N3A motifs in RIβ mediate allosteric crosstalk between cAMP and ATP in PKA activation

Abstract

The RIβ subunit of cAMP-dependent protein kinase (PKA) is highly expressed in the brain, yet it remains the least studied of the PKA regulatory subunits (R). As pathologic variants of its gene are increasingly implicated in neurodevelopmental disorders, neurodegeneration, and cancer, gaining more information about the structure/function of RIβ, and how it differs from RIα, has become increasingly important. We previously reported the structure of the RIβ₂C₂ holoenzyme, which revealed a novel conformation where ATP binding was stabilized by a head-to-head anti-parallel packing of the C-tail wrapped around the N-lobe of the catalytic subunit (C). Although visible, the Dimerization/Docking Domain was poorly folded and reduced. Since RIβ is oxidized in brain tissues, we asked if oxidation or binding of an A Kinase Anchoring Protein (AKAP) would affect the holoenzyme structure. Oxidation or addition of an AKAP peptide to crystals led to the release of nucleotide. To capture this at higher resolution we crystallized RIβ₂C₂ in the presence of an AKAP peptide. This new structure represents an RIβ:C heterodimer. Density for the D/D domain was missing; ATP was absent, the kinase adopted an open conformation, and the C-terminus of the RIβ subunit was no longer resolved. Because the crosstalk between ATP and cAMP in the R:C complex appears to be mediated by the two N3A motifs (N3A^A and N3A^B) as well as by the linker, which in free RIβ is intrinsically disordered, we describe the conserved features of these two motifs as well as the linker and show how each contributes in a unique but coordinated way to allosteric activation of RIβ holoenzymes by cAMP. A key difference in our RIβ:C structure is the rotation of the side chain of W260 at the N-terminus of the αA Helix in N3A^B. W260, at the R:C interface in the holoenzyme, is also the capping residue for cAMP bound to CNB-A, so we may have actually captured the first step in cAMP activation.

Keywords: PKA; PRKAR1B; heterodimer structure; holoenzyme.

FIGURE 1

Overall structure of RIβ:C heterodimer. (a) Cartoon representation of the RIβ:C heterodimer structure. (b) Surface representation of the RIβ:C heterodimer structure, with a 180° rotation on the far right. The color coding is the same as (a). (c) G‐loop is in a partially open conformation, and the ATP is missing in RIβ:C structure. The C‐subunit are shown in a surface representation; N‐lobe is in white and C‐lobe in tan. The C‐tail, the residues T300 to F350, and the G‐loop are shown in red. Two key residues F327 and Y330 are highlighted. (d) Comparison of the G‐loop in RIβ:C (red) with a fully closed (dark blue, PDB: 1ATP) and open (cyan, PDB: 1J3H) conformations. The same color coding was applied for all the following figures: The N‐ and C‐lobe of C‐subunit are shown in white and tan, the CNB‐A and CNB‐B domains of RIβ are in dark blue and light blue, the Inhibitor site in red, the PBC motif in golden, the Linker in dark yellow, αB/C/N helix in dark red, respectively. To highlight the main focus of each figure, we also colored those elements in red while keeping the other elements in this color code.

FIGURE 2

RIβ Inhibitor site docks into the active site cleft of C‐subunit. (a) The Inhibitor site (red) docks to the C‐subunit. The C‐subunit is shown as a surface representation, with N‐ and C‐lobes in white and tan, respectively. The P‐2 Arg and αB/C/N helix (dark blue) are also shown. (b) Zoom‐in view of C‐linker. The C‐linker wraps around the αB/C/N helix, some key residues from linker to N3A^A are highlighted. (c) Sequence alignment of the Inhibitor site. The green arrow marks the first residue, A91, in this RIβ:C structure. Four key C‐linker residues are labeled in red circles. (d) Structural alignment of Inhibitor sites. The N‐linkers have different conformations in RIα:C (purple) and RIβ:C (red), whereas the Inhibitor sites and C‐linkers are superimposed well. (e) The same view as (d) when docking into the C‐subunit. (f) The Inhibitor site of RIβ:C, some key residues are also shown.

FIGURE 3

The N3A motifs nucleate multiple protein: protein interfaces and are stable rigid bodies. (a) Crystal packing of RIβ:C heterodimer structure. RIβ:C is shown in cartoon, and RIβ:C′ in surface representation. The color coding are the same as Figure 1a. (b) The N3A^A:N3A^A′ dimer interface. Four residues on the interface, Y120, K121, R144, and S145 are labeled. The sequence alignment of the N3A^A in RIα and RIβ are shown on the bottom. (c) The interface between N3A^A and PBC^A. The hydrophobic shells are colored in sand, and H‐bonds are labeled with black lines. (d) N3A^A is a stable rigid body. The hydrophobic shell within N3A^A is colored in sand. The black arrows show the different domain interfaces of N3A^A. (e) The interface between N3A^A and C‐subunit. V134 and H138 (red shell) hydrophobically pack to αG helix of C‐subunit (sand shell). (f) The interface between N3A^B and PBC^B. (g) N3A^B is also a stable rigid body, the hydrophobic shells are in sand.

FIGURE 4

C‐terminal Tail in RIβ subunit is disordered. (a) The C‐tail of RIβ and ATP in C‐subunit are ordered in the structure of RIβ₂C₂ holoenzyme (PDB: 4DIN). C‐subunit is shown as surface representation with its C‐tail and ATP in black. The CNB‐A domain is colored in dark blue with N3A^A in dark red, and the CNB‐B domain in light blue with N3A^B in red, respectively. (b) RIβ:C heterodimer in the same view and color coding as (a). Residues 367–379 at the C‐terminus is not visible, marked by a yellow arrow. (c) Secondary structure elements near N3A^B are shown in cartoon. (d) Zoom‐in view of N3A^B motif in RIβ₂C₂ structure. R370 from C″‐helix forms several H‐bonds to 3₁₀ loop of N3A^B. PBC^B is colored in golden. W260 (sand shell), as well as E261 H‐bonding to R366 is also shown. (e) N3A^B motif in RIβ:C structure, the same view as (d).

FIGURE 5

Different functional roles of N3A motifs. N3A^A motif (dark red) serves as a dimer interface. N3A^B motif (red) is on the R:C interface with the Activation loop of C‐subunit. The yellow arrows show the binding sites of cAMP and ATP (black) in RIβ₂C₂ structure. Top inset shows the ATP is missing from the RIβ:C structure. Bottom inset (right) shows the R:C interface of N3A^B in RIβ₂C₂, whereas (left) shows the same interface in RIβ:C structure. W260 (sand shell) has a side‐chain flip, and R194‐D267 interactions were broken.

FIGURE 6

The extended BCN helix joining the two CNB‐domains. (left) The RIβ:C structure shown in surface representation (H‐form). The N‐ and C‐lobe of C‐subunit are shown in white and tan, the CNB‐A and CNB‐B domains of RIβ are in dark blue and light blue, respectively. The extended BCN helix is in red. (right) The cAMP bound RIα structure shown in surface representation (B‐form). (middle) The structural comparison of BCN helices in two states.