While the cAMP-bound structures of RIα and RIIβ revealed many conserved features of the R subunits, there were other conserved residues that were not explained. These structures also did not explain how the C-subunit is inhibited by the R-subunit, nor did they reveal the mechanism for cAMP-induced activation of the holoenzymes. Finally, they did not explain the fundamental differences between the isoforms. To appreciate these features, it was necessary to solve structures of holoenzyme complexes. While the CBDs are quite stable in their cAMP-bound conformation, as is the D/D domain, the linker region containing the inhibitor site is very mobile in solution [24
]. The linker region, for example, was always present but disordered in the crystal structures of cAMP-bound RIα(91-379) and RIIβ(91-400) where both R-subunits were monomeric and lacked the D/D domain. Crystallization of a full length R-subunit dimer has never been achieved. To obtain a structure of a complex between the R and C subunit required some novel strategies. Previous studies showed that the smallest stable deletion mutant of RIα that bound both cAMP and C-subunit with high affinity was RIα(91-244) [25
]. This construct contained the inhibitor site in the linker as well as most of Domain A. Hydrogen/deuterium exchange, coupled with mass spectrometry (H/DMS) allowed us to map the interface between the R and C-subunits [26
], but this did not provide a detailed picture of the molecular interactions. The structure of this complex, shown in , crystallized in the presence of a non-hydrolyzable analog of ATP, AMP-PNP, allowed us for the first time to understand how the C-subunit is actually inhibited by the R-subunit [28
]. This holoenzyme complex showed two striking features, in addition to the disorder/order transition of the inhibitor site in the linker region which now becomes embedded at the active site in the cleft between the two lobes of the C-subunit. First is that the C-subunit serves as a stable scaffold for binding of the R-subunit using a large surface area that derives mostly from its large lobe. Second is that the R-subunit undergoes major conformational reorganization as it releases cAMP and wraps around the C-subunit. This change involves a reorganization of the two CBD domains as well as major changes within the α subdomain of each CBD. The details of this interaction are summarized below.
Models of the catalytic subunit bound to different inhibitors
The RIα subunit, like PKI, requires Mg2
ATP to bind tightly to the C-subunit (Kd = 0.1 nM). As in the case of PKI [15
], the C-subunit in the holoenzyme complex with RIα(91-244) assumes a fully closed conformation where the C-tail is folded over onto the core and the inhibitor site is wedged tightly in the cleft between the small and large lobes of the kinase core. Docking of the inhibitor peptide (Arg-Arg-Gly-Ala-Ile) to the active site cleft is thus a critical event that not only occludes the active site cleft thereby preventing binding of other substrates but also nucleates the large binding interface between the R and C subunits. For RIα, which is a pseudosubstrate with an Ala at the P Site, docking of this peptide also forces the molecule into a fully closed conformation. Essentially, the Ala pulls the entire N-lobe with its bound ATP into a closed conformation because there is no place to transfer the γ-phosphate of ATP. The C-tail, anchored to ATP through Phe327 and Tyr330, is dragged along with the N-lobe. The central portion of this interface is hydrophobic and ordering of the P+1 Ile is critical for bringing together two important hydrophobic motifs, one from the RIα-subunit and one from the C-subunit (). From the C-subunit, it is the αG Helix that provides a critical tyrosine, Tyr247. This αG-helix is exposed to solvent in the free C-subunit but protected from H/D exchange in the holoenzyme complex [27
]. The H/D studies thus provided the first clue that this motif was part of the R/C interface. The PBC in RIα also contributes to this interface. Tyr205 at the tip of the PBC provides the other essential contact. The convergence of Tyr205 in RIα, Tyr247 in the C-subunit, and Ile99 in the Inhibitor Site of RIα defines an essential triad that distorts the PBC in Domain A. cAMP, a small ligand, and the C-subunit, a large protein, are thus both competing for the PBC in Domain A of RIα.
The active site cleft of the catalytic subunit is filled by the pseudosubstrate inhibitor segment of RIα
The surface of the C-subunit, especially the large lobe, serves as an extended stable scaffold for binding of the R-subunit. With the exception of closing the active site cleft, the conformation of the C-subunit remains mostly unchanged. The surface that is masked by the binding of RIα extends from the Activation Loop (residues 191-197) through the αG Helix (residues 244 – 252). The Activation Loop thus serves many roles. Not only does it stabilize the active conformation of the kinase so that it is optimal for catalysis [29
], it also provides a docking surface for the RIα subunit. This is likely to be a general feature for most kinases – one side of the Activation Loop reaches out to stabilize the active site through phosphorylation of a residue that is equivalent to Thr197 while the other side faces outward towards the solvent where it serves as a docking site for binding to other proteins, either substrates or inhibitors. We believe that the αG Helix will also be a docking motif for most protein kinases [31
]. Unlike the αE, αF, and αH Helices, which are buried in the hydrophobic core of the large lobe and not readily accessible to deuterium exchange, the αG Helix is solvent exposed in the free C-subunit but shielded from solvent in the holoenzyme.