Although individual structures of cAMP-dependent protein kinase (PKA) catalytic (C) and regulatory (R) subunits have been determined at the atomic level, our understanding of the effects of cAMP activation on protein dynamics and intersubunit communication of PKA holoenzymes is very limited. To delineate the mechanism of PKA activation and structural differences between type I and II PKA holoenzymes, the conformation and structural dynamics of PKA holoenzymes Iα and IIβ were probed by amide hydrogen-deuterium exchange coupled with Fourier transform infrared spectroscopy (FT-IR) and chemical protein footprinting. Binding of cAMP to PKA holoenzymes Iα and IIβ leads to a downshift in the wavenumber for both the α-helix and β-strand bands, suggesting that R and C subunits become overall more dynamic in the holoenzyme complexes. This is consistent with the H-D exchange results showing a small change in the overall rate of exchange in response to the binding of cAMP to both PKA holoenzymes Iα and IIβ. Despite the overall similarity, significant differences in the change of FT-IR spectra in response to the binding of cAMP were observed between PKA holoenzymes Iα and IIβ. Activation of PKA holoenzyme Iα led to more conformational changes in β-strand structures, while cAMP induced more apparent changes in the α-helical structures in PKA holoenzyme IIβ. Chemical protein footprinting experiments revealed an extended docking surface for the R subunits on the C subunit. Although the overall subunit interfaces appeared to be similar for PKA holoenzymes Iα and IIβ, a region around the active site cleft of the C subunit was more protected in PKA holoenzyme Iα than in PKA holoenzyme IIβ. These results suggest that the C subunit assumes a more open conformation in PKA holoenzyme IIβ. In addition, the chemical cleavage patterns around the active site cleft of the C subunit were distinctly different in PKA holoenzymes Iα and IIβ even in the presence of cAMP. These observations provide direct evidence that the R subunits may be partially associated with the C subunit with the pseudosubstrate sequence docked in the active site cleft in the presence of cAMP.
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