Probing cAMP-dependent protein kinase holoenzyme complexes I alpha and II beta by FT-IR and chemical protein footprinting.

Although individual structures of cAMP-dependent protein kinase 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 holoenzymes is very limited. To delineate the mechanism of duanyu1529 activation and structural differences between type I and II duanyu1529 holoenzymes, the conformation and structural dynamics of duanyu1529 holoenzymes Ialpha and IIbeta were probed by amide hydrogen-deuterium exchange coupled with Fourier transform infrared spectroscopy (FT-IR) and chemical protein footprinting. Binding of cAMP to duanyu1529 holoenzymes Ialpha and IIbeta leads to a downshift in the wavenumber for both the alpha-helix and beta-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 duanyu1529 holoenzymes Ialpha and IIbeta. Despite the overall similarity, significant differences in the change of FT-IR spectra in response to the binding of cAMP were observed between duanyu1529 holoenzymes Ialpha and IIbeta. Activation of duanyu1529 holoenzyme Ialpha led to more conformational changes in beta-strand structures, while cAMP induced more apparent changes in the alpha-helical structures in duanyu1529 holoenzyme IIbeta. 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 duanyu1529 holoenzymes Ialpha and IIbeta, a region around the active site cleft of the C subunit was more protected in duanyu1529 holoenzyme Ialpha than in duanyu1529 holoenzyme IIbeta. These results suggest that the C subunit assumes a more open conformation in duanyu1529 holoenzyme IIbeta. In addition, the chemical cleavage patterns around the active site cleft of the C subunit were distinctly different in duanyu1529 holoenzymes Ialpha and IIbeta 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.

KEYWORDS: {{ getKeywords(articleDetailText.words) }}