Compartmentalization of the cAMP-dependent protein kinase (PKA) is coordinated through association with A-kinase anchoring proteins (AKAPs). A defining characteristic of most AKAPs is a 14-to 18-aa sequence that binds to the regulatory subunits (RI or RII) of the kinase. Cellular delivery of peptides to these regions disrupts PKA anchoring and has been used to delineate a physiological role for AKAPs in the facilitation of certain cAMP-responsive events. Here, we describe a bioinformatic approach that yields an RIIselective peptide, called AKAP-in silico (AKAP-IS), that binds RII with a Kd of 0.4 nM and binds RI with a Kd of 277 nM. AKAP-IS associates with the type II PKA holoenzyme inside cells and displaces the kinase from natural anchoring sites. Electrophysiological recordings indicate that perfusion of AKAP-IS evokes a more rapid and complete attenuation of ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor currents than previously described anchoring inhibitor peptides. Thus, computerbased and peptide array screening approaches have generated a reagent that binds PKA with higher affinity than previously described AKAPs.

The structure of an AKAP docked to the dimerization/docking (D/D) domain of the type II (RIIa) isoform of protein kinase A (PKA) has been well characterized, but there currently is no detailed structural information of an AKAP docked to the type I (RIa) isoform. Dual-specific AKAP2 (D-AKAP2) binds in the nanomolar range to both isoforms and provided us with an opportunity to characterize the isoform-selective nature of AKAP binding using a common docked ligand. Hydrogen/ deuterium (H/D) exchange combined with mass spectrometry (DXMS) was used to probe backbone structural changes of an a-helical A-kinase binding (AKB) motif from D-AKAP2 docked to both RIa and RIIa D/D domains. The region of protection upon complex formation and the magnitude of protection from H/D exchange were determined for both interacting partners in each complex. The backbone of the AKB ligand was more protected when bound to RIa compared to RIIa, suggesting an increased helical stabilization of the docked AKB ligand. This combined with a broader region of backbone protection induced by the AKAP on the docking surface of RIa indicated that there were more binding constraints for the AKB ligand when bound to RIa. This was in contrast to RIIa, which has a preformed, localized binding surface. These distinct modes of AKAP binding may contribute to the more discriminating nature of the RIa AKAP-docking surface. DXMS provides valuable structural information for understanding binding specificity in the absence of a high-resolution structure, and can readily be applied to other protein-ligand and protein-protein interactions.

The first direct NMR determination of the conformation of a conformationally flexible heparin-like hexasaccharide bound to a key receptor, FGF-1, is described. The determination has been based on the use of a 13C-labeled protein and a regular 12C sugar. FGF-1 recognizes several conformations of the iduronic moieties of the hexasaccharide. Therefore, this case is different than that described for the controversial recognition of heparin-like saccharides by AT-III, which seems to recognize just one conformation of the iduronic acid residues.