Distinct interaction modes of an AKAP bound to two regulatory subunit isoforms of protein kinase A revealed by amide hydrogen/deuterium exchange (original) (raw)
Structure of D-AKAP2:PKA RI Complex: Insights into AKAP Specificity and Selectivity
Structure, 2010
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 RIa 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 RIIa:D-AKAP2 complex structure makes the mode of binding to RIa novel. Finally, the comparison allows us to deduce a molecular explanation for the sequence and spatial determinants of AKAP specificity.
Journal of Molecular Biology, 2000
Compartmentalization of cAMP-dependent protein kinase (PKA) is in part mediated by specialized protein motifs in the dimerization domain of the regulatory (R)-subunits of PKA that participate in protein-protein interactions with an amphipathic helix region in A-kinase anchoring proteins (AKAPs). In order to develop a molecular understanding of the subcellular distribution and speci®c functions of PKA isozymes mediated by association with AKAPs, it is of importance to determine the apparent binding constants of the R-subunit-AKAP interactions. Here, we present a novel approach using surface plasmon resonance (SPR) to examine directly the association and dissociation of AKAPs with all four R-subunit isoforms immobilized on a modi®ed cAMP surface with a high level of accuracy. We show that both AKAP79 and S-AKAP84/D-AKAP1 bind RIIa very well (apparent K D values of 0.5 and 2 nM, respectively). Both proteins also bind RIIb quite well, but with three-to fourfold lower af®nities than those observed versus RIIa. However, only S-AKAP84/D-AKAP1 interacts with RIa at a nanomolar af®nity (apparent K D of 185 nM). In comparison, AKAP95 binds RIIa (apparent K D of 5.9 nM) with a tenfold higher af®nity than RIIb and has no detectable binding to RIa. Surface competition assays with increasing concentrations of a competitor peptide covering amino acid residues 493 to 515 of the thyroid anchoring protein Ht31, demonstrated that Ht31, but not a proline-substituted peptide, Ht31-P, competed binding of RIIa and RIIb to all the AKAPs examined (EC 50-values from 6 to 360 nM). Furthermore, RIa interaction with S-AKAP84/D-AKAP1 was competed (EC 50 355 nM) with the same peptide. Here we report for the ®rst time an approach to determine apparent rate-and equilibria binding constants for the interaction of all PKA isoforms with any AKAP as well as a novel approach for characterizing peptide competitors that disrupt PKA-AKAP anchoring.
Biochemical Journal, 2006
PKA (protein kinase A) is tethered to subcellular compartments by direct interaction of its regulatory subunits (RI or RII) with AKAPs (A kinase-anchoring proteins). AKAPs preferentially bind RII subunits via their RII-binding domains. RII-binding domains form structurally conserved amphipathic helices with unrelated sequences. Their binding affinities for RII subunits differ greatly within the AKAP family. Amongst the AKAPs that bind RIIα subunits with high affinity is AKAP7δ [AKAP18δ; K d (equilibrium dissociation constant) value of 31 nM]. An N-terminally truncated AKAP7δ mutant binds RIIα subunits with higher affinity than the full-length protein presumably due to loss of an inhibitory region [Henn, Edemir, Stefan, Wiesner, Lorenz, Theilig, Schmidtt, Vossebein, Tamma, Beyermann et al. (2004) J. Biol. Chem. 279, 26654-26665]. In the present study, we demonstrate that peptides (25 amino acid residues) derived from the RII-binding domain of AKAP7δ bind RIIα subunits with higher affinity (K d = 0.4 + − 0.3 nM) than either fulllength or N-terminally truncated AKAP7δ, or peptides derived from other RII binding domains. The AKAP7δ-derived peptides and stearate-coupled membrane-permeable mutants effectively disrupt AKAP-RII subunit interactions in vitro and in cell-based assays. Thus they are valuable novel tools for studying anchored PKA signalling. Molecular modelling indicated that the high affinity binding of the amphipathic helix, which forms the RIIbinding domain of AKAP7δ, with RII subunits involves both the hydrophobic and the hydrophilic faces of the helix. Alanine scanning (25 amino acid peptides, SPOT technology, combined with RII overlay assays) of the RII binding domain revealed that hydrophobic amino acid residues form the backbone of the interaction and that hydrogen bond-and salt-bridge-forming amino acid residues increase the affinity of the interaction.
A Dynamic Mechanism for AKAP Binding to RII Isoforms of cAMP-Dependent Protein Kinase
Molecular Cell, 2006
A kinase-anchoring proteins (AKAPs) target PKA to specific microdomains by using an amphipathic helix that docks to N-terminal dimerization and docking (D/D) domains of PKA regulatory (R) subunits. To understand specificity, we solved the crystal structure of the helical motif from D-AKAP2, a dual-specific AKAP, bound to the RIIa D/D domain. The 1.6 Å structure reveals how this dynamic, hydrophobic docking site is assembled. A stable, hydrophobic docking groove is formed by the helical interface of two RIIa protomers. The flexible N terminus of one protomer is then recruited to the site, anchored to the peptide through two essential isoleucines. The other N terminus is disordered. This asymmetry provides greater possibilities for AKAP docking. Although there is strong discrimination against RIa in the N terminus of the AKAP helix, the hydrophobic groove discriminates against RIIa. RIa, with a cavity in the groove, can accept a bulky tryptophan, whereas RIIa requires valine.
Domain Organization of D-AKAP2 Revealed by Enhanced Deuterium Exchange-Mass Spectrometry (DXMS
Journal of Molecular Biology, 2002
Dual specific A-kinase anchoring protein 2 (D-AKAP2) is a scaffold protein that coordinates cAMP-mediated signaling complexes by binding to type I and type II protein kinase A (PKA). While information is unfolding regarding specific binding motifs, very little is known about the overall structure and dynamics of these scaffold proteins. We have used deuterium exchange-mass spectrometry (DXMS) and limited proteolysis to probe the folded regions of D-AKAP2, providing for the first time insight into the intra-domain dynamics of a scaffold protein. Deuterium on-exchange revealed two regions of low deuterium exchange that were surrounded by regions of high exchange, suggestive of two distinctly folded regions, flanked by disordered or solvent accessible regions. Similar folded regions were detected by limited proteolysis. The first folded region contained a putative regulator of G-protein signaling (RGS) domain. A structural model of the RGS domain revealed that the more deuterated regions mapped onto loops and turns, whereas less deuterated regions mapped onto a-helices, consistent with this region folding into an RGS domain. The second folded region contained a highly protected PKA binding site and a more solvent-accessible PDZ binding motif, which may serve as a potential targeting domain for D-AKAP2. DXMS has verified the multi-domain architecture of D-AKAP2 implied by sequence homology and has provided unique insight into the accessibility of the PKA binding site.
Journal of Biological Chemistry, 1996
Compartmentalization of the type II cAMP-dependent protein kinase is conferred by interaction of the regulatory subunit (RII) with A-Kinase Anchoring Proteins (AKAPs). The AKAP-binding site involves amino-terminal residues on each RII protomer and is formed through dimerization. A site-directed mutagenesis strategy was utilized to assess the contribution of individual residues in either RII isoform, RII␣ or RII, for interaction with various anchoring proteins. Substitution of long-chain or bulky hydrophobic groups (leucines or phenylalanines) for isoleucines at positions 3 and 5 in RII␣ decreased AKAP-binding up to 24 ؎ 3 (n ؍ 8)-fold, whereas introduction of valines had minimal effects. Replacement with hydrophilic residues (serine or asparigine) at both positions abolished AKAP binding. Mutation of proline 6 in RII␣ reduced binding for four AKAPs (Ht31, MAP2, AKAP79, and AKAP95) from 2.3 to 20-fold (n ؍ 4) whereas introduction of an additional proline at position 6 in RII increased or conferred binding toward these anchoring proteins. Therefore, we conclude that -branched side chains at positions 3 and 5 are favored determinants for AKAP-binding and prolines at positions 6 and 7 increase or stabilize RII␣ interaction with selected anchoring proteins.
Novel Isoform-Specific Interfaces Revealed by PKA RIIβ Holoenzyme Structures
Journal of Molecular Biology, 2009
The PKA catalytic (C) subunit is inhibited by two classes of functionally non-redundant regulatory subunits, RI and RII. Unlike RI-subunits, RII-subunits are both substrates and inhibitors. Because RIIβ knockout mice have important disease phenotypes, the RIIβ holoenzyme is a target for developing isoform-specific agonists and/or antagonists. We also know little about the linker region that connects the inhibitor site to the N-terminal dimerization domain although this linker determines the unique globular architecture of the RIIβ holoenzyme. To understand how RIIβ functions as both an inhibitor and substrate and to elucidate the structural role of the linker, we engineered different RIIβ constructs. In the absence of nucleotide RIIβ(108-268) that contains a single cyclic nucleotide binding domain bound C-subunit poorly whereas with AMP-PNP, a nonhydrolyzable ATP analog, the affinity was 11nM. The RIIβ(108-268) holoenzyme structure (1.62Å) with AMP-PNP/Mn 2+ , showed that we trapped the RIIβ-subunit in an enzyme:substrate complex with the C-subunit in a closed conformation. The enhanced affinity afforded by AMP-PNP/Mn 2+ may be a useful strategy for increasing affinity and trapping other protein substrates with their cognate protein kinase. Because mutagenesis predicted that the region N-terminal to the inhibitor site might dock differently to RI and RII, we also engineered RIIβ(102-265) that contained six additional linker residues. The additional linker residues in RIIβ(102-265) increased the affinity to 1.6 nM suggesting that docking to this surface may also enhance catalytic efficiency. In the corresponding holoenzyme structure this linker docks as an extended strand onto the surface of the large lobe. This hydrophobic pocket, formed by the αF-αG loop and conserved in many protein kinases, also provides a docking site for the amphipathic helix of PKI. This novel orientation of the linker peptide provides the first clues as to how this region contributes to the unique organization of the RIIβ holoenzyme.
We employ ensemble docking simulations to characterize the interactions of two enantiomeric forms of a Ru-complex compound (1-R and 1-S) with three protein kinases, namely PIM1, GSK-3β, and CDK2/cyclin A. We show that our ensemble docking computational protocol adequately models the structural features of these interactions and discriminates between competing conformational clusters of ligand-bound protein structures. Using the determined X-ray crystal structure of PIM1 complexed to the compound 1-R as a control, we discuss the importance of including the protein flexibility inherent in the ensemble docking protocol, for the accuracy of the structure prediction of the bound state. A comparison of our ensemble docking results suggests that PIM1 and GSK-3β bind the two enantiomers in similar fashion, through two primary binding modes: conformation I, which is very similar to the conformation presented in the existing PIM1/compound 1-R crystal structure; conformation II, which represents a 180°flip about an axis through the NH group of the pyridocarbazole moiety, relative to conformation I. In contrast, the binding of the enantiomers to CDK2 is found to have a different structural profile including a suggested bound conformation, which lacks the conserved hydrogen bond between the kinase and the ligand (i.e., ATP, staurosporine, Ru-complex compound). The top scoring conformation of the inhibitor bound to CDK2 is not present among the topscoring conformations of the inhibitor bound to either PIM1 or GSK-3β and vice-versa. Collectively, our results help provide atomic-level insights into inhibitor selectivity among the three kinases.
Molecular Basis of AKAP Specificity for PKA Regulatory Subunits
Molecular Cell, 2006
Localization of cyclic AMP (cAMP)-dependent protein kinase (PKA) by A kinase-anchoring proteins (AKAPs) restricts the action of this broad specificity kinase. The high-resolution crystal structures of the docking and dimerization (D/D) domain of the RIIa regulatory subunit of PKA both in the apo state and in complex with the high-affinity anchoring peptide AKAP-IS explain the molecular basis for AKAP-regulatory subunit recognition. AKAP-IS folds into an amphipathic a helix that engages an essentially preformed shallow groove on the surface of the RII dimer D/D domains. Conserved AKAP aliphatic residues dominate interactions to RII at the predominantly hydrophobic interface, whereas polar residues are important in conferring R subunit isoform specificity. Using a peptide screening approach, we have developed SuperAKAP-IS, a peptide that is 10,000-fold more selective for the RII isoform relative to RI and can be used to assess the impact of PKA isoform-selective anchoring on cAMPresponsive events inside cells.