Abstract
The activation of protein kinase A involves the synergistic binding of cAMP to two cAMP binding sites on the inhibitory R subunit, causing release of the C subunit, which subsequently can carry out catalysis. We used NMR to structurally characterize in solution the RIalpha-(98-381) subunit, a construct comprising both cyclic nucleotide binding (CNB) domains, in the presence and absence of cAMP, and map the effects of cAMP binding at single residue resolution. Several conformationally disordered regions in free RIalpha become structured upon cAMP binding, including the interdomain alphaC:A and alphaC':A helices that connect CNB domains A and B and are primary recognition sites for the C subunit. NMR titration experiments with cAMP, B site-selective 2-Cl-8-hexylamino-cAMP, and A site-selective N(6)-monobutyryl-cAMP revealed that cyclic nucleotide binding to either the B or A site affected the interdomain helices. The NMR resonances of this interdomain region exhibited chemical shift changes upon ligand binding to a single site, either site B or A, with additional changes occurring upon binding to both sites. Such distinct, stepwise conformational changes in this region reflect the synergistic interplay between the two sites and may underlie the positive cooperativity of cAMP activation of the kinase. Furthermore, nucleotide binding to the A site also affected residues within the B domain. The present NMR study provides the first structural evidence of unidirectional allosteric communication between the sites. Trp(262), which lines the CNB A site but resides in the sequence of domain B, is an important structural determinant for intersite communication.
Highlights
Comparing the crystal structures of RI␣-(103–376) with cyclic adenosine monophosphate (cAMP) bound in both the A and B domains [10] with the structure of RI␣-(91–379) (R333K) complexed with the C subunit [4] revealed pronounced differences in the two cyclic nucleotide binding (CNB) domains, in particular with respect to their relative positioning (Fig. 1)
The conformational change of cAMP-ligated RI␣ in the area that interacts with the C subunit may explain why C binds to apoRI␣ with several orders of magnitude higher affinity than to cAMP-saturated RI␣ [15, 16]
Conformational changes observed for the ␣C:A helix, as well as the regions immediately preceding and following this helix, provided direct evidence for communication between the two binding sites and for additional structural effects that ensue only when both sites are occupied by cyclic nucleotide
Summary
We present an NMR study of RI␣-(98 –381), a construct that includes both CNB domains and the N-terminal region between the inhibitory sequence and the A domain, in the absence (apo-form) and presence of cAMP. We show that cAMP binding induces conformational stabilization of residues close to the binding sites as well as of regions that contact the C subunit of PKA. The conformational change of cAMP-ligated RI␣ in the area that interacts with the C subunit may explain why C binds to apoRI␣ with several orders of magnitude higher affinity than to cAMP-saturated RI␣ [15, 16]. Titration of apoRI␣ with site-selective cAMP analogs permitted us to map the effects caused by single site occupancy at the amino acid level. Conformational changes observed for the ␣C:A helix, as well as the regions immediately preceding and following this helix, provided direct evidence for communication between the two binding sites and for additional structural effects that ensue only when both sites are occupied by cyclic nucleotide
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
