Mutations of PKA cyclic nucleotide-binding domains reveal novel aspects of cyclic nucleotide selectivity

Abstract

Cyclic AMP and cyclic GMP are ubiquitous second messengers that regulate the activity of effector proteins in all forms of life. The main effector proteins, the 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) and the 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG), are preferentially activated by cAMP and cGMP, respectively. However, the molecular basis of this cyclic nucleotide selectivity is still not fully understood. Analysis of isolated cyclic nucleotide-binding (CNB) domains of PKA regulatory subunit type Iα (RIα) reveals that the C-terminal CNB-B has a higher cAMP affinity and selectivity than the N-terminal CNB-A. Here, we show that introducing cGMP-specific residues using site-directed mutagenesis reduces the selectivity of CNB-B, while the combination of two mutations (G316R/A336T) results in a cGMP-selective binding domain. Furthermore, introducing the corresponding mutations (T192R/A212T) into the PKA RIα CNB-A turns this domain into a highly cGMP-selective domain, underlining the importance of these contacts for achieving cGMP specificity. Binding data with the generic purine nucleotide 3',5'-cyclic inosine monophosphate (cIMP) reveal that introduced arginine residues interact with the position 6 oxygen of the nucleobase. Co-crystal structures of an isolated CNB-B G316R/A336T double mutant with either cAMP or cGMP reveal that the introduced threonine and arginine residues maintain their conserved contacts as seen in PKG I CNB-B. These results improve our understanding of cyclic nucleotide binding and the molecular basis of cyclic nucleotide specificity.

Keywords: CNB domain; cAMP; cGMP; cyclic nucleotide; protein kinase A; protein kinase G.

Figure 1. Structural alignment of CNB domains from PKG and PKA

(A) Co-crystal structure of the C-terminal CNB domain (CNB-B) of PKG Iβ with cGMP (PDB code 4KU7) [20]. Key cGMP contact residues are highlighted (sites 1–3). Y351 at the C-terminal loop provides a capping interaction for cGMP. (B) Crystal structure of human PKA RIα CNB-B. Corresponding residues of sites 1–3 differ from PKG Iβ. Tyrosine 373 (Y373) is homologous to Y351 of PKG Iβ and caps the bound cAMP. (C) Structural alignment of the CNB-Bs of PKG Iβ and PKA RIα. (D) Sequence alignment from the β5 strands to the end of the PBC (box). Sequences were aligned using Clustal Omega [46,47]. Identical residues are shown in yellow. The three differing sites are shaded in red and marked with asterisks. All structure figures were generated using PyMOL (DeLano Scientific, Palo Alto, CA, U.S.A.).

Figure 2. SPR solution competition experiments

(A) SPR solution competition data of PKA hRIα CNB-A (115–274) with cAMP. hRIα CNB-A (2 nM) was preincubated with a cAMP concentration series ranging from 0.6 nM to 1 mM. Additionally, the protein sample was injected without cAMP. The samples were injected over a high-density 6-AH-cAMP surface for 150 s (association phase) before the dissociation was monitored for 100 s using a Biacore T100/T200 instrument (GE Healthcare, Chalfont St Giles, U.K.). A report point was set 3 s before the end of the association (red-dashed line) to monitor the SPR signal of each sample. RU: resonance unit. (B) The SPR signal was plotted against the logarithmic cAMP concentration and the data were fitted with a sigmoidal dose–response curve employing Prism 6.01 (GraphPad, La Jolla, CA, U.S.A.). The EC₅₀ is the cAMP concentration which gives half-maximal competition of the SPR signal (1/2 max. SPR signal). The EC₅₀ for this experiment was 184 nM.

Figure 3. Cyclic nucleotide binding of individual CNB domains

Competition curves were derived from SPR experiments as exemplified in Figure 2 (normalized data). (A) Binding competition of PKA hRIα CNB-A (residues 115–274). Cyclic AMP and cyclic IMP bind to CNB-A with high nanomolar affinities, while cGMP binds with a micromolar affinity. CNB-A shows a more than 30-fold preference for cAMP. (B) CNB-B (residues 234–381) has a significantly higher affinity for cAMP compared with CNB-A, but still binds cGMP with micromolar affinity, resulting in an 110-fold selectivity. Cyclic IMP is bound with an intermediate affinity between cAMP and cGMP.

Figure 4. Cyclic nucleotide binding of PKA RIα CNB-B mutants

Binding competition experiments were performed as described in Figure 2. The following mutants were characterized: (A) V315L, (B) G316R, (C) A336T, (D) V315L/G316R, (E) G316R/A336T, and (F) V315L/G316R/A336T. Dashed lines show binding competition curves of wild-type CNB-B for cAMP (black) and cGMP (red).

Figure 5. Crystal structure of PKA hRIα CNB-B (234–381) G316R/A336T bound with cAMP

(A) Overall structure of the PKA RIα CNB-B G316R/A336T double mutant bound with cAMP. The protein is shown in cartoon representation with the secondary structure elements labeled, while cAMP is shown as sticks. (B) Detailed view of the CNB pocket with the bound cAMP. The introduced A336T does not interact with cAMP. The side chain of G316R moves away from the cAMP, indicating a steric clash between the position 6 amino group and the arginine side chain. Therefore, R316 is a negative determinant for binding of cAMP as recently described for PKG Iβ [21]. However, a hydrogen bond with the N7 of the purine ring can still be formed. Residues are labeled and hydrogen bond interactions are shown as dashed lines. (C) Structural alignment of the PKA RIα CNB-B G316R/A336T: cAMP complex (gray) with the PKG Iβ CNB-B: cAMP complex structure (red; PDB code 4QX5) [21].

Figure 6. Crystal structure of PKA hRIα 234–381 G316R/A336T bound with cGMP

(A) Overall structure of the PKA RIα CNB-B G316R/A336T mutant bound with cGMP. The same representations and labels are used as described in Figure 5. (B) Detailed view of the CNB pocket and the bound cGMP. The side chain of T336 forms a hydrogen bond with the 2 amino group of cGMP. Additionally, the hydroxyl group of the threonine residue hydrogen bonds the axial oxygen of the cyclic phosphate. The cGMP co-crystal structure reveals that both hydrogen bonds seen in the PKG I CNB-B: cGMP complex (PDB code 4KU7) can be formed with cGMP. The guanidinium side chain of G316R hydrogen bonds both the N7 and the 6 carbonyl of the guanine nucleobase. (C) Structural alignment of the binding pockets of the PKA RIα CNB-B G316R/A336T: cGMP structure (gray) with the PKG Iβ CNB-B: cGMP complex (red; PDB code 4KU7) [20].

Figure 7. Cyclic nucleotide binding of PKA RIα CNB-A mutants

Binding curves as derived from SPR solution competition experiments. (A) The PKA hRIα 115–274 double-mutant T192R/A212T has a switched selectivity compared with the wild-type CNB-A (black- and red-dashed lines for cAMP and cGMP, respectively). (B) The T192R mutant and (C) the A212T single mutants have a reduced affinity for cAMP and an increased affinity for cGMP compared with the wild-type CNB-A.