Dynamics of cAPK Type IIβ Activation Revealed by Enhanced Amide H/ 2H Exchange Mass Spectrometry (DXMS)

• Published in: Journal of molecular biology Vol. 327; no. 5; pp. 1065 - 1076
• Main Authors: Hamuro, Yoshimoto, Zawadzki, Kerri M., Kim, Jack S., Stranz, David D., Taylor, Susan S., Woods, Virgil L.
• Format: Journal Article
• Language: English
• Published: Elsevier Ltd 01.04.2003
• Subjects: amide hydrogen exchange; cAPK; mass spectrometry; PKA; regulatory subunit; C-subunit, catalytic subunit; apo R-subunit, R-subunit devoid of cAMP and C-subunit; cA, cAMP-binding domain A; cAMP, adenosine 3′,5′-cyclic monophosphate; PKA; cAPK, cAMP-dependent protein kinase; R-subunit, regulatory subunit; amide hydrogen exchange; cGMP, guanosine 3′,5′-cyclic monophosphate; regulatory subunit; DD, dimerization/docking domain; cAPK; PBC, phosphate-binding cassette; mass spectrometry; cB, cAMP-binding domain B; TFA, trifluoroacetic acid
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/S0022-2836(03)00234-1

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 IIβ 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 RIIβ 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.