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A dynamic hydrophobic core orchestrates allostery in protein kinases

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Science Advances  07 Apr 2017:
Vol. 3, no. 4, e1600663
DOI: 10.1126/sciadv.1600663
  • Fig. 1 The architecture of the hydrophobic core.

    (A) Hydrophobic motifs defining active and inactive states of the kinase: C-spine (yellow), R-spine (red), and shell (teal). (B) X-ray structures of PKA-C showing the architecture of the hydrophobic core of the enzyme along the major conformational states in the catalytic cycle and the inhibited state with the pseudosubstrate. Enclosed in the box is the transition from the apo (open, uncommitted) to the ATP-bound (intermediate, committed) conformation. The synchronous motions associated with this transition are captured here with methyl-TROSY relaxation dispersion NMR spectroscopy.

  • Fig. 2 Methyl CSPs define the conformational transition between open and closed states.

    (A) Portions of the methyl-TROSY spectra showing the linear chemical shift trajectories upon ligand binding along the major states of the catalytic cycle. The residues with highly correlated chemical shift changes (R2 > 0.97) are shown with purple spheres, whereas residues with lower correlation coefficients (R2 < 0.97) are shown in white. A comprehensive map of the cross-correlation is shown in fig. S5. (B) Zoom-in of the core of the enzyme showing that the chemical shift changes with the highest correlation cluster in the proximity of the C-spine reporting on the open-to-closed conformational transition.

  • Fig. 3 Changes of the fast conformational dynamics of the hydrophobic core upon nucleotide and pseudosubstrate binding.

    (A) Order parameters of the methyl groups mapped onto the apo, binary (ATPγC-bound), and ternary (ATPγN/PKI5–24) forms of the enzyme, showing an increasing rigidification of the hydrophobic core. (B) Ordering of the C-spine, αF, and the catalytic loop upon nucleotide and PKI binding. (C). Structural details of PKA-C showing the C-spine and the αE helix, highlighting the rigidification of I150, I180, and V182 upon ligand binding and bridging β1-β2 in the N-lobe with β7-β8 in the C-lobe.

  • Fig. 4 Synchronization of motions within the hydrophobic core upon nucleotide binding.

    (A) 13C CPMG relaxation dispersion curves carried out at two different magnetic fields (700 MHz, black symbols; 850 MHz, red symbols) for selected residues in the hydrophobic core for the apo, binary (ATPγC-bound), and ternary (ATPγN/PKI5–24) complexes. (B) Mapping of the methyl groups showing conformational dynamics in the C-spine, R-spine, and bridging residues of the hydrophobic core. V104 bridges the R-spine and C-spine together with the adenine ring of ATP, whereas I150 bridges the C-spine to the αE helix.

  • Fig. 5 β structure of the kinase core is anchored to the hydrophobic core and recruited for catalysis by ATP binding.

    (A) The β sheet of the N-lobe (β1 to β5) is anchored to the adenine ring of ATP through V57 (β2) and A70 (β3). In the C-lobe, β7 and β8 are anchored to the adenine ring through L173 (β7). The catalytic loop that spans β6 and β7 is anchored to the F helix by L167; the Mg loop (red) bridges β8 and β9. (B) Another conserved structural element in the N-lobe is the αC-β4 loop that is characterized by a stable β turn. This loop also contains many key residues with highly correlated chemical shift changes (black spheres).

  • Fig. 6 Dynamic ordering of the hydrophobic core.

    (A) Most of the elements of the hydrophobic core (teal and gray) are stable in both active and inactive conformational states. This includes β1 through β8 and the catalytic loop. These elements, as well as the C-spine, are anchored to the F helix by hydrophobic interactions. The assembly of the R-spine completes the hydrophobic core and positions the Mg loop and β9 (red). (B) This active conformation, particularly the β8-β9 sheet, is further stabilized by phosphorylation at Thr197 in the activation loop.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/4/e1600663/DC1

    fig. S1. [1H-13C] methyl-TROSY spectra of apo, nucleotide-bound (ATPγC), and ternary (ATPγN/PKI5-24 and ATPγN/PLN1–19) forms of 2H,13C-isoleucine, leucine, and valine (IVL)–labeled PKA-C.

    fig. S2. Plots of the IVL methyl group CSPs upon ligand binding.

    fig. S3. Mapping of the CSPs of the IVL methyl side-chain groups onto the crystal structure of PKA-C (PDB: 1ATP).

    fig. S4. Statistical analysis of the chemical shift changes.

    fig. S5. CHESCA (39) correlation matrix showing the degree of correlated chemical shift changes for the methyl side chains of IVL residues.

    fig. S6. Global conformational transitions mapped via linear CSPs probed by amide backbone resonances and methyl group resonances.

    fig. S7. Fast time scale (picosecond to nanosecond) conformational dynamics of the kinase upon ligand binding.

    fig. S8. Thermodynamics of ligand binding for PKA-C as measured by isothermal titration calorimetry (ITC).

    fig. S9. Slow time scale (microsecond to millisecond) conformational dynamics of the kinase.

    fig. S10. Slow time scale conformational dynamics of the kinase.

    fig. S11. Synchronous dynamics occurring in the highly conserved hydrophobic core.

    fig. S12. Location of I150 at the interface between different communities of PKA-C.

    fig. S13. Bridging residues connect the R-spine and C-spine at the PKA hydrophobic core.

    fig. S14. Western blot–based activity assay.

    fig. S15. Assembly of the R-spine for active protein kinases.

    fig. S16. Expression and purification of recombinant 2H, 15N, 13CH3-ILV, and PKA-C from E. coli bacteria.

    table S1. Classification of residues undergoing correlated chemical shift changes and their respective location in a specific community as identified by community map analysis.

    table S2. The dynamic light scattering data for three different forms of PKA-C.

    table S3. T2 and S2 values for methyl side-chain groups of apo PKA-C.

    table S4. T2 and S2 values for methyl side-chain groups of the ATPγC-bound state of PKA-C.

    table S5. T2 and S2 values for methyl side-chain groups of the ATPγN/PKI5-24-bound state of PKA-C.

    table S6. Group fits of CPMG dispersion curves measured at 700 and 850 MHz of the apo form of PKA-C.

    table S7. Group fits of the CPMG relaxation dispersion curves measured at 700 and 850 MHz of the ATPγC form of PKA-C.

    table S8. Single-quantum individual site fits of CPMG relaxation dispersion curves measured at 700 and 850 MHz of the apo form of PKA-C.

    table S9. Individual fits of the CPMG dispersion curves measured at 700 and 850 MHz of the ATPγC-bound state of PKA-C.

    table S10. Single-quantum individual site fits of CPMG curves at 700 and 850 MHz of the ATPγN/PKI5-24-bound state of PKA-C.

    table S11. Approximate Rex values from two points of the CPMG experiment for the ATPγN/PLN1–19-bound form of PKA-C.

    Reference (54)

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. 1H-13C methyl-TROSY spectra of apo, nucleotide-bound (ATPγC), and ternary (ATPγN/PKI5-24 and ATPγN/PLN1–19) forms of 2H,13C-isoleucine, leucine, and valine (IVL)–labeled PKA-C.
    • fig. S2. Plots of the IVL methyl group CSPs upon ligand binding.
    • fig. S3. Mapping of the CSPs of the IVL methyl side-chain groups onto the crystal structure of PKA-C (PDB: 1ATP).
    • fig. S4. Statistical analysis of the chemical shift changes.
    • fig. S5. CHESCA (39) correlation matrix showing the degree of correlated chemical shift changes for the methyl side chains of IVL residues.
    • fig. S6. Global conformational transitions mapped via linear CSPs probed by amide backbone resonances and methyl group resonances.
    • fig. S7. Fast time scale (picosecond to nanosecond) conformational dynamics of the kinase upon ligand binding.
    • fig. S8. Thermodynamics of ligand binding for PKA-C as measured by isothermal titration calorimetry (ITC).
    • fig. S9. Slow time scale (microsecond to millisecond) conformational dynamics of the kinase.
    • fig. S10. Slow time scale conformational dynamics of the kinase.
    • fig. S11. Synchronous dynamics occurring in the highly conserved hydrophobic core.
    • fig. S12. Location of I150 at the interface between different communities of PKA-C.
    • fig. S13. Bridging residues connect the R-spine and C-spine at the PKA hydrophobic core.
    • fig. S14. Western blot–based activity assay.
    • fig. S15. Assembly of the R-spine for active protein kinases.
    • fig. S16. Expression and purification of recombinant 2H, 15N, 13CH3-ILV, and PKA-C from E. coli bacteria.
    • table S1. Classification of residues undergoing correlated chemical shift changes and their respective location in a specific community as identified by community map analysis.
    • table S2. The dynamic light scattering data for three different forms of PKA-C.
    • table S3. T2 and S2 values for methyl side-chain groups of apo PKA-C.
    • table S4. T2 and S2 values for methyl side-chain groups of the ATPγC-bound state of PKA-C.
    • table S5. T2 and S2 values for methyl side-chain groups of the ATPγN/PKI5-24-bound state of PKA-C.
    • table S6. Group fits of CPMG dispersion curves measured at 700 and 850 MHz of the apo form of PKA-C.
    • table S7. Group fits of the CPMG relaxation dispersion curves measured at 700 and 850 MHz of the ATPγC form of PKA-C.
    • table S8. Single-quantum individual site fits of CPMG relaxation dispersion curves measured at 700 and 850 MHz of the apo form of PKA-C.
    • table S9. Individual fits of the CPMG dispersion curves measured at 700 and 50 MHz of the ATPγC-bound state of PKA-C.
    • table S10. Single-quantum individual site fits of CPMG curves at 700 and 850 MHz of the ATPγN/PKI5-24-bound state of PKA-C.
    • table S11. Approximate Rex values from two points of the CPMG experiment for the ATPγN/PLN1–19-bound form of PKA-C.
    • Reference (54)

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