A model for agonism and antagonism in an ancient and ubiquitous cAMP-binding domain.
The cAMP-binding domain (CBD) is an ancient and conserved regulatory motif that allosterically modulates the function of a group of diverse proteins, thereby translating the cAMP signal into a controlled biological response. The main receptor for cAMP in mammals is the ubiquitous regulatory (R) subunit of protein kinase A. Despite the recognized significant potential for pharmacological applications of CBDs, currently only one group of competitive inhibitor antagonists is known: the (R(p))-cAMPS family of phosphorothioate cAMP analogs, in which the equatorial exocyclic oxygen of cAMP is replaced by sulfur. It is also known that the diastereoisomer (S(p))-cAMPS with opposite phosphorous chirality is a cAMP agonist, but the molecular mechanism of action of these analogs is currently not fully understood. Previous crystallographic and unfolding investigations point to the enhanced CBD dynamics as a key determinant of antagonism. Here, we investigate the (R(p))- and (S(p))-cAMPS-bound states of R(CBD-A) using a comparative NMR approach that reveals a clear chemical shift and dynamic NMR signature, differentiating the (S(p))-cAMPS agonist from the (R(p))-cAMPS antagonist. Based on these data, we have proposed a model for the (R(p)/S(p))-cAMPS antagonism and agonism in terms of steric and electronic effects on two main allosteric relay sites, Ile(163) and Asp(170), respectively, affecting the stability of a ternary inhibitory complex formed by the effector ligand, the regulatory and the catalytic subunits of protein kinase A. The proposed model not only rationalizes the existing data on the phosphorothioate analogs, but it will also facilitate the design of novel cAMP antagonists and agonists.
Das
Rahul
R
Department of Chemistry, McMaster University, Hamilton, Ontario L8S 4M1, Canada.
Melacini
Giuseppe
G
D000495:Allosteric Site; D020816:Amino Acid Motifs; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001667:Binding, Competitive; D002417:Cattle; D000242:Cyclic AMP / Q000737:chemistry* / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D007532:Isoleucine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013696:Temperature
D007532:Isoleucine; D001224:Aspartic Acid; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases