Distinct interaction modes of an AKAP bound to two regulatory subunit isoforms of protein kinase A revealed by amide hydrogen/deuterium exchange.
The structure of an AKAP docked to the dimerization/docking (D/D) domain of the type II (RIIalpha) 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 (RIalpha) 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 alpha-helical A-kinase binding (AKB) motif from D-AKAP2 docked to both RIalpha and RIIalpha 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 RIalpha compared to RIIalpha, 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 RIalpha indicated that there were more binding constraints for the AKB ligand when bound to RIalpha. This was in contrast to RIIalpha, which has a preformed, localized binding surface. These distinct modes of AKAP binding may contribute to the more discriminating nature of the RIalpha 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.
Burns-Hamuro
Lora L
LL
Department of Medicine, University of California at San Diego, Department 0656, 9500 Gilman Drive, La Jolla, CA 92093-0656, USA.
Hamuro
Yoshitomo
Y
Kim
Jack S
JS
Sigala
Paul
P
Fayos
Rosa
R
Stranz
David D
DD
Jennings
Patricia A
PA
Taylor
Susan S
SS
Woods
Virgil L
VL
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D000577:Amides / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D041961:Deuterium Exchange Measurement; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; D000577:Amides; D008024:Ligands; D008565:Membrane Proteins; C111169:Pakap protein, mouse; D020033:Protein Isoforms; D021122:Protein Subunits; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases