Amide H/2H exchange reveals communication between the cAMP and catalytic subunit-binding sites in the R(I)alpha subunit of protein kinase A.
The changes in backbone hydrogen/deuterium (H/2H) exchange in the regulatory subunit (R(I)alpha(94-244)) of cyclic AMP-dependent protein kinase A (PKA) were probed by MALDI-TOF mass spectrometry. The three naturally occurring states of the regulatory subunit were studied: (1) free R(I)alpha(94-244), which likely represents newly synthesized protein, (2) R(I)alpha(94-244) bound to the catalytic (C) subunit, or holoenzyme, and (3) R(I)alpha(94-244) bound to cAMP. Protection from amide exchange upon C-subunit binding was observed for the helical subdomain, including the A-helix and B-helix, pointing to regions adjacent to those shown to be important by mutagenesis. In addition, C-subunit binding caused changes in observed amide exchange in the distal cAMP-binding pocket. Conversely, cAMP binding caused protection in the cAMP-binding pocket and increased exchange in the helical subdomain. These results suggest that the mutually exclusive binding of either cAMP or C-subunit is controlled by binding at one site transmitting long distance changes to the other site.
Anand
Ganesh S
GS
Howard Hughes Medical Institute, University of California, San Diego 9500 Gilman Drive, La Jolla, CA 92093-0359, USA.
Hughes
Carrie A
CA
Jones
John M
JM
Taylor
Susan S
SS
Komives
Elizabeth A
EA
D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D000242:Cyclic AMP / Q000737:chemistry / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D006859:Hydrogen / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D012997:Solvents; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000577:Amides; D021122:Protein Subunits; D012997:Solvents; D006859:Hydrogen; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases