Crystal Structures of RIα Subunit of Cyclic Adenosine 5‘-Monophosphate (cAMP)-Dependent Protein Kinase Complexed with (R_p)-Adenosine 3‘,5‘-Cyclic Monophosphothioate and (S_p)-Adenosine 3‘,5‘-Cyclic Monophosphothioate, the Phosphothioate Analogues of cAMP^,

Abstract

Cyclic adenosine 5'-monophosphate (cAMP) is an ancient signaling molecule, and in vertebrates, a primary target for cAMP is cAMP-dependent protein kinase (PKA). (R(p))-adenosine 3',5'-cyclic monophosphothioate ((R(p))-cAMPS) and its analogues are the only known competitive inhibitors and antagonists for cAMP activation of PKA, while (S(p))-adenosine 3',5'-cyclic monophosphothioate ((S(p))-cAMPS) functions as an agonist. The crystal structures of a Delta(1-91) deletion mutant of the RIalpha regulatory subunit of PKA bound to (R(p))-cAMPS and (S(p))-cAMPS were determined at 2.4 and 2.3 A resolution, respectively. While the structures are similar to each other and to the crystal structure of RIalpha bound to cAMP, differences in the dynamical properties of the protein when (R(p))-cAMPS is bound are apparent. The structures highlight the critical importance of the exocyclic oxygen's interaction with the invariant arginine in the phosphate binding cassette (PBC) and the importance of this interaction for the dynamical properties of the interactions that radiate out from the PBC. The conformations of the phosphate binding cassettes containing two invariant arginine residues (Arg209 on domain A, and Arg333 on domain B) are somewhat different due to the sulfur interacting with this arginine. Furthermore, the B-site ligand together with the entire domain B show significant differences in their overall dynamic properties in the crystal structure of Delta(1-91) RIalpha complexed with (R(p))-cAMPS phosphothioate analogue ((R(p))-RIalpha) compared to the cAMP- and (S(p))-cAMPS-bound type I and II regulatory subunits, based on the temperature factors. In all structures, two structural solvent molecules exist within the A-site ligand binding pocket; both mediate water-bridged interactions between the ligand and the protein. No structured waters are in the B-site pocket. Owing to the higher resolution data, the N-terminal segment (109-117) of the RIalpha subunit can also be traced. This strand forms an intermolecular antiparallel beta-sheet with the same strand in an adjacent molecule and implies that the RIalpha subunit can form a weak homodimer even in the absence of its dimerization domain.

Signaling through dynamic linkers as revealed by PKA

“…S5 A-D showing that several residues in the 141-150 region are more exposed in the Rp-cAMPS-bound form than in the presence of cAMP, similarly to what is observed for R113. However, it would have been challenging to establish the B-state selectivity of the linker/CBD-A contacts solely based on the crystal structures, because R113 is affected by crystal packing (28,33) and R113 interacts with CBD-A also in the R A :C complex (Fig. S6 C and D) (31).…”

“…The C subunit exhibits higher affinity for the H-rather than the B state, whereas cAMP binds the B state more tightly than the H state, i.e., H and B represent the inactive and active forms of R A , respectively. As a result of the B selectivity of cAMP, cAMP binding to apo R A lowers the population of the H state, causing an effective weakening of the R:C interface and leading to the activation of PKA.Whereas the crystal structures of the H and B states of R A (28, 31, 32) have been pivotal in understanding the cAMP-dependent activation of PKA, the allosteric role of the flexible R A linker [i.e., RIα (99-118)] remains elusive due to lack of electron density and to crystal packing for several linker residues (28,32,33). Does the R A linker control the cAMP-dependent activation of PKA?…”

“…Whereas the crystal structures of the H and B states of R A (28, 31, 32) have been pivotal in understanding the cAMP-dependent activation of PKA, the allosteric role of the flexible R A linker [i.e., RIα (99-118)] remains elusive due to lack of electron density and to crystal packing for several linker residues (28,32,33). Does the R A linker control the cAMP-dependent activation of PKA?…”

Crystallization and preliminary X-ray crystallographic characterization of a cyclic nucleotide-binding homology domain from the mouse EAG potassium channel

“…A molecular-replacement solution (log-likelihood gain = 28, Z score = 5.1) was only obtained when an ensemble of six superimposed models created by the Phyre server was provided to Phaser. The six search homology models were created from CNB-domain structures with PDB codes 1q3e, 1vp6 (21% identity; Clayton et al, 2004), 2ptm (17% identity; Flynn et al, 2007), 1cx4 (13% identity; Diller et al, 2001), 1ne6 (18% identity; Wu et al, 2004) and 2pqq (18% identity; Midwest Center for Structural Genomics, unpublished work). Electron-density maps calculated after partial refinement of the molecular-replacement solution in PHENIX (Adams et al, 2010) showed features that were not present in the search models, confirming the validity of the solution.…”

Assay Principle for Modulators of Protein−Protein Interactions and Its Application to Non-ATP-Competitive Ligands Targeting Protein Kinase A

“…To prove probe specificity and demonstrate that this assay is not reporting artifacts such as protein or peptide aggregation, a truncated version of IP20, PKI (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24), was shown to compete with FAM-IP20 for binding to C-subunit in a dose dependent manner, with an IC 50 of 2.7 µM (Figure 2c). …”