Two PKA RIα holoenzyme states define ATP as an isoform-specific orthosteric inhibitor that competes with the allosteric activator, cAMP.

Protein kinase A (PKA) holoenzyme, comprised of a cAMP-binding regulatory (R)-subunit dimer and 2 catalytic (C)-subunits, is the master switch for cAMP-mediated signaling. Of the 4 R-subunits (RIα, RIß, RIIα, RIIß), RIα is most essential for regulating PKA activity in cells. Our 2 RIα2C2 holoenzyme states, which show different conformations with and without ATP, reveal how ATP/Mg2+ functions as a negative orthosteric modulator. Biochemical studies demonstrate how the removal of ATP primes the holoenzyme for cAMP-mediated activation. The opposing competition between ATP/cAMP is unique to RIα. In RIIß, ATP serves as a substrate and facilitates cAMP-activation. The isoform-specific RI-holoenzyme dimer interface mediated by N3A-N3A' motifs defines multidomain cross-talk and an allosteric network that creates competing roles for ATP and cAMP. Comparisons to the RIIß holoenzyme demonstrate isoform-specific holoenzyme interfaces and highlights distinct allosteric mechanisms for activation in addition to the structural diversity of the isoforms.

Implementing fluorescence anisotropy screening and crystallographic analysis to define PKA isoform-selective activation by cAMP analogs.

Cyclic AMP (cAMP) is a ubiquitous second messenger that regulates many proteins, most notably cAMP-dependent protein kinase (PKA). PKA holoenzymes (comprised of two catalytic (C) and two regulatory (R) subunits) regulate a wide variety of cellular processes, and its functional diversity is amplified by the presence of four R-subunit isoforms, RIα, RIß, RIIα, and RIIß. Although these isoforms all respond to cAMP, they are functionally nonredundant and exhibit different biochemical properties. In order to understand the functional differences between these isoforms, we screened cAMP derivatives for their ability to selectively activate RI and RII PKA holoenzymes using a fluorescence anisotropy assay. Our results indicate that RIα holoenzymes are selectively activated by C8-substituted analogs and RIIß holoenzymes by N6-substituted analogs, where HE33 is the most prominent RII activator. We also solved the crystal structures of both RIα and RIIß bound to HE33. The RIIß structure shows the bulky aliphatic substituent of HE33 is fully encompassed by a pocket comprising of hydrophobic residues. RIα lacks this hydrophobic lining in Domain A, and the side chains are displaced to accommodate the HE33 dipropyl groups. Comparison between cAMP-bound structures reveals that RIIß, but not RIα, contains a cavity near the N6 site. This study suggests that the selective activation of RII over RI isoforms by N6 analogs is driven by the spatial and chemical constraints of Domain A and paves the way for the development of potent noncyclic nucleotide activators to specifically target PKA iso-holoenyzmes.

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.

Signaling through cAMP and cAMP-dependent protein kinase: diverse strategies for drug design.

The catalytic subunit of cAMP-dependent protein kinase has served as a prototype for the protein kinase superfamily for many years while structures of the cAMP-bound regulatory subunits have defined the conserved cyclic nucleotide binding (CNB) motif. It is only structures of the holoenzymes, however, that enable us to appreciate the molecular features of inhibition by the regulatory subunits as well as activation by cAMP. These structures reveal for the first time the remarkable malleability of the regulatory subunits and the CNB domains. At the same time, they allow us to appreciate that the catalytic subunit is not only a catalyst but also a scaffold that mediates a wide variety of protein:protein interactions. The holoenzyme structures also provide a new paradigm for designing isoform-specific activators and inhibitors of PKA. In addition to binding to the catalytic subunits, the regulatory subunits also use their N-terminal dimerization/docking domain to bind with high affinity to A Kinase Anchoring Proteins using an amphipathic helical motif. This targeting mechanism, which localizes PKA near to its protein substrates, is also a target for therapeutic intervention of PKA signaling.

PKA-I holoenzyme structure reveals a mechanism for cAMP-dependent activation.

Protein kinase A (PKA) holoenzyme is one of the major receptors for cyclic adenosine monophosphate (cAMP), where an extracellular stimulus is translated into a signaling response. We report here the structure of a complex between the PKA catalytic subunit and a mutant RI regulatory subunit, RIalpha(91-379:R333K), containing both cAMP-binding domains. Upon binding to the catalytic subunit, RI undergoes a dramatic conformational change in which the two cAMP-binding domains uncouple and wrap around the large lobe of the catalytic subunit. This large conformational reorganization reveals the concerted mechanism required to bind and inhibit the catalytic subunit. The structure also reveals a holoenzyme-specific salt bridge between two conserved residues, Glu261 and Arg366, that tethers the two adenine capping residues far from their cAMP-binding sites. Mutagenesis of these residues demonstrates their importance for PKA activation. Our structural insights, combined with the mutagenesis results, provide a molecular mechanism for the ordered and cooperative activation of PKA by cAMP.

Repeat motions and backbone flexibility in designed proteins with different numbers of identical consensus tetratricopeptide repeats.

The tetratricopeptide repeat (TPR) is a 34-residue helix-turn-helix motif that occurs as three or more tandem repeats in a wide variety of proteins. We have determined the repeat motions and backbone fluctuations of proteins containing two or three consensus TPR repeats (CTPR2 and CPTR3, respectively) using 15N NMR relaxation measurements. Rotational diffusion tensors calculated from these data for each repeat within each TPR protein indicate that there is a high degree of motional correlation between different repeats in the same protein. This is consistent with the prevailing view that repeat proteins, such as CTPR2 and CTPR3, behave as single cooperatively folded domains. The internal motions of backbone NH groups were determined using the Lipari-Szabo model-free formalism. For most residues, there was a clear separation between the influence of internal motion and the influence of global rotational tumbling on the observed magnetic relaxation. The local internal motions are highly restricted in most of the helical elements, with slightly greater flexibility in the linker elements. Comparisons between CTPR2 and CTPR3 indicate that an addition of a TPR repeat to the C-terminus (before the solvation helix) of CTPR2 slightly reduces the flexibility of the preceding helix.