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PKA-C^L205R shows a marked loss of binding cooperativity between ATP and PKI

To determine the thermodynamics of nucleotide binding (ATPγN) to both PKA-C^WT and PKA-C^L205R, we used isothermal titration calorimetry (ITC). We found that PKA-C^L205R exhibits a threefold lower binding affinity compared to PKA-C^WT [K_d (dissociation constant) of 215 ± 15 μM versus 83 ± 8 μM, respectively]. Fitting of the titration curves shows that the binding of ATPγN to PKA-C^L205R is enthalpically favorable and entropically unfavorable, whereas both contributions are favorable for ATPγN binding to PKA-C^WT. To evaluate the binding cooperativity between nucleotide and substrate (17, 18), we analyzed the binding thermodynamics of the pseudo-substrate peptide inhibitor, PKI_5–24, to both PKA-C^WT and PKA-C^L205R in the presence and absence of nucleotide. ΔH, −TΔS, ΔG, and K_d, as well as the cooperativity coefficients (σ) derived from ITC titrations, are summarized in table S1. The binding thermodynamics of PKI_5–24 to the apo forms of the two kinases is comparable, with similar enthalpy changes (favorable). When saturated with ATPγN, PKA-C^WT binds PKI_5–24 with higher affinity (K_d = 0.16 ± 0.02 μM) (18). In contrast, PKA-C^L205R displays a 62-fold reduction in binding affinity (K_d = 10 ± 3 μM), with a net decrease in both ΔH and ΔS. The binding of PKI_5–24 to the PKA-C^WT/ATPγN complex is highly cooperative (σ = 106 ± 18), whereas its binding cooperativity to the PKA-C^L205R/ATPγN complex decreases 18-fold (σ = 6 ± 2). Therefore, this single mutation affects both the affinity for ligands and the binding cooperativity between nucleotide and pseudo-substrate.

L205R mutation causes a reduction in phosphorylation kinetics

To evaluate the catalytic efficiency of PKA-C^L205R, we carried out steady-state coupled assays using the standard substrate, Kemptide. As expected from the binding thermodynamics and previous kinetic assays, the Leu-to-Arg substitution at the P+1 loop increases the K_M (Michaelis constant) 11-fold (table S2). The k_cat for the mutant increases from 19 to 41 s⁻¹, which results in an overall decrease in catalytic efficiency (k_cat/K_M) by over fivefold compared to PKA-C^WT. These results are in quantitative agreement with experiments carried out by two other independent groups (16, 19).

PKI binding to PKA-C^L205R falls short in shifting the enzyme to a fully closed state

To analyze the structural changes of PKA-C upon binding PKI and the effects of the L205R mutation, we mapped the amide backbone fingerprint of the enzyme using [¹H, ¹⁵N]–TROSY (transverse relaxation-optimized spectroscopy)–HSQC (heteronuclear single-quantum coherence) experiments (20). The amide signatures of the kinases in different ligated forms are displayed in fig. S1. The binding of ATPγN to both PKA-C^WT and PKA-C^L205R gives rise to similar chemical shift perturbations (CSPs), with the exception of the residues in the Gly-rich, catalytic, and Mg²⁺positioning loops that show larger chemical shift changes for PKA-C^WT (fig. S2). To evaluate the global response to ligand binding for the kinase and its mutant, we used CONCISE (coordinated chemical shifts behavior), which performs a statistical analysis on linear chemical shift trajectories of each amide resonance to identify the position of each state along the conformational equilibrium (21). Following ligand binding, the amide resonances of PKA-C display linear chemical shift trajectories, with the amide resonances corresponding to the apo enzyme populating one extreme and those of the closed state (ternary complex with PKI) populating the opposite extreme of the linear correlations. This behavior exemplifies a conformational equilibrium along the three major conformational states of the kinase (open, intermediate, and closed), whose relative populations are modulated by ligand binding. A total of 55 residues exhibit linear chemical shift trajectories for both PKA-C^WT and PKA-C^L205R, indicating a fast exchange limit in the NMR chemical shift time scale. These residues are distributed throughout the kinase. We found that the binding of ATPγN and PKI shifts the conformational equilibrium of PKA-C^WT toward a fully closed state. In contrast, these ligands drive the mutant to a partially closed state, which is located between the intermediate and closed conformation (table S3). This is supported by thermostability data obtained by circular dichroism (CD) measurements (table S4). CD melting curves show that the PKA-C^WT/ATPγN/PKI complex has a higher melting temperature (T_m) than the corresponding complex with PKA-C^L205R, further suggesting that this complex may adopt a more open and unstable conformation. Since the extent of the closed state is correlated with binding affinity and cooperativity (18), these results are consistent with the loss in allosteric cooperativity revealed by thermocalorimetric data.

L205R disrupts the allosteric network of the kinase upon PKI binding

To determine a possible correlation between the binding cooperativity and the intramolecular allosteric network, we analyzed the correlated CSPs for both PKA-C^WT and PKA-C^L205R using chemical shift covariance analysis (CHESCA) (22). This method identifies the allosteric networks of residues that are involved in concerted responses to ligand binding (23). The [¹H, ¹⁵N]-TROSY-HSQC spectra of the three forms of wild-type and PKA-C mutant (apo, ATPγN bound, and ATPγN/PKI bound) were used for the analysis. The CHESCA matrices for PKA-C^WT and PKA-C^L205R reported in Fig. 2 (A and B) reveal remarkable differences in their allosteric networks. There is an overall reduction in the number of correlations throughout PKA-C^L205R (Fig. 2, C and D), with specific domains of the enzyme more affected by this single mutation. In particular, residues belonging to the Gly-rich, activation loop and peptide-positioning loop experience the most substantial reductions in both number and extent of chemical shift covariation. In addition, several interresidue correlations within the activation loop (W196), αF helix (K217, V219, D220, G225, V226, and A233), and αG helix (Q245) are also ablated. The loss of interresidue correlations for the αF helix is especially important as this motif spans the hydrophobic core of the C-lobe and is conserved throughout the AGC kinase family. The function of the αF helix is to anchor all the hydrophobic motifs within the kinase core and to orchestrate catalysis (24, 25). As highlighted before, all Cushing’s driver mutations, with the exception of E31V, are located near L205R and are likely to affect the enzyme in a similar manner, disrupting these important allosteric nodes.

Binding of VPS36 substrate to PKA-C^L205R rewires the intramolecular allosteric network and reestablishes binding cooperativity

In addition to affecting the regulation of PKA-C by the R-subunits, L205R changes the kinase’s downstream substrate specificity (14, 15). Specifically, it was found that in Escherichia coli, PKA-C^L205R favors noncanonical substrates with negatively charged residues (Glu or Asp) at positions P+1, P+2, and P+3. From our own phosphoproteomic studies on endogenous substrates in human cells (14), we identified several substrates that are preferentially phosphorylated by the mutated kinase. Among those substrates, we selected the top target hyperphosphorylated by PKA-C^L205R: the vacuolar protein-sorting–associated protein 36 (VPS36), a protein that plays a role in endosomal sorting of ubiquinated cargo proteins by the endosomal sorting complex. The recognition sequence of VPS36 (RRLSEEM) for PKA-C has two Glu residues in the P+1 and P+2 positions. Using steady-state coupled enzyme assays with Kemptide and a VPS36-derived peptide (residues 115 to 130 encompassing the PKA-C recognition sequence and denoted VPS36 from hereon), we found that PKA-C^L205R reduces the kinase’s catalytic efficiency for Kemptide, which contains a hydrophobic residue at the P+1 position. In contrast, the mutation causes an increase in k_cat (from 5 to ~18 s⁻¹) for VPS36, leading to a threefold increase in catalytic efficiency (Fig. 3Aand table S2). In addition, the ITC analysis carried out with VPS36 indicates that PKA-C^L205R/ATPγN has an affinity typical of other substrates (K_d = 3.5 ± 0.1 μM), while the binding to wild type cannot be detected reliably, suggesting a notably lower binding affinity (table S1). A possible explanation is that the acidic residues in positions P+1 and P+2 of VPS36 hamper the intermolecular interactions between the peptide and residues lining the binding pocket of PKA-C^WT, while they might favor the interactions with PKA-C^L205R. These results support the proposed altered substrate specificity for PKA-C^L205R detected in intact cells.

To further investigate the underlying mechanism for the change in substrate specificity of PKA-C^L205R, we analyzed the trajectories of the amide resonances for the wild-type and mutated kinase upon binding VPS36. Although VPS36 binding causes linear chemical shift changes similar to PKI binding, the number of residues involved in the binding response is substantially higher for PKA-C^L205R. In addition, VPS36 binding causes an overall attenuation of the peak intensities throughout the entire fingerprint of the enzyme, with some resonances broadened beyond detection (fig. S3). For instance, resonances associated with the αF helix and Gly-rich loop are noticeably broadened, suggesting an increase in protein dynamics in the microsecond to millisecond time scale. The CONCISE analysis shows that the conformation of the PKA-C^WT/ATPγN/VPS36 complex only reaches the intermediate state. In contrast, the PKA-C^L205R/ATPγN/VPS36 complex reaches a state lying between the intermediate and closed states (Fig. 3B). This position in between the intermediate and closed states was also observed in the crystal structure of PKA-C^WT bound to a substrate peptide (26), suggesting that the ternary complex of PKA-C^L205R with VPS36 adopts a catalytically committed state.

The CHESCA maps for VPS36 binding to PKA-C^WT and PKA-C^L205R are radically different (Fig. 3, C and D). While the PKA-C^WT/ATPγN/PKI complex shows an intense cross-talk between the small and large lobes, the corresponding map for PKA-C^WT/ATPγN/VPS36 reveals a complete loss of allosteric interactions. Notably, correlations between the nucleotide binding site and the substrate binding hub are completely missing. In particular, the interresidue correlations for the activation loop, peptide-positioning loop, and the αF and αG helices are either reduced in their number or completely missing. In contrast, a number of correlations between the N- and C-lobes for PKA-C^L205R, which are absent for the complex with PKI, are regained upon VPS36 binding. Note that 10 of the resonances corresponding to residues located in and around the Gly-rich loop are broadened beyond detection and could not be included in the CHESCA analysis; therefore, the extent of coordinated changes in chemical shifts is largely underestimated. Nonetheless, the higher density of correlations found in the PKA-C^L205R/ATPγN/VPS36 complex underscores an extensive rewiring of the internal allosteric network, supporting the reestablished binding cooperativity.

PKA-C^WT and PKA-C^L205R explore different conformational states

To determine the effects of the L205R mutation on the conformational energy landscape of PKA-C, we carried out parallel MD simulations of the ternary complexes in explicit water environments starting from their respective x-ray coordinates [Protein Data Bank (PDB) IDs: 4WB6 and 1ATP] (16). The dominant motion of the kinase core was imaged using principal components analysis (PCA) of the C^α atoms encompassing residues 50 to 300. PC1 depicts the global opening-closing motion of the small and large lobes (fig. S4A), while PC2 describes the shearing motion of the two lobes that can be envisioned as an “asymmetric bite.” To describe the opening-closing motion of the active site, we monitored the time dependence of the distance between the C^α atoms of S53 in the Gly-rich loop and G186 (fig. S4, B and C). The binary and ternary complexes of PKA-C^WT and PKA-C^L205R sample similar conformational energy basins; however, the apo form of PKA-C^L205Rexplores a considerably broader basin than the wild type. The Gly-rich loop of PKA-C^WT toggles between the open and closed conformations, featuring a transient hydrogen bond between S53 and the γ-phosphate of ATP, whereas the Gly-rich loop of PKA-C^L205R exhibits larger motions with the S53-G186 distance ranging from 5 to 18 Å and, more frequently, samples a conformation that hinders nucleotide binding, which might explain its weaker binding affinity (Fig. 4A). R205 causes steric hindrance with the C-terminal tail of PKI, disrupting the hydrophobic pocket required to anchor the Ile residue in the P+1 position, which likely contributes to the loss of PKI binding affinity.

To visualize the alterations of the protein-protein interaction interface that may influence binding cooperativity, we analyzed the probability distribution of the inter-residue contacts between the substrate and the kinase binding pocket. Overall, PKA-C^L205R shows a contact map with PKI similar to the wild-type enzyme (fig. S5, A and B). The high-affinity binding region (HAR) and the consensus binding sequences remain bound throughout the entire MD trajectories with a probability of contact greater than 0.8. However, the L205R mutation reduces the probability of interactions between the Ile (at position P+1 in PKI) and the P+1 loop from 0.5 to 0.2. In contrast, VPS36 shows stable interactions with the P+1 loop with a probability greater than 0.8 for Glu at the P+1 position and R205 (fig. S5C). Moreover, the N terminus of VPS36 forms transient interactions with the C-terminal tail located in the small lobe of PKA-C^L205R, which are absent in the corresponding complex with PKI. As a result, the peptide is more dynamic, a feature that might explain the dramatic exchange-broadening in the NMR spectrum of the PKA-C^L205R/ATPγN/VPS36 complex.

As observed previously (27), the backbone of the tertiary complex PKA-C^WT/ATP/PKI is mostly rigid (fig. S5, D and F). The activation loop adopts a stable conformation as in the x-ray structure (PDB ID: 1ATP) (28) throughout the entire MD simulation with an RMSD of ~1.5 Å. In contrast, the activation loop and the adjacent loop that is anchored to the αF helix (residues 210 to 220) in the corresponding PKA-C^L205R/ATP/PKI complex are more flexible, adopting two distinct conformations in which the most populated is similar to the x-ray structure (PDB ID: 1ATP) and a second minor flipped conformation (Fig. 4B). In this conformation, the activation loop no longer interacts with the C-lobe and forms a salt bridge with E86 of the αC helix (Fig. 4, D and E). For the PKA-C^L205R/ATP/VPS36 complex, the activation loop becomes more ordered and the flipped configuration becomes dominant. The existence of these two conformations is corroborated by chemical shift trajectories observed for the indole resonance of W196 in the activation loop (Fig. 4C). Upon ligand binding, the indole resonance of W196 follows a linear chemical shift trajectory from the apo to the ternary form. This conformational change is highly correlated with the other major allosteric nodes (CHESCA plots), indicating that it follows the cooperative structural changes of the enzyme. In contrast, the CHESCA plots for PKA-C^L205R show no correlations for these loops/residues, suggesting that the L205R mutation disrupts the internal allosteric network and may dislodge the activation loop from the large lobe.

The disruption of the allosteric network is mirrored by the pairwise mutual information plots (29) obtained from the analysis of the MD trajectories. For the wild-type enzyme, there are strong inter-residue correlations throughout the entire core of the kinase, especially among key catalytic motifs such as the Gly-rich loop (S53), the activation loop (R194, W196), and the αF (Y215), αG (D241), and αH (N283) helices, as well as the PIF motif at the C-terminal tail (F347) (fig. S6A). In contrast, PKA-C^L205R lacks a number of allosteric interactions within the N-lobe, with only a few correlations between the activation loop and the C-lobe (fig. S6B). As for the CHESCA correlation maps, the mutual information plots reveal that the allosteric network between the N-lobe and C-terminal tail of PKA-C^L205R is partially recovered upon VPS36 binding (fig. S6C) with the engagement of E91 in the αC helix and S53 of the Gly-rich loop.