Cyclic AMP- and (Rp)-cAMPS-induced conformational changes in a complex of the catalytic and regulatory (RI{alpha}) subunits of cyclic AMP-dependent protein kinase

Abstract

We took a discovery approach to explore the actions of cAMP and two of its analogs, one a cAMP mimic ((S(p))-adenosine cyclic 3':5'-monophosphorothioate ((S(p))-cAMPS)) and the other a diastereoisomeric antagonist ((R(p))-cAMPS), on a model system of the type Iα cyclic AMP-dependent protein kinase holoenzyme, RIα(91-244)·C-subunit, by using fluorescence spectroscopy and amide H/(2)H exchange mass spectrometry. Specifically, for the fluorescence experiments, fluorescein maleimide was conjugated to three cysteine single residue substitution mutants, R92C, T104C, and R239C, of RIα(91-244), and the effects of cAMP, (S(p))-cAMPS, and (R(p))-cAMPS on the kinetics of R-C binding and the time-resolved anisotropy of the reporter group at each conjugation site were measured. For the amide exchange experiments, ESI-TOF mass spectrometry with pepsin proteolytic fragmentation was used to assess the effects of (R(p))-cAMPS on amide exchange of the RIα(91-244)·C-subunit complex. We found that cAMP and its mimic perturbed at least parts of the C-subunit interaction Sites 2 and 3 but probably not Site 1 via reduced interactions of the linker region and αC of RIα(91-244). Surprisingly, (R(p))-cAMPS not only increased the affinity of RIα(91-244) toward the C-subunit by 5-fold but also produced long range effects that propagated through both the C- and R-subunits to produce limited unfolding and/or enhanced conformational flexibility. This combination of effects is consistent with (R(p))-cAMPS acting by enhancing the internal entropy of the R·C complex. Finally, the (R(p))-cAMPS-induced increase in affinity of RIα(91-244) toward the C-subunit indicates that (R(p))-cAMPS is better described as an inverse agonist because it decreases the fractional dissociation of the cyclic AMP-dependent protein kinase holoenzyme and in turn its basal activity.

Fig. 1.

Overview of PKA structure and cAMP analogs. A, domain organization of RIα showing the domain boundaries of RIα(91–244) where the pseudosubstrate in green is connected to CNB-A domain in blue by a linker segment. B, structure of RIα(91–244) in the C-subunit-bound conformation (Protein Data Bank code 1U7E (23)) showing the pseudosubstrate in green, linker in yellow, and helical subdomain comprising helices αN, αA, αB, and αC in blue and β-subdomain in tan. The PBC is in red. C, structure of the C·RIα(91–244) holoenzyme showing the C-subunit in tan and RIα(91–244) in blue. Sites for introduction of cysteines by site-directed mutagenesis are represented by red circles. The cAMP binding site (PBC) is in red. D, structure of cAMP showing the 2′-OH group and 3′–5′ phosphodiester bond. The exocyclic oxygens upon replacement with sulfur atoms to generate the (S_p)-cAMPS and (R_p)-cAMPS diastereomers are highlighted.

Fig. 2.

Effects of cAMP, (R_p)-cAMPS, and (S_p)-cAMPS on the stopped-flow kinetics of FM-R92C RIα(91–244) binding to the C-subunit. Top left, control. Top right, in the presence of 10 μm cAMP. Bottom left, in the presence of 100 μm (R_p)-cAMPS. Bottom right, in the presence of 100 μm (S_p)-cAMPS. Stopped-flow traces of FM-R92C-RIα(91–244) rapidly mixed with C-subunit in the absence of cAMP are shown. The final concentration of FM-RIα(91–244) R92C was 100 nm, and the concentration of the C-subunit was varied from 1 to 6 μm. The decreasing emission follows the time course of binding. The solid lines denote the best fit of the data to a single exponential equation. Samples were suspended in a buffer containing 50 mm MOPS, pH 7.0, 50 mm NaCl, 1 mm DTT, 2 mm MgCl₂, and 0.2 mm ATP at 22 °C. A.U., absorbance units.

Fig. 3.

Effects of cAMP, (R_p)-cAMPS, and (S_p)-cAMPS on stopped-flow kinetics of FM-R92C RIα(91–244) dissociating from C-subunit. A, control and in the presence of 100 μm (R_p)-cAMPS. B, in the presence of 10 μm cAMP and 100 μm (S_p)-cAMPS. Stopped-flow traces of FM-R92C-RIα(91–244)·C-subunit complexes rapidly mixed with excess unlabeled RIα(91–244) are shown. The final concentration of FM-RIα(91–244) R92C was 100 nm, and the concentration of the unlabeled RIα(91–244) was 1 μm. The increasing emission follows the time course of dissociation of the labeled RIα(91–244). The solid lines denote the best fit of the data to a single exponential equation. Please see the legend to Fig. 2 for additional details. A.U., absorbance units.

Fig. 4.

Time-resolved anisotropy decay of FM-R92C, FM-T104C, and FM-R239C RIα(91–244)·C-subunit free in solution (A) and complexed to C-subunit (B). The smooth line through each decay plot was generated with best fit parameters for a biexponential function (Equation 4). Measurements were made with the samples suspended in 50 mm MOPS, pH 7.0 with 50 mm NaCl, 1 mm DTT, 0.2 mm ATP, and 2 mm MgCl₂ at 22 °C. IRF, instrument response function.

Fig. 5.

ESI-TOF spectra of one of the peptides spanning PBC (residues 204–221) in RIα(91–244). The spectra are expanded to show the isotopic distribution for the peptide of interest (m/z = 644.39). The higher mass peaks in the envelope are caused by naturally occurring isotopes. The isotopic envelope for the same peptide after 10 min of deuteration from C·RIα(91–244) bound to (R_p)-cAMPS (A), (R_p)-cAMPS ligand-free (B), and undeuterated sample (C).

Fig. 6.

Time course of deuterium incorporation into backbone amides in regions of RIα(91–244) subunit in C·RIα(91–244) in absence (○) and presence of (R_p)-cAMPS (●) for peptides. A, m/z = 797.86, z = 2 (residues 136–148). B, m/z = 846.89, z = 2 (residues 157–172). C, m/z = 567.32, z = 2 (residues 202–212). D, m/z = 523.81, z = 2 (residues 230–238). The solid lines denote the best fit of the data to a single exponential equation. Error bars were calculated from 2 replicate experiments.

Fig. 7.

Effects of (R_p)-cAMPS on time course of deuterium incorporation into backbone amides in regions of C-subunit complexed to RIα(91–244). Data are plotted in the absence (○) and presence of (R_p)-cAMPS (●) for the peptides. A, m/z = 700.32, z = 2 (residues 7–18). B, m/z = 732.72, z = 3 (residues 14–31); C, 673.35, z = 5 (173–178). D, m/z = 728.36, z = 2 (residues 335–346). The solid lines denote the best fit of the data to a single exponential equation. Error bars were calculated from 2 replicate experiments.

Fig. 8.

Effects of (R_p)-cAMPS binding to PKA holoenzyme (RIα(91–244)·C-subunit by amide H/²H exchange displayed on structure of C·RIα(91–244) complex (Protein Data Bank code 1U7E). Segments are colored based on the effects of (R_p)-cAMPS binding on amide exchange: segments showing increased exchange are red, segments showing no differences are tan, and segments showing decreased exchange are blue. Segments missing in the analysis are gray.