Abstract
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.
Highlights
Of proteins, including protein kinase A (PKA) in eukaryotes [1], transcription factors in bacteria [2,3,4,5,6,7,8,9,10,11], guanine exchange factors (EPAC) [12,13,14,15], and ion channel proteins [16, 17]
(Fig. 5) suggests [77] that the residual dynamics observed for the cAMP-bound state of cAMP-binding domain (CBD)-A may assist the early recognition of the C-subunit even before cAMP is released from the R-subunit, leading to the transient formation of the cAMP1⁄7R1⁄7C ternary intermediate, as hypothesized above
We have mapped by Nz exchange NMR spectroscopy and 15N relaxation measurements the interactions and the dynamics of the binary R(CBD-A)1⁄7(Sp)-cAMPS and R(CBD-A)1⁄7(Rp)cAMPS complexes
Summary
NMR Sample Preparation of RI␣-(119 –244) Bound to Different Ligands—Samples of uniformly 15N-labeled cAMP-bound RI␣-(119 –244) in MES buffer (50 mM MES, pH 6.5, 100 mM NaCl, and 0.02% NaN3) were prepared as previously discussed [29, 30]. Hydrodynamic Simulations—The contributions of the overall tumbling and the effect of diffusional anisotropy on the relaxation rates and the reduced spectral densities were modeled through bead method-based hydrodynamic simulations using the HYDRONMR program [56, 57] For this purpose, the coordinates for the 119 –244 fragment of the 1RGS Protein Data Bank structure of RI␣ were employed with hydrogen atoms added through the program Molmol [58] and with an atomic element radius of 3.3 Å, which represents the optimal average value that has been previously found to best fit several hydrodynamic properties (i.e. translational diffusion, sedimentation coefficients, rotational diffusion, and intrinsic viscosity) for a set of model proteins [57]. Secondary Structure Analyses—The secondary structure elements of 1RGS were identified based on the hydrogen bonding patterns according to the Kabsch/Sander algorithm [63], whereas the solution secondary structure probabilities were predicted based on the sequence of RI␣-(119 –244) and on the measured chemical shifts using the program PECAN [64]
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