference point), 0.33 s (red), 1 min (orange), 10 min (cyan), 60 min (blue), and 120 min (black)), the mass increase of each peptide is tracked and compared with those derived of a different protein variant in a head-to-head analysis. B–F, H/DX-MS results for comparison of exchange kinetics between different states of c-Src full-length and the c-Src kinase domain; every time point was measured in duplicate. Left panels, the color-coded structure. Red indicates more uptake and blue less uptake of the second-named compared with the first-named state. Regions showing no difference are marked in gray, and undetermined regions are shown in white. Right panels, the sum of differences plots for the different comparisons. Colored lines describe mass differences for each labeling time point, and gray bars represent the sum of differences over all investigated labeling time points for every single peptide (N to C terminus from left to right). The limit of significance (±1 Da), referring to the sum of differences, is indicated as vertical dashed lines (48). Elements important for kinase activation are highlighted. Shown are the active-site KER residues (Lys²⁹⁵, Glu³¹⁰, and Arg⁴⁰⁹), the activation loop (Asp⁴¹³-Arg⁴¹⁹), and the HRD motif (His³⁸⁴-Arg³⁸⁵-Asp³⁸⁶).

Figure 4.

Molecular dynamics simulations of c-Src in different ligand states. A, solvent-accessible surface area of the KER residues and the HRD motif for all constructs. Strong changes are indicated by arrows. B, interaction probability between phosphorylated and unphosphorylated states. Only changes larger than 10% are shown for clarity. C and D, tilting of the αC-helix in both KD and FL constructs. The reorientation of Arg⁴⁰⁹ facilitates interaction with Glu³⁰⁵. E–H, difference in interaction probability between phosphorylated and unphosphorylated states. For the FL constructs, only the KD domain is shown. G, contacts between the RDs and the KD are present in FLU for the complete 1-μs trajectory, whereas in the FLP state, rapid dissociation is observed. H, snapshot of the arginine cluster formed upon phosphorylation.

Figure 5.

A and B, conformational sampling of the central Glu³¹⁰-Lys²⁹⁵ ion pair distance (d_E310-K295) and the extent of the A-loop (<d_A-loop>). Both FL and KD constructs with and without ATP are shown.

Figure 6.

Schematic model of c-Src activation upon Tyr⁴¹⁶ phosphorylation. The different states during the final c-Src activation process are shown. In the unphosphorylated apo-state (FLU), Lys²⁹⁵ and Glu³¹⁰ interact strongly, whereas Arg⁴⁰⁹ in the activation loop binds and stabilizes Tyr⁴¹⁶ and the SH3 domain. Upon ATP binding in the FLU/ATP-state, Lys²⁹⁵ is dragged out of its interaction with Glu³¹⁰, which concomitantly becomes coordinated by Arg⁴⁰⁹. This leaves Tyr⁴¹⁶ free for phosphorylation by another c-Src molecule. In the pTyr⁴¹⁶ state (FLP), pTyr⁴¹⁶ is complexed by an arginine cluster consisting of Arg⁴⁰⁹, Arg³⁸⁵, and Arg⁴¹⁹, strongly stabilizing the A-loop in an extended conformation. Arg⁴¹⁹ fixes Glu³⁰⁵ in the αC-helix, which contributes to global stabilization of the kinase domain. Within this state, the Lys²⁹⁵-Glu³¹⁰ bond is prone to flickering, which, in combination with a stabilized ATP-binding site, contributes to higher ATP affinity. The interaction between Arg⁴⁰⁹ and pTyr⁴¹⁶ prevents binding to the SH3 domain, leading to increased exposure and partial destabilization of the regulatory domains.