A novel mechanism of PKA anchoring revealed by solution structures of anchoring complexes

Abstract

The specificity of intracellular signaling events is controlled, in part, by compartmentalization of protein kinases and phosphatases. The subcellular localization of these enzymes is often maintained by protein- protein interactions. A prototypic example is the compartmentalization of the cAMP-dependent protein kinase (PKA) through its association with A-kinase anchoring proteins (AKAPs). A docking and dimerization domain (D/D) located within the first 45 residues of each regulatory (R) subunit protomer forms a high affinity binding site for its anchoring partner. We now report the structures of two D/D-AKAP peptide complexes obtained by solution NMR methods, one with Ht31(493-515) and the other with AKAP79(392-413). We present the first direct structural data demonstrating the helical nature of the peptides. The structures reveal conserved hydrophobic interaction surfaces on the helical AKAP peptides and the PKA R subunit, which are responsible for mediating the high affinity association in the complexes. In a departure from the dimer-dimer interactions seen in other X-type four-helix bundle dimeric proteins, our structures reveal a novel hydrophobic groove that accommodates one AKAP per RIIalpha D/D.

Fig. 1. Backbone fold and AKAP peptide orientation of RIIα(1–44) with either Ht31(493–515) (views A–D) or AKAP79(392–413) (views E–H) bound. (A–D) Views of RIIα(1–44) (protomers in red and blue) and Ht31(493–515) in green are depicted. These views highlight two orientations of the best fit superposition of the backbone heavy atoms of the 13 lowest energy structures (A and B) or the lowest energy structure (C and D). The views (B) and (D) clarify the AKAP binding surface. (E–H) Similar views of RIIα(1–44) in red and blue and AKAP79(392–413) in yellow are shown. The best fit superposition of the 10 lowest energy structures (E and F) and the lowest energy structure (G and H) are represented. The two different views are shown in (A), (C), (E) and (G), and (B), (D), (F) and (H).

Fig. 2. Representation of the hydrophobic core of RIIα(1–44) in both the free and AKAP-bound structures. The anchoring peptides have been removed for clarity and the first eight or four residues of RIIα are removed. Superposition of the 17, 13 and 10 lowest energy structures of free, Ht31(493–515)- and AKAP79(392–413)-bound RIIα(1–44), respectively. In all views, RIIα(1–44) is colored gray. Hydrophobic residues in free RIIα(1–44) are colored yellow and in both AKAP complexes are colored green. (A) This view emphasizes both the organization of the hydrophobic core and the hydrophobic groove of RIIα(1–44). (B) This view highlights the AKAP binding surface and is a 90° rotation of the view presented in (A). (C) This view is identical to (A) although the backbone atoms of residues 5–8 and 5′–8′ are indicated in either purple or red for the complex or apo-form of RIIα(1–44), respectively. Additionally, the side chain of Ile5 is also depicted and colored.

Fig. 3. Similarity of the AKAP structure and binding region on RIIα(1–44) in the Ht31 and AKAP79 complexes. The superposition in RIIα(1–44), with the protomers of RIIα D/D shown in gray and blue, reveals the similarity in the backbone fold of the two complexes. The first 17 residues of the RII binding region in both Ht31(493–515) (red) and AKAP79(392–413) (yellow) are indicated, and bind to identical regions with similar helical structures to RIIα(1–44).

Fig. 4. AKAP sequence alignment, helical wheel analysis of Ht31 and AKAP79 and structural helical overlay of Ht31 and AKAP79. (A) The alignment of the AKAP binding region (18 residues) of 13 known AKAPs is shown. The first two, Ht31 and AKAP79, are shown in red for emphasis. Conserved hydrophobic positions are shown in green. (B) Helical wheel analysis of the AKAP binding region of both Ht31 and AKAP79. The conserved hydrophobic positions as highlighted in (A) are colored green and map to a well defined space. (C) An overlay of the structures of Ht31 (red) and AKAP79 (yellow) when in RIIα are deleted for clarity. The conserved hydrophobic positions, as shown in (A) and (B), are colored either blue (Ht31) or green (AKAP79).

Fig. 5. NOE contacts between RIIα and Ht31 and AKAP79. (A and B) The NOE contacts for each residue on the Ht31 (green) or AKAP79 (purple) to RIIα (A) or on RIIα to each of the AKAPs (B). (C and D) Structural view of the NOE contacts. RIIα is colored yellow and gray for each protomer. Ht31 (C) is colored green and AKAP79 (D) is colored purple. All of the residues that have observed NOE contacts are indicated.

Fig. 6. Stereoview representation of the AKAP binding surface of RIIα using Insight software (MSI, San Diego, CA). RIIα(1–44) protomers are colored yellow and pink, respectively. All hydrophobic residues in RIIa, Ht31 and AKAP79 are colored green. Acidic and basic residues are red and blue, respectively. (A) Ht31 lies above RIIα, with the hydrophobic face that contacts RIIα facing the hydrophobic face of RIIα. Additional residues in Ht31 are colored pink. (B) AKAP79 is shown in this figure with the hydrophobic face in similar orientation to RIIα to that of Ht31 in (A). Additional residues in Ht31 are colored pink.

Fig. 7. Schematic diagram of protein–protein interactions mediated by X-type four-helix bundles. RIIα is unique in binding one protomer per dimer at a surface hydrophobic groove. HNF-1α presents a hydrophobic face and forms a dimer of dimers. Likewise, the stoichiometry of P53 binding to S100 is two peptides per dimer. The binding interactions with SAM are under investigation.