Dynamics of cAPK type IIbeta activation revealed by enhanced amide H/2H exchange mass spectrometry (DXMS).
cAMP-dependent protein kinase (cAPK) is a key component in numerous cell signaling pathways. The cAPK regulatory (R) subunit maintains the kinase in an inactive state until cAMP saturation of the R-subunit leads to activation of the enzyme. To delineate the conformational changes associated with cAPK activation, the amide hydrogen/deuterium exchange in the cAPK type IIbeta R-subunit was probed by electrospray mass spectrometry. Three states of the R-subunit, cAMP-bound, catalytic (C)-subunit bound, and apo, were incubated in deuterated water for various lengths of time and then, prior to mass spectrometry analysis, subjected to digestion by pepsin to localize the deuterium incorporation. High sequence coverage (>99%) by the pepsin-digested fragments enables us to monitor the dynamics of the whole protein. The effects of cAMP binding on RIIbeta amide hydrogen exchange are restricted to the cAMP-binding pockets, while the effects of C-subunit binding are evident across both cAMP-binding domains and the linker region. The decreased amide hydrogen exchange for residues 253-268 within cAMP binding domain A and for residues 102-115, which include the pseudosubstrate inhibitory site, support the prediction that these two regions represent the conserved primary and peripheral C-subunit binding sites. An increase in amide hydrogen exchange for a broad area within cAMP-binding domain B and a narrow area within cAMP-binding domain A (residues 222-232) suggest that C-subunit binding transmits long-distance conformational changes throughout the protein.
Hamuro
Yoshimoto
Y
Department of Medicine, University of California, 9500 Gilman Drive, San Diego, La Jolla, CA 92093-0656, USA.
Zawadzki
Kerri M
KM
Kim
Jack S
JS
Stranz
David D
DD
Taylor
Susan S
SS
Woods
Virgil L
VL
D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D006868:Hydrolysis; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D000577:Amides; D011994:Recombinant Proteins; D017868:Cyclic AMP-Dependent Protein Kinases