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.
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.
Anand
Ganesh S
GS
Department of Biological Sciences, National University of Singapore, Singapore.
Krishnamurthy
Srinath
S
Bishnoi
Tanushree
T
Kornev
Alexandr
A
Taylor
Susan S
SS
Johnson
David A
DA
D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D019793:Fluorescein / Q000737:chemistry; D005454:Fluorescence Polarization; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization
D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D019793:Fluorescein