Cyclic AMP- and (Rp)-cAMPS-induced Conformational Changes in a Complex of the Catalytic and Regulatory (RIα) Subunits of Cyclic AMP-dependent Protein Kinase

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 I alpha cyclic AMP-dependent protein kinase holoenzyme, RI alpha (91-244).C-subunit, by using fluorescence spectroscopy and amide H/H-2 exchange mass spectrometry. Specifically, for the fluorescence experiments, fluorescein maleimide was conjugated to three cysteine single residue substitution mutants, R92C, T104C, and R239C, of RI alpha(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 RI alpha(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 alpha C of RI alpha(91-244). Surprisingly, (R-p)-cAMPS not only increased the affinity of RI alpha(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 RI alpha(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. Molecular & Cellular Proteomics 9:2225-2237, 2010.