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1 Departments of Chemistry & Biochemistry, 2 Department of Medicine and Biomedical Sciences Graduate Program, and 3 Howard Hughes Medical Institute, University of California at San Diego, La Jolla, California 92093, USA
4 Sierra Analytics, Modesto, California 95356, USA
(RECEIVED July 7, 2005; FINAL REVISION September 8, 2005; ACCEPTED September 12, 2005)
The structure of an AKAP docked to the dimerization/docking (D/D) domain of the type II (RII
) 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 (RI) 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 back-bone structural changes of an -helical A-kinase binding (AKB) motif from D-AKAP2 docked to both RI and RII 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 RI compared to RII, 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 RI indicated that there were more binding constraints for the AKB ligand when bound to RI. This was in contrast to RII, which has a preformed, localized binding surface. These distinct modes of AKAP binding may contribute to the more discriminating nature of the RI 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.
Keywords: DXMS; PKA; D-AKAP2; isoform diversity; hydrogen/deuterium exchange; mass spectrometry
Abbreviations: PKA, protein kinase A • AKAP, A kinase anchoring protein • DAKAP, dual specific A kinase anchoring protein • AKB, A kinase binding • R, regulatory subunit of protein kinase A • D/D, dimerization/docking domain of regulatory subunit • RGS, regulators of G-protein signaling • DXMS, deuterium exchange mass spectrometry • GdnHCl, guanidine hydrochloride • TFA, trifluoracidic acid.
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051687305.
Reprint requests to: Virgil L. Woods Jr., Department of Medicine, University of California San Diego, Department 0656, 9500 Gilman Drive, La Jolla, CA 92093-0656, USA; e-mail: vwoods{at}ucsd.edu; fax: (858) 534-2606.
5 These authors contributed equally to this work.
6 Present addresses: L.L. Burns-Hamuro, Provid Pharmaceuticals, 671 US Route 1 South, North Brunswick, NJ 08902, USA
7 Y. Hamuro, ExSAR Corporation, 11 Deer Park Drive, Suite 103, Monmouth Junction, NJ 08852, USA.
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