Structural and catalytic motifs of PKA-C.

(A) Surface representation of the X-ray structure of PKA-C bound to the endogenous inhibitor, PKI (PDB: 4WB5). (B) Hydrophobic organization of the PKA-C core, with the R-spine (gold), C-spine (blue), shell residues (cyan), and the αC-β4 loop (hot pink) that locks into αE helix.

Free energy landscape (FEL) of PKA-C in various ligated forms obtained from replica-averaged metadynamics (RAM) simulations.

(A) Convergence of the bias deposition along the first three collective variables (CVs). The free energy (expressed in kcal/mol) of the different CVs were averaged over the last 100 ns of RAM simulations. The standard deviations are reported as red error bars. (B-D) FEL along the first two principal components (PC1 and PC2) of PKA-C in the apo, ATP-bound, and ATP and the model substrate PKI bound forms. PC1 and PC2 are projected from the first three CVs. The vertices represent conformational states. In the apo form, multiple states have comparable free energy with ΔG < 5 kcal/mol, whereas in the binary form, fewer states have ΔG < 5 kcal/mol, whereas for the ternary form only a major ground state is populated.

The apo, ATP-bound, and ATP/PKI-bound PKA-C reveal distinct free energy surface (FES) and dynamics, as determined by a Markov State Model (MSM).

(A) Free energy landscape projected along the first two time-lagged independent components (tICs) of the apo PKA-C, the projections of known crystal structures, and characteristic features of GS, ES1, and ES2. The transition from GS to ES1 highlights the changes around the αB-αC loop, where the salt bridges between K72-E91 and H87-T197 and the PIF pocket (V80-I85-F347) are all disrupted. The transition from GS to ES2 highlights the rearrangement around the αC-β4 loop, with distinct local hydrophobic packing. (B and C) FES projected along the first two tICs for the ATP-bound PKA-C (B), ATP/PKI bound PKA-C (C), and the projections of known crystal structures.

Conformational transition between GS and ES1, ES2 along the kinetic Monte Carlo trajectory in apo (A) and ATP-bound (B) forms. (A, B)

The transition from GS to ES1, revealed as breaking of the K72-E91 salt bridge, is frequently found in both forms, whereas the transition to ES2, revealed as the contact between F100 and V104, only occurs in the apo form and in concert with allosteric changes between D166-N171, K168-T201, and W222-A206-P207. The darker colors in (A) and (B) highlight moving averages over every 10 frames. (C) GS conformation reveals the assembly of key catalytic features across the core region. (D) ES2 conformation revealed disruption of key structure motifs across the core region, indicative of inactivation.

Transition from GS to ES2 shown in the apo PKA-C recapitulated the structural changes near the αC- β4 loop probed by NMR experiments.

(A and B) Distribution of predicted 13C CS of selected methyl groups in ES (magenta) and GS (blue) of the apo PKA-C for Val104- Cγ1 (A) and Ile150-Cδ1 (B). The experimental CS is shown in dotted lines for GS (black) and ES (red). (C). Correlation of the predicted chemical shift differences | ΔωPred| and the experimental result | ΔωExp| for a set of hydrophobic residues near the αC-β4 loop. The fitted linear correlation has a slope of 0.86 and R2 of 0.82.

Increased dynamics at the αC-β4 loop upon F100A mutation, perturbing the local hydrophobic packing and its anchoring to the αE helix.

(A) Time series of the αC-β4 loop, H-bond occurrence for the β− and γ-turns, F102 χ1 angle, and N99 and Y156 for WT (black) and F100A (red) in the ATP-bound state. (B) Representative structural snapshots showing the formation of the β-turn for the PKA-CWT (green) and γ-turn for the PKA-CF100A mutant.

Distinct global structural response to ATP binding in the F100A mutant.

(A) Structure superposition of the C Spine, R Spine, and Shell residues between WT (lime) and F100A (hot pink), highlighting the differences between Shell and R Spine. (B) Change of RMSD upon ATP binding at C Spine, Shell, and R spine, for WT and F100A, respectively. (C) Structural illustration of the first principal component (PC1), i.e., the breathing motion of the two lobes. (D) Structural illustration of the second principal component (PC2), i.e., the shearing motion of the two lobes. (E) Comparison of the 2D projection and distribution along PC1 and PC2 for WT and F100A, highlighting their dramatic differences along both axes.

Mutual information of dihedral angles for the (A) WT and (B) F100A upon binding ATP.

This analysis reveals the prominent loss of allosteric communication of F100A, especially at multiple key motifs as is highlighted by purple strips.

Structural response of PKA-CF100A binding to nucleotide and protein kinase inhibitor.

(A) Histogram shows the chemical shift perturbation (CSP) of the amide fingerprint for PKA- CF100A (black) in response to ATPγN binding compared to the CSP obtained for the wild-type protein (cyan). The dashed line on the histogram indicates one standard deviation from the average CSP. (B) CSPs of PKA-CF100A/ATPγN amide resonances mapped onto the structure (PDB: 4WB5). (C) CSP of amide fingerprint for PKA-CF100A bound to ATPγN and PKI5-24 (black), compared to the CSP of the wild-type protein obtained in the same conditions. (D) CSP for the F100A/ATPγN/PKI complex mapped onto the crystal structure (PDB: 4WB5).

Changes of the intramolecular allosteric network in F100A as mapped by correlated chemical shift changes.

(A) Comparison of the CHESCA matrices obtained from the analysis of the amide chemical shifts of PKA-CWT (top diagonal, blue) and PKA-CF100A (bottom diagonal, black) in the apo, ADP-bound, ATPγN-bound, and ATPγN/PKI5-24-bound states. Only correlations with Rij > 0.98 are reported. The enlarged CHESCA map of F100A is available in Figure 10 – figure supplement 1 while the data for the PKA-CWT matrix are taken from Walker et al.15. (B) Community CHESCA analysis of PKA-CWT (top diagonal, blue) and PKA-CF100A (bottom diagonal, black). Only correlations with RA,B > 0.98 are shown. (C) Community CHESCA matrix plotted on its corresponding structures. The size of each node is independent of the number of residues it encompasses while the weight of each line indicates the strength of coupling between the individual communities.

Illustration of the collective variables (CVs) used in the RAM simulations.

(A) The ψ angles of the backbone of all the loops not in contact with ATP (Back-far), where the Cα atoms of the residues involved are highlighted in the blue sphere. (B) The ψ angles of the backbone of all the loops in contact with ATP (Back-close), where the Cα atoms of the residues involved are highlighted in magenta sphere. (C) The χ1 angles of side chains of all the loops that are in contact with ATP (Side-close), where the side chains of the residues involved are highlighted in magenta stick. (D) The radius of gyration is calculated over the rigid part of the protein (rgss), where the residues involved are colored in cyan.

Distribution of the Root-Mean-Square-Error (RMSE) of the chemical shifts in different simulation schemes.

(A) RMSE of CS for the apo PKA-C from standard MD (left), REX (middle), and RAM (right). (B) RMSE of CS for PKA-C/ATP from standard MD (left), REX (middle), and RAM (right). (C) RMSE of CS for PKA-C/ATP/PKI5-24 from standard MD (left), REX (middle), and RAM (right). Color codes for different backbone atoms (C, Cα, CO, H and N) are shown in the left figures.

Replica-averaged metadynamics (RAM) simulations explore a larger conformational space than standard MD and replica exchange (REX) simulations.

(A) Comparison of conformational space sampled by RAM Replica 1, standard MD, and REX Replica 1 of the apo PKA-C, along the CV1 and CV2. (B) Comparison of conformational space sampled by RAM Replica 1, standard MD, and REX Replica 1 of the apo PKA-C, along the CV3 and CV2.

Accumulative deposition of history-dependent biases along the first three CVs for the RAM simulation of the apo PKA-C.

The accumulative biased converged after around 300 ns along all three CVs.

Residues of the regulatory spine and shell are chosen as the metrics for two time-lagged independent components (tICA) and Markov State Model (MSM) analysis.

(A) Atom motions of key residues that define tIC1 of the apo PKA-C, colored by the superposition deviations. Backbone atoms of Val104 show the largest change in tIC1. (B) Atom motions of key residues that define tIC2 of the apo PKA-C, colored by the superposition deviations. Backbone atoms of Phe185 and Val104 show largest change in tIC2.

ES and GS in the apo PKA-C show distinct hydrophobic packing for residues around the αC-β4 loop.

(A) Projections of randomly selected conformations for ES (magenta) and GS (blue) onto the conformational landscape of the apo PKA-C. To best separate ES from GS, snapshots with tIC1 < 1.2 were clustered as ES, whereas those with tIC1 > 0.2 were clustered as GS. (B,C) Representative structure of ES (B) reveals different hydrophobic packing from that of GS (C), highlighted by the distinction at Leu103, Val104, Ile150, Leu172, and Ile180, where all show slow chemical exchanges in CPMG experiment of the apo PKA-C.

Distribution of predicted 13C CS of selected methyl groups.

(A-C) ES (magenta) and GS (blue) of the apo PKA-C for Leu103-Cδ2 (A), Leu172-Cδ1 (B) and Ile180-Cδ1 (C). The experimental CS are shown in dotted line for GS (black) and ES (red).

figure supplement 1. NMR fingerprints of PKA-CF100A.

(A) [1H,15N]-WADE-TROSY spectrum of apo PKA-CF100A and bound to bound to ADP, ATPγN, and e ATPγN/PKI5-24. (B) Change in chemical shift perturbation (CSP) between PKA-CWT and PKA-CF100A upon binding ATPγN. (C) Change in CSP (ΔδWT - ΔδF100A) upon binding ATPγN and PKI5-24.

title supplement 1: CONCISE plot showing the probability distribution of the amide resonances as a function of ligand binding.

The per-residue information is averaged into the average principal component (PC) score indicative of the position of each conformational state of the kinase along the equilibrium.

figure supplement 2. Changes of the intermolecular allosteric network in F100A as mapped by correlated chemical shift changes.

(A) CHESCA matrix obtained from the amide chemical shifts of PKA-CF100A in the apo, ADP-bound, ATPγN-bound, and ATPγN/PKI5-24-bound states. Only correlations with Rij > 0.98 are reported. (B) Plot of the correlation score vs. residue calculated for PKA-CWT (blue) and PKA-CF100A (black). (C) Community CHESCA analysis of and PKA-CF100A (bottom diagonal, black). Only correlations with RA,B > 0.98 are shown. (D) Community CHESCA matrices of PKA-CF100A and PKA-CWT plotted on their corresponding structures. The size of each node is independent of the number of residues it encompasses, meanwhile the weight of each line indicates the strength of coupling between the individual communities.

ΔG (kcal/mol) and relative population of ground state and the first 6 excited states in different forms of PKA-C by the RAM simulations.

Kinetic parameters of Kemptide phosphorylation by PKA-CWT and PKA-CF100A.

The KM and Vmax values were obtained from a nonlinear least squares analysis of the concentration-dependent initial phosphorylation rates using a standard coupled enzyme activity. Error in kcat/KM was propagated from the error in KM and kcat.

Changes in enthalpy, entropy, free energy, and dissociation constant for the binding of nucleotide to PKA-CWT and PKA-CF100A.

All errors were calculated using triplicate measurements. Values for PKA-CWT are re-printed for clarity but were originally published in Walker et al. 15

Changes in enthalpy, entropy, free energy, and dissociation constant for the binding of PKI5-24 to apo and nucleotide-saturated PKA-CWT and PKA-CF100A.

All errors were calculated using triplicate measurements. The error in σ was propagated from the error in Kd. Values for PKA-CWT are re-printed for clarity but were originally published in Walker et al. 15.