PDBsum entry 3iia

Transferase

PDB id: 3iia

Contents

Protein chain

137 a.a. *

Ligands

GOL

Waters ×6

* Residue conservation analysis

PDB id:

3iia

Name:

Transferase

Title:

Crystal structure of apo (91-244) ria subunit of camp-dependent protein kinase

Structure:

Camp-dependent protein kinase type i-alpha regulatory subunit. Chain: a. Fragment: the ria subunit: unp residues 92-245. Engineered: yes

Source:

Bos taurus. Bovine. Organism_taxid: 9913. Gene: prkar1a. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.70Å R-factor: 0.235 R-free: 0.285

Authors:

T.J.Sjoberg,C.Kim,A.P.Kornev,S.S.Taylor

Key ref:

S.Badireddy et al. (2011). 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. PubMed id: 21081668

Date:

31-Jul-09 Release date: 11-Aug-10

Protein chain

P00514  (KAP0_BOVIN) -  cAMP-dependent protein kinase type I-alpha regulatory subunit from Bos taurus

Seq: 380 a.a.

Struc: 137 a.a.

Mol Cell Proteomics 10:M110.004390 (2011)

PubMed id: 21081668

Cyclic AMP analog blocks kinase activation by stabilizing inactive conformation: conformational selection highlights a new concept in allosteric inhibitor design.

S.Badireddy, G.Yunfeng, M.Ritchie, P.Akamine, J.Wu, C.W.Kim, S.S.Taylor, L.Qingsong, K.Swaminathan, G.S.Anand.

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.