R-subunit isoform specificity in protein kinase A: distinct features of protein interfaces in PKA types I and II by amide H/2H exchange mass spectrometry.
The two isoforms (RI and RII) of the regulatory (R) subunit of cAMP-dependent protein kinase or protein kinase A (PKA) are similar in sequence yet have different biochemical properties and physiological functions. To further understand the molecular basis for R-isoform-specificity, the interactions of the RIIbeta isoform with the PKA catalytic (C) subunit were analyzed by amide H/(2)H exchange mass spectrometry to compare solvent accessibility of RIIbeta and the C subunit in their free and complexed states. Direct mapping of the RIIbeta-C interface revealed important differences between the intersubunit interfaces in the type I and type II holoenzyme complexes. These differences are seen in both the R-subunits as well as the C-subunit. Unlike the type I isoform, the type II isoform complexes require both cAMP-binding domains, and ATP is not obligatory for high affinity interactions with the C-subunit. Surprisingly, the C-subunit mediates distinct, overlapping surfaces of interaction with the two R-isoforms despite a strong homology in sequence and similarity in domain organization. Identification of a remote allosteric site on the C-subunit that is essential for interactions with RII, but not RI subunits, further highlights the considerable diversity in interfaces found in higher order protein complexes mediated by the C-subunit of PKA.
Anand
Ganesh S
GS
Howard Hughes Medical Institute, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0359, USA.
Hotchko
Matthew
M
Brown
Simon H J
SH
Ten Eyck
Lynn F
LF
Komives
Elizabeth A
EA
Taylor
Susan S
SS
D000255:Adenosine Triphosphate / Q000378:metabolism; D000577:Amides / Q000737:chemistry*; D020134:Catalytic Domain; D000242:Cyclic AMP / Q000378:metabolism; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit / Q000737:chemistry* / Q000378:metabolism*; D003903:Deuterium / Q000737:chemistry*; D041961:Deuterium Exchange Measurement; D020035:Holoenzymes / Q000737:chemistry* / Q000378:metabolism*; D013058:Mass Spectrometry; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance
D000577:Amides; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D020035:Holoenzymes; D010446:Peptide Fragments; D021122:Protein Subunits; D000255:Adenosine Triphosphate; D003903:Deuterium; D000242:Cyclic AMP