Realizing the Allosteric Potential of the Tetrameric Protein Kinase A RIα Holoenzyme

Abstract

Summary PKA holoenzymes contain two catalytic (C) and a regulatory (R) subunit dimer where the two R-subunits are linked by an N-terminal Dimerization/Docking (D/D) domain. Cooperative binding of four cAMP molecules induces major structural changes in the R-subunits that cause kinase activation. While cooperativity exists between the two tandem cAMP binding domains, additional levels of cooperativity are associated with the tetramer. This allostery cannot be appreciated by studying heterodimers formed between C-subunit and deletion mutants of R that lack the D/D domain. Of critical importance is the flexible linker in the R-subunit that contains the Inhibitor Site (IS) that mimics the PKA substrate sequence and binds to the active site of the C-subunit. Two flexible linkers connect the IS to the D/D domain (N-Linker) and to the cAMP binding domains (C-Linker). In the cAMP-bound conformation IS and the C-Linker are disordered but become structured at the R:C interface when RIα binds to the C-subunit. The overall conformation of the tetramer, however, is mediated in large part by the N-Linker. To probe the function of the N-Linker in RIα and specifically to determine how the N-Linker contributes to assembly of the tetrameric holoenzyme, we engineered a monomeric form of RIα that contains most of the N-Linker, RIα(73-244), and crystallized a holoenzyme complex. Our previous RIα constructs began with the inhibitor site. RIα(73-244):C holoenzyme does not form stable dimers in solution without the dimerization domain; however, part of the extended linker is now ordered by interactions with a symmetry-related-dimer in the crystal. This complex of two symmetry-related dimers forms a tetramer that not only reveals novel mechanisms for allosteric regulation but also has many features that are consistent with known properties of the full-length holoenzyme. A model of the tetrameric RIα holoenzyme, based on this structure, is also consistent with small angle X-ray and neutron scattering data reported earlier. The model has been validated with new SAXS data and with a mutant of RIα that is localized to a novel interface that is unique to the tetramer.

Localization and quaternary structure of the PKA RIβ holoenzyme

“…S7D) (17). In summary, a portion of the D/ D domain docks in a trans manner onto a conserved docking site on the C-subunit and appears to be an integral part of the quaternary structure.…”

“…The structure of the RIα 1 :C 1 heterodimer that includes its N-linker region shows the importance of this linker for the assembly of the quaternary structure (17). This N-linker docks onto the R-subunit of the symmetry-related dimer and thus creates an interface that enables us to build an RIα holoenzyme model that is a dimer of a heterodimer (17). All R isoforms dock in a similar manner to the active site cleft of the C-subunit, and the overall architecture of the R 1 :C 1 heterodimers is remarkably similar (Fig.…”

“…Small-angle Xray/neutron scattering (SAXS/SANS) showed profound differences in the overall shape of the full-length RIα, RIIα, and RIIβ holoenzymes (15,16). A recently described structure of a deletion mutant of RIα (75-244):C containing an extended linker shows how the linker interacts with the symmetry-related dimer (17) and thereby creates a twofold axis of symmetry between the two heterodimers. This structure provides a plausible model explaining how a dimer of heterodimers can be assembled.…”

Structural insights into mis-regulation of protein kinase A in human tumors

“…We further investigated possibilities that mis-regulation of PKA signaling is caused by mis-localization of the PKA fusion, a mechanism known to be associated with disease states (18). We did not expect interactions with canonical A-kinase anchoring proteins (AKAPs), which localize PKA through binding to the N-terminal dimerization domain of R subunits (7), to be affected by the fusion because this region is readily accessible in the structures of PKA holoenzymes (3)(4)(5)(6). AKIP1a, on the other hand, is an unconventional AKAP that binds to PKA near the N terminus of the C subunit (19).…”

“…Four isoforms of the R subunit (RIα, RIβ, RIIα, and RIIβ) are known. Structures of the R 2 :C 2 holoenzyme provide insight into mechanisms of allosteric control and reveal distinct, isoform-specific assemblies of the tetramers (3)(4)(5)(6), even though all R:C heterodimer associations are similar. Specificity of PKA activity is also controlled by localization.…”