Domain organization of D-AKAP2 revealed by enhanced deuterium exchange-mass spectrometry (DXMS).

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 alpha-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.

Isoform specific differences in binding of a dual-specificity A-kinase anchoring protein to type I and type II regulatory subunits of PKA.

Dual-specificity AKAPs bind to type I (RI) and type II (RII) regulatory subunits of cAMP-dependent protein kinase A (PKA), potentially recruiting distinct cAMP responsive holoenzymes to a given intracellular location. To understand the molecular basis for this "dual" functionality, we have examined the pH-dependence, the salt-dependence, and the kinetics of binding of the A-kinase binding (AKB) domain of D-AKAP2 to the regulatory subunit isoforms of PKA. Using fluorescence anisotropy, we have found that a 27-residue peptide corresponding to the AKB domain of D-AKAP2 bound 25-fold more tightly to RIIalpha than to RIalpha. The higher affinity for RIIalpha was the result of a slower off-rate as determined by surface plasmon resonance. The high-affinity interaction for RIalpha and RIIalpha was pH-independent from pH 7.4 to 5.0. At pH 4.0, both isoforms had a reduction in binding affinity. Additionally, binding of the AKB domain to RIalpha was independent of solution ionic strength, whereas RIIalpha had an increased binding affinity at higher ionic strength. This suggests that the relative energetic contribution of the charge stabilization is different for the two isoforms. This prediction was confirmed by mutagenesis in which acidic mutations, primarily of E10 and D23, in the AKB domain affected binding to RIalpha but not to RIIalpha. These isoform-specific differences provide a foundation for developing isoform-specific peptide inhibitors of PKA anchoring by dual-specificity AKAPs, which can be used to evaluate the physiological significance of dual-specificity modes of PKA anchoring.

Induction of flexibility through protein-protein interactions.

The dimerization/docking (D/D) domain of the cyclic AMP-dependent protein kinase (PKA) holoenzyme mediates important protein-protein interactions that direct the subcellular localization of the enzyme. A kinase anchoring proteins (AKAPs) provide the molecular scaffold for the localization of PKA. The recent solution structures of two D/D AKAP complexes revealed that the AKAP binds to a surface-exposed, hydrophobic groove on the D/D. In the present study, we present an analysis of the changes in hydrogen/deuterium exchange protection and internal motions of the backbone of the D/D when free and bound to the prototype anchoring protein, Ht31(pep). We observe that formation of the complex results in significant, but small, increases in H/D exchange protection factors as well as increases in backbone flexibility, throughout the D/D, and in particular, in the hydrophobic binding groove. This unusual observation of increased backbone flexibility and marginal H/D exchange protection, despite high affinity protein-ligand interactions, may be a general effect observed for the stabilization of hydrophobic ligand/hydrophobic pocket interactions.