Local and distal changes in dynamics are caused by an L205R Cushing's syndrome mutant in PRKACA

Abstract

Cushing's syndrome (CS) is an abnormal condition characterized by elevated cortisol levels, often resulting from genetic alterations in the PRKACA gene, which encodes the catalytic subunit of cAMP-dependent protein kinase A (PKA-C). The most common CS mutation, L205R, lies at the P + 1 loop. Understanding how this mutation alters the internal allosteric network within PKA-C and changes nucleotide and substrate cooperativity is a major goal. Using molecular dynamics (MD) simulations and protein residue networks based on local spatial pattern (LSP) method, we compare crystal structures of wild-type PKA-C and L205R. Our findings indicate that L205R not only locally disrupts the P + 1 hydrophobic pocket, leading to the displacement of the P + 1-residue and altered substrate specificity, but also has long-range effects in the linker connecting the A helix to β strand 1. The MD simulations and LSP analyses also reveal critical changes at the phosphoryl transfer site. Some of these changes are captured in the L205R crystal structure while others are not. With this strategy, we also show how the dynamics of local and distal allosteric networks are differentially influenced by backbone and side-chain dynamics.

Keywords: Cushing’s syndrome; L205R mutation; PKA; local spatial pattern; molecular dynamic simulations.

Fig. 1.

Structural changes caused by an L205R Cushing’s syndrome mutation in the protein kinase A-C subunit (PKA-C). The crystal structure of wt PKA-C bound to IP20 and Mg₂ATP (Left, PDB: 1ATP) is compared to a crystal structure of the L205R CS mutant (Right, PDB: 4WB6). The N and C termini of IP20 are bound through hydrophobic motifs (red shells) to wt PKA-C (center). The P + 1-site in IP20 (I22, red shell) binds to the hydrophobic P + 1 Loop (residues L198–L205) formed by L198, P202 (sand shell), and L205 (cyan shell), which is part of the Activation Segment in the C-Lobe. The P-2 and P-3 arginines in IP20 interact with E230 and E127 and the ribose of ATP, respectively. Binding of I22(PKI) to the P + 1 Loop is severed by the L205R mutation (Right). The N- and C-lobes of PKA-C are colored in white and tan, the N-linker and β4 in cyan, and IP20 in red, respectively. All PKI residues are also italicized.

Fig. 2.

Mapping changes in dynamics of the L205R mutation in PKA-C using LSP alignment. Panel (A) shows the structure of the IP20 peptide docked into the active site of wt PKA-C. The hydrophobic docking sites are indicated by shells. Tan shells are from PKA-C while red shells are from IP20. The L205R shell is in teal. The LSP-based PRN maps are shown in (B). The IP20 residues are indicated by red dots while PKA residues are in green. The red circle highlights communication between the N- and C-Lobes while F54 in the G-Loop of the N-Lobe and F187 following the DFG motif in the C-Lobe are labeled in red. Panel (C) highlights the structure of the IP20 peptide docked into the active site of the L205R mutant. Panel (D) is a graphic representation of the DC changes when the wt and mutant are compared. The positive (Left, red) and negative (Right, blue) ΔDC_WT-L205R values are mapped onto the PKA-C structure on the left and right, respectively. Negative ΔDC_WT-L205R values correlate with gain of stability or loss of dynamics in L205R compared to wt PKA-C while positive ΔDC_WT-L205R values correlate with enhanced dynamics and loss of stability. Key residues (T37, S53, F187, G186) with higher mobility and loss stability in L205R are highlighted by arrows on the graph. The IP20 peptide is highlighted by the red box.

Fig. 3.

Anchoring and stability of F350 at the C-terminus of PKA-C. Top (Left): Environment of F350 in the crystal structure of wt PKA-C. Top (Right): Environment of F350 in the crystal structure of PKA-C(L205R). Bottom (Middle): Close up of the interactions of F350 in the crystal structure of wt PKA-C. Hydrophobic interactions are highlighted by tan shells. The β4 strand and the flexible linker are in teal. Panels (A–F) compare MD simulations of residues that interact with F350 in wt PKA C and the L205R mutant. The location of each site of interaction is indicated on the Middle panel. Triplicate runs of the MD simulations are in SI Appendix, Fig. S1.

Fig. 4.

Order/disorder changes at the N-Tail linker with the kinase core are induced by the L205R mutant. Top panel highlights the electrostatic and hydrogen bonds that anchor the N-linker to β4 in wt PKA-C and also shows how the linker (residues 34–36) is anchored at each end to hydrophobic motifs in the kinase core and to the A-Helix. Coloring is the same as in Fig. 3. L40 at the end of the linker segment is anchored to F110 and K111 in the loop joining β4 and β5, while P33 that precedes the flexible linker segment is anchored to R93 in the C Helix. R93 is part of the hydrophobic node that fuses the N-and C-lobe interface to W30 in the A Helix (23). Panels (A–F) compare MD simulations of residues in the linker that interact with β4 and the two hydrophobic motifs. Each specific interaction is highlighted in red on the Upper panel. Triplicate runs of the MD simulations are in SI Appendix, Fig. S1.

Fig. 5.

Displacement of I22 in the P + 1 site of IP20 due to the L205R mutation is captured in the crystal structures and in the MD simulations. (A and B) The free energy landscape (FEL) plotted from MD simulations for the distance L205@CG-I22@CD1 is shown for WT (A) and the L205R mutant (B). (C) MD simulations for WT and L205R show that the position of I22 in IP20 is altered in the L205R mutant. (D) The probability density plot (PDF) of WT and L205R from MD simulations. (E) Position of I22 in the crystal structure of wt PKA-C. (F) Position of I22 in the crystal structure of L205R. Triplicate runs of the MD simulations are in SI Appendix, Fig. S1.

Fig. 6.

Interactions of IP20 residues that flank the P-site with the active site of PKA-C and the L205R mutant. Middle: Crystal structures of wt PCA-C (Left) and the L205R mutant (Right) showing changes in the residues in IP20 that flank the P-site in IP20. (A) MD simulations capture the dynamic features of the P + 2 residue (H23) in IP20 in wt PKA-C and the L205R mutant. (B) MD simulations of the interactions of the P-2 arginine (R19) with E230 in wt PKA-C and L205R. (C) MD simulations of interactions of the P-2 arginine (R19) with E203 in wt PKA-C and L205R. (D) MD simulations of interactions of the P-3 arginine (R18) with E127 in wt PKA-C and L205R. (E and F) MD simulations of interactions of the P-6 arginine (R15) with E203 in wt PKA-C and L205R. Triplicate runs of the MD simulations are in SI Appendix, Fig. S1.

Fig. 7.

Changes in the dynamics at the active site of PKA-C are induced by the L205R CS mutant. Top: Interactions of A21 in IP20 with the G-Loop in wt PKA-C (Left) and in L205R (Right) are altered based on the MD simulations. MD simulations for interaction of A21 with the γ-phosphate of ATP vs. the side chain of S53 in the G-Loop are shown in A and B, respectively. Middle: MD simulations of interactions of K72 with the α and β phosphates of ATP in wt PKA-C (Left) and L205R (Right). MD simulations are shown in C and D, respectively. Bottom: MD simulation of interactions of the guanidinium side chain of the P-3 Arg in IP20 with E127 and the γ-phosphate of ATP in wt PKA-C (Left) and in the L205 mutant (Right). MD simulations are shown in E and F. Triplicate runs of the MD simulations are in SI Appendix, Fig. S3.

Fig. 8.

The local and long-distance allosteric effects resulted from the L205R mutation alter the dynamics of the opening and closing of the catalytic cleft. The allosteric range of changes induced by the L205R mutation are indicated on the left while the interactions made by residues in the P + 1 loop, in addition to anchoring of the P + 1 Ile in IP20, are shown on the right. Movies of the active site cleft in the center show how closing of the cleft, mediated by interactions of F54 in the G-Loop with F187 that follows the DFG motif in the C-lobe, is altered by the mutation. In SI Appendix, Fig. S4 we show how signal integration motifs (SIMs) in β3 and C Helix weave together the hydrophobic core of the N-lobe of PKA-C.