3IIA

Crystal structure of apo (91-244) RIa subunit of cAMP-dependent protein kinase

Summary for 3IIA

• Entry DOI: 10.2210/pdb3iia/pdb
• Related: 1NE4 1NE6 1RGS 1RL3 3FHI
• Descriptor: cAMP-dependent protein kinase type I-alpha regulatory subunit, GLYCEROL (3 entities in total)
• Functional Keywords: protein kinase a, cyclic amp, ria subunit, camp, camp-binding, disulfide bond, nucleotide-binding, phosphoprotein, transferase
• Biological source: Bos taurus (bovine)
• Cellular location: Cell membrane : P00514
• Total number of polymer chains: 1
• Total formula weight: 17431.66

Authors

Sjoberg, T.J.,Kim, C.,Kornev, A.P.,Taylor, S.S. (deposition date: 2009-07-31, release date: 2010-08-11, Last modification date: 2023-09-06)

Primary citation

Badireddy, S.,Yunfeng, G.,Ritchie, M.,Akamine, P.,Wu, J.,Kim, C.W.,Taylor, S.S.,Qingsong, L.,Swaminathan, K.,Anand, G.S.
Cyclic AMP analog blocks kinase activation by stabilizing inactive conformation: conformational selection highlights a new concept in allosteric inhibitor design.
Mol.Cell Proteomics, 10:M110.004390-M110.004390, 2011

PubMed Abstract: The regulatory (R) subunit of protein kinase A serves to modulate the activity of protein kinase A in a cAMP-dependent manner and exists in two distinct and structurally dissimilar, end point cAMP-bound "B" and C-subunit-bound "H"-conformations. Here we report mechanistic details of cAMP action as yet unknown through a unique approach combining x-ray crystallography with structural proteomics approaches, amide hydrogen/deuterium exchange and ion mobility mass spectrometry, applied to the study of a stereospecific cAMP phosphorothioate analog and antagonist((Rp)-cAMPS). X-ray crystallography shows cAMP-bound R-subunit in the B form but surprisingly the antagonist Rp-cAMPS-bound R-subunit crystallized in the H conformation, which was previously assumed to be induced only by C-subunit-binding. Apo R-subunit crystallized in the B form as well but amide exchange mass spectrometry showed large differences between apo, agonist and antagonist-bound states of the R-subunit. Further ion mobility reveals the apo R-subunit as an ensemble of multiple conformations with collisional cross-sectional areas spanning both the agonist and antagonist-bound states. Thus contrary to earlier studies that explained the basis for cAMP action through "induced fit" alone, we report evidence for conformational selection, where the ligand-free apo form of the R-subunit exists as an ensemble of both B and H conformations. Although cAMP preferentially binds the B conformation, Rp-cAMPS interestingly binds the H conformation. This reveals the unique importance of the equatorial oxygen of the cyclic phosphate in mediating conformational transitions from H to B forms highlighting a novel approach for rational structure-based drug design. Ideal inhibitors such as Rp-cAMPS are those that preferentially "select" inactive conformations of target proteins by satisfying all "binding" constraints alone without inducing conformational changes necessary for activation.

PubMed: 21081668
DOI: 10.1074/mcp.M110.004390

Experimental method

X-RAY DIFFRACTION (2.7 Å)