Sensing Domain Dynamics in Protein Kinase A-Iα 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 (RI alpha, RI beta, RII alpha, and RII beta) that all interact with the C-subunit in different ways. Here, we investigate the dynamic behavior of protein complexes between RI alpha and C-subunits using small angle x-ray scattering. We show that a single point mutation in RI alpha, 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 RI alpha, RI alpha(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 RI alpha Domain B and the C-subunit in the RI alpha.C complex involve large scale dynamics that can be disrupted by single point mutations in both proteins. In contrast to RI alpha.C complexes. Domain B in the RII beta.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.