Sensing domain dynamics in protein kinase A-I{alpha} complexes by solution X-ray scattering.
The catalytic (C) and regulatory (R) subunits of protein kinase A are exceptionally dynamic proteins. Interactions between the R- and C-subunits are regulated by cAMP binding to the two cyclic nucleotide-binding domains in the R-subunit. Mammalian cells express four different isoforms of the R-subunit (RIalpha, RIbeta, RIIalpha, and RIIbeta) that all interact with the C-subunit in different ways. Here, we investigate the dynamic behavior of protein complexes between RIalpha and C-subunits using small angle x-ray scattering. We show that a single point mutation in RIalpha, R333K (which alters the cAMP-binding properties of Domain B) results in a compact shape compared with the extended shape of the wild-type R.C complex. A double mutant complex that disrupts the interaction site between the C-subunit and Domain B in RIalpha, RIalpha(AB)R333K.C(K285P), results in a broader P(r) curve that more closely resembles the P(r) profiles of wild-type complexes. These results together suggest that interactions between RIalpha Domain B and the C-subunit in the RIalpha.C complex involve large scale dynamics that can be disrupted by single point mutations in both proteins. In contrast to RIalpha.C complexes. Domain B in the RIIbeta.C heterodimer is not dynamic and is critical for both inhibition and complex formation. Our study highlights the functional differences of domain dynamics between protein kinase A isoforms, providing a framework for elucidating the global organization of each holoenzyme and the cross-talk between the R- and C-subunits.
Cheng
Cecilia Y
CY
Departments of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92037-0654, USA.
Yang
Jie
J
Taylor
Susan S
SS
Blumenthal
Donald K
DK
D000818:Animals; D000242:Cyclic AMP / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D020836:Protein Structure, Quaternary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D014961:X-Ray Diffraction
D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D007527:Isoenzymes; D000242:Cyclic AMP