Research ArticleBIOPHYSICS

Cushing’s syndrome driver mutation disrupts protein kinase A allosteric network, altering both regulation and substrate specificity

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Science Advances  28 Aug 2019:
Vol. 5, no. 8, eaaw9298
DOI: 10.1126/sciadv.aaw9298
  • Fig. 1 Architecture of PKA-C and locations of the Cushing’s syndrome mutations.

    (A) Structure of PKA-C [Protein Data Bank (PDB) ID: 1ATP] in complex with pseudo-substrate PKI depicting the location of Cushing’s mutations (yellow spheres) in relation to structural elements of the kinase. The E248Q mutation includes an additional deletion (del243-247), and the S212R mutation includes an insertion (insIILR) not depicted. (B) X-ray structure of PKA-CL205R (PDB ID: 4WB6) with the overlay of PKI5–24 (PKIWT from PDB ID: 1ATP) describing the architecture of the peptide binding site and steric clash between kinase (PKA-CL205R) and pseudo-substrate. (C) Structure of the R/C complex (PDB ID: 2QCS) depicting locations of Cushing’s mutations in relation to the pseudo-substrate inhibitory sequence of the R-subunit. (D) Primary sequence comparison of common regulators (RIα, RIIβ, and PKI) and peptide substrates of the catalytic subunit (Kemptide and VPS36).

  • Fig. 2 Allosteric network of interactions observed upon pseudo-substrate binding.

    The CHESCA correlation matrix for (A) PKA-CWT upon binding PKI and (B) PKA-CL205R upon binding PKI. (C) Correlations corresponding to the binding of PKI to PKA-CWT plotted on the structure of PKA-C. Residues that are commonly correlated for both PKA-CWT and PKA-CL205R are highlighted. (D) Correlations corresponding to the binding of PKI to PKA-CL205R plotted on the structure of PKA-C. Specific residues that are correlated for only PKA-CL205R are highlighted. Only correlations with rij > 0.98 are shown throughout.

  • Fig. 3 Rewiring of the allosteric network of PKA-CL205R upon binding VPS36.

    (A) Steady-state phosphorylation kinetics of Kemptide and VPS36 peptides for PKA-CWT and PKA-CL205R. Corresponding values can be found in table S2. (B) CONCISE analysis on the apo, ATPγN, ATPγN/PKI, and ATPγN/VPS36 states of PKA-CWT and PKA-CL205R. (C) The CHESCA correlation matrix for PKA-CWT upon binding VPS36. (D) The CHESCA correlation matrix for PKA-CL205R upon binding VPS36.

  • Fig. 4 Conformational dynamics of the activation loop of PKA-CWT and PKA-CL205R upon binding substrate.

    (A) Distinct opening-closing motions of the Gly-rich loop highlighting the hindered conformation that occludes the entering of ATP in PKA-CL205R. (B) Probability density describing the conformation of the activation loop in response to different substrates and pseudo-substrates. (C) [1H, 15N]-TROSY-HSQC spectra showing the backbone amide chemical shift changes of the W196 indole amide (located on the activation loop) in response to binding PKI or VPS36. (D) X-ray structure of PKA-CWT (gray) (PDB ID: 1ATP) with the overlay PKA-CL205R in complex with PKI (light blue) (PDB ID: 4WB6) describing the architecture of the peptide binding site and activation loop flip. (E) X-ray structure of PKA-CWT (gray) (PDB ID: 1ATP) with the overlay PKA-CL205R in complex with VPS36 (light blue) (MD simulations) describing the architecture of the peptide binding site and activation loop flip.

  • Fig. 5 Cushing’s mutations are located in allosteric nodes identified via CHESCA.

    (A) Correlation matrix of PKA-CWT when bound to PKI emphasizing locations of Cushing’s mutation in relation to allosteric nodes. (B) Missing correlations for PKA-CL205R upon binding PKI colored according to the community map of PKA-CWT. (C) CHESCA correlation matrices for PKA-CWT + PKI, PKA-CL205R + PKI, and PKA-CL205R + VPS36, respectively, plotted on the structure of PKA-C with colors specific to the community map analyses completed previously for PKA-CWT (35). Specific communities are emphasized to highlight elements that experience the most dramatic changes in the number and extent of chemical shift covariance for PKA-CL205R upon binding PKI and VPS36.

  • Fig. 6 Schematic of the energy landscape for PKA-CWT and PKA-CL205R, combining thermodynamics and MD simulations data.

    Relative free energy for the binding of ATPγN, PKI, and VPS36 to PKA-CWT and PKA-CL205R derived from ITC data. (1) Apo PKA-CL205R samples mostly uncommitted states, with the Gly-rich loop partially occluded and the αC helix turned outward. (2) Binary PKA-CL205R features a wired allosteric network for substrate binding, i.e., committed state. (3) Ternary PKA-CL205R complex with lower affinity for PKI (i.e., higher free energy relative to PKA-CWT). The conformation of the activation loop of PKA-CL205R is in equilibrium between an unflipped and a sparsely populated flipped conformation. (4) PKA-CL205R/ATPγN/VPS36 ternary complex features a flipped conformation of the activation loop with the electrostatic interactions between E86 and R194.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/8/eaaw9298/DC1

    Fig. S1. [1H, 15N]-TROSY-HSQC spectra for PKA-CWT and PKA-CL205R in apo, ATPγN, and ATPγN/PKI-bound and ATPγN/VPS36-bound forms.

    Fig. S2. CSPs observed upon ligand binding for PKA-CWT and PKA-CL205R.

    Fig. S3. Intensity plot for the binding of VPS36 to ATPγN-saturated PKA-CL205R.

    Fig. S4. PCA of the catalytic lobes in PKA-CWT and PKA-CL205R.

    Fig. S5. Probability of the formation of inter-residue contact and ΔRMSF of PKA-C upon forming ternary complexes with PKI5–24 or VPS36.

    Fig. S6. Allosteric changes upon peptide binding revealed by MD simulation and mutual information (MutInf) analysis.

    Table S1. Changes in enthalpy, entropy, free energy, and dissociation constant of binding ATPγN, PKI5–24, and VPS36 for PKA-CWT and PKA-CL205R.

    Table S2. Kinetic parameters of Kemptide and VPS36 phosphorylation by PKA-CWT and PKA-CL205R.

    Table S3. PCA and SD of the CONCISE analysis of the structural states analyzed.

    Table S4. Tm as determined using CD.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. 1H, 15N-TROSY-HSQC spectra for PKA-CWT and PKA-CL205R in apo, ATPγN, and ATPγN/PKI-bound and ATPγN/VPS36-bound forms.
    • Fig. S2. CSPs observed upon ligand binding for PKA-CWT and PKA-CL205R.
    • Fig. S3. Intensity plot for the binding of VPS36 to ATPγN-saturated PKA-CL205R.
    • Fig. S4. PCA of the catalytic lobes in PKA-CWT and PKA-CL205R.
    • Fig. S5. Probability of the formation of inter-residue contact and ΔRMSF of PKA-C upon forming ternary complexes with PKI5–24 or VPS36.
    • Fig. S6. Allosteric changes upon peptide binding revealed by MD simulation and mutual information (MutInf) analysis.
    • Table S1. Changes in enthalpy, entropy, free energy, and dissociation constant of binding ATPγN, PKI5–24, and VPS36 for PKA-CWT and PKA-CL205R.
    • Table S2. Kinetic parameters of Kemptide and VPS36 phosphorylation by PKA-CWT and PKA-CL205R.
    • Table S3. PCA and SD of the CONCISE analysis of the structural states analyzed.
    • Table S4. Tm as determined using CD.

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