Abstract
Different isoforms of the full-length protein kinase A (PKA) regulatory subunit homodimer (R2) and the catalytic (C) subunit-bound holoenzyme (R2C2) have very different global structures despite similar molecular weights and domain organization within their primary sequences. To date, it has been the linker sequence between the R subunit dimerization/docking domain and cAMP-binding domain A that has been implicated in modulating domain interactions to give rise to these differences in global structure. The small angle solution scattering data presented here for three different isoforms of PKA heterodimer (deltaR-C) complexes reveal a role for another conformationally dynamic sequence in modulating inter-subunit and domain interactions, the C helix that connects the cAMP-binding domains A and B of the R subunit. The deltaR-C heterodimer complexes studied here were each formed with a monomeric N-terminal deletion mutant of the R subunit (deltaR) that contains the inhibitor sequence and both cAMP-binding domains. The scattering data show that type IIalpha and type IIbeta deltaR-C heterodimers are relatively compact and globular, with the C subunit contacting the inhibitor sequence and both cAMP-binding domains. In contrast, the type Ialpha heterodimer is significantly more extended, with the C subunit interacting with the inhibitor sequence and cAMP-binding domain A, whereas domain B extends out such that its surface is almost completely solvent exposed. These data implicate the C helix of RIalpha in modulating isoform-specific interdomain communication in the PKA holoenzyme, adding another layer of structural complexity to our understanding of signaling dynamics in this multisubunit, multidomain protein kinase.
Highlights
The catalytic (C) subunits are responsible for catalyzing phosphoryl transfer, whereas the regulatory (R) subunits confer cAMP dependence and localize the holoenzyme to discrete subcellular locations via interactions with protein kinase A anchoring proteins [5]
The results reported here further explore the conformational diversity of R subunit isoforms using small angle solution scattering to study N-terminal-truncated monomeric versions of RI␣, RII␣, and RII that contain the inhibitor sequence and both cAMP-binding domains (A and B) in heterodimeric complexes with the C subunit
The current study shows unequivocally that both cAMP-binding domains interact with C in the type II isoforms, but only domain A does so in the case of RI␣
Summary
These isoforms have different biological functions, as determined by genetic studies using mice [6, 7] Despite their different cellular functions, the different R isoforms share the same domain organization within their sequences: starting from the N terminus is a dimerization/docking (D/D) domain that anchors the R subunits to PKA anchoring proteins, a linker region that contains an inhibitor sequence, and two tandem cAMP-binding domains (designated A and B). The results reported here further explore the conformational diversity of R subunit isoforms using small angle solution scattering to study N-terminal-truncated monomeric versions of RI␣, RII␣, and RII that contain the inhibitor sequence and both cAMP-binding domains (A and B) in heterodimeric complexes with the C subunit. The first crystal structure showing an R-C interaction was recently obtained for a complex of the C subunit with a deletion mutant of RI␣ that includes just the pseudo-substrate inhibitor region and cAMP-binding domain A [22]. The results reported here show that there is a dramatic conformational change in the disposition of the cAMP-binding domains of RI␣ upon C subunit binding and that there are significant isoform differences in the cAMP-binding domain and C subunit interactions in the heterodimeric complexes studied
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