Structure of D-AKAP2:PKA RI complex: insights into AKAP specificity and selectivity

Abstract

A-kinase anchoring proteins (AKAPs) regulate cyclic AMP-dependent protein kinase (PKA) signaling in space and time. Dual-specific AKAP 2 (D-AKAP2) binds to the dimerization/docking (D/D) domain of both RI and RII regulatory subunits of PKA with high affinity. Here we have determined the structures of the RIalpha D/D domain alone and in complex with D-AKAP2. The D/D domain presents an extensive surface for binding through a well-formed N-terminal helix, and this surface restricts the diversity of AKAPs that can interact. The structures also underscore the importance of a redox-sensitive disulfide in affecting AKAP binding. An unexpected shift in the helical register of D-AKAP2 compared to the RIIalpha:D-AKAP2 complex structure makes the mode of binding to RIalpha novel. Finally, the comparison allows us to deduce a molecular explanation for the sequence and spatial determinants of AKAP specificity.

Figure 1. The functional domain structures of D-AKAP2 and RIα

The domain structures of RIα (A) and D-AKAP2 (B) are shown. In RI, the Dimerization/Docking (D/D) domain, the inhibitor sequence and the two cAMP-binding domains are labeled. In D-AKAP2, the RGS-like domains, the A-kinase Binding (AKB) region and the C-terminal PDZ motif are indicated. The sequences of the RIα D/D domain and the D-AKAP2 AKB region used for crystallization are shown and numbered. Residues involved in α-helices are indicated by a solid bar and every tenth residue is indicated by a dot. In RIα D/D, green and red stars indicate residues crucial for dimerization and docking to AKAP, respectively. The conserved Cys residues are highlighted in yellow. In D-AKAP2, residues not modeled and modeled as Ala in the structure are indicated in black and blue respectively.

Figure 2. Overview of Apo RIα D/D and RIα D/D:D-AKAP2 structures

(A) Overall structure of Apo RIα D/D showing the anti-parallel, four-helix bundle. The D/D domain monomer is colored gold with the other monomer, generated by the crystallographic 2-fold symmetry, colored brown. The termini and the helices are labeled. The intermolecular disulfide bonds between residues Cys16 and Cys37 are shown as ball-and-stick models and indicated by a black arrow. (B) Stereoview of the 2F_o–F_c density map contoured at 1 ρ_rms (root-mean-square electron density of map often reported as σ) shows clear density for the partially reduced disulfide bond and packing residues. The secondary structure elements and the residues are labeled. (C) Overall structure of the RIα D/D domain complexed with the AKB region of D-AKAP2. The monomers of the D/D domain are depicted in gold and brown and D-AKAP2 is shown in red. The locations of the disulfide bonds are indicated by black arrows. (D) Stereoview of overlay of the complex RIα D/D dimer with the apo RIα D/D. RIα D/D:D-AKAP2 structure is colored as Figure 2C and apo RIα D/D is colored gray. Secondary structural elements and the termini are labeled. Black arrows indicate the position of the disulfide bonds. (E) Correlation of crystallographic B-factors with H/D-exchange protection data. The average main chain B-factors of the two monomers of RIα D/D and the AKB region of D-AKAP2 are plotted against residue number. The sequence of the crystallized proteins and the location of the secondary structural elements are indicated below the plot. Residues not modeled or modeled as Ala in the structure are in black and blue respectively. In previous H/D MS experiments, the core of the AKB region showed increased protection from solvent upon binding to the D/D domain consistent with lower B-factors. At the termini, the lack of protection is consistent with the higher B-factors and weak electron density.

Figure 3. Interactions between RIα D/D and D-AKAP2

(A) Schematic figure detailing the interactions between the side chains of D-AKAP2 and RIα D/D monomers. Solid and dashed arrows indicate hydrophobic and polar interactions respectively. Pockets I-IV are indicated by blue-colored boxes and numbered. (B) Overall structure showing the interaction. The structures are colored as panel (A). The side chains of D-AKAP2 are also shown to indicate the interface of binding. The pockets are indicated by blue-colored boxes and numbered. (C) Close-up views of pockets I through IV correlated with peptide array data. The coloring scheme is similar to Figure 3A. Previous peptide array data (Burns-Hamuro et al., 2003) are shown to highlight the stringent requirements for D-AKAP2 binding to RIα D/D (Copyright 2003, National Academy of Sciences, U.S.A). The surfaces of residues involved in each pocket are shown to highlight the tight packing interactions at the interface. (D) View of additional hydrophobic and polar interactions that stabilize RIα D/D:D-AKAP2 interaction. The coloring scheme is similar to Figure 3A. The residues involved in the hydrophobic interaction are represented as surfaces and circled. Hydrogen bonds between residues are indicated by dashed lines. For orientation, pocket IV residues, V646 and M647, are also shown.

Figure 4. Comparison of RIα D/D:D-AKAP2 and RIIα D/D:D-AKAP2 structures revealing D-AKAP2 specificity

(A) Structural overlay of D-AKAP2 bound to RIα D/D (in red) and to RIIα D/D (in teal; PDB code: 2HWN) (Kinderman et al., 2006) highlighting the shift in the helical register. Pocket residues are labeled. Residues Trp636 and Asp650 at the termini are also shown to facilitate visualization of the register shift. D-AKAP2 bound to RIα (left) and RIIα (right) are also shown to indicate the pocket positions. Pocket residues are represented as surfaces. (B) Structural overlay of RIα D/D:D-AKAP2 complex with the RIIα D/D:D-AKAP2 complex. The RIα D/D complex is colored as Figure 3A. The D/D domain of RIIα is colored light blue and the D-AKAP2 peptide is colored teal. The helices and the termini are labeled with the respective colors. The N-terminal region that houses the disulfide bond in RIα is boxed. (C) A close-up view of the N-terminal region showing the disulfide bond region of RIα D/D and the equivalent residues from RIIα D/D. For clarity, the D-AKAP2 peptides bound to the two structures are not shown. Cys37 is structurally equivalent to Leu21 of RIIα whereas Cys16 does not have any structurally equivalent counterparts. (D) Structure-based alignment of D-AKAP2 sequence bound to RIα and RIIα. The residue numbers of the N- and C-termini of D-AKAP2 are shown and every tenth residue indicated by a black dot. Due to the shift in the helical register, different residues occupy structurally equivalent pockets of RIα and RIIα. Binding pockets are boxed and labeled. Residues that can replace the pocket residues without disrupting the interaction are shown above (for RIα) and below (for RIIα) the alignment in order of preference. (E–G) Alignment of dual-specific (E), RI-specific (F) and RII-specific (G) AKAPs to the aligned D-AKAP2 sequences according to their expected mode of binding to both RIα and RIIα. Dual-specific AKAPs satisfy the requirements for binding to both R subunits whereas RI- or RII-specific AKAPs satisfy criteria to binding to RI or RII subunits.