Allosteric Communication between cAMP Binding Sites in the RI Subunit of Protein Kinase A Revealed by NMR

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 RI␣-(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 RI␣ become structured upon cAMP binding, including the interdomain ␣C:A and ␣C: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 Protein kinase A (PKA)3 is a primary receptor for cyclic adenosine monophosphate (cAMP) in eukaryotic cells (1, 2). In the absence of cAMP, the enzyme is an inactive, tetrameric holoenzyme complex, composed of two regulatory (R) and two catalytic (C) subunits (R 2 C 2 ). The catalytic site of the C subunit is occluded by a short inhibitory sequence in the R subunit (residues 94 -99 in bovine RI␣) that connects the N-terminal dimerization domain to the two cyclic nucleotide binding (CNB) domains. Multiple contacts exist between the CNB domains and the C subunit. The enzyme is allosterically activated by cAMP (3, 4), whose binding to the R subunits causes dissociation of the C subunits from the holoenzyme complex, thereby rendering C catalytically active (5). Two CNB domains (A and B) are present in all four isoforms (RI␣, RI␤, RII␣, and RII␤) of mammalian PKA, and both need to be occupied by cAMP to achieve PKA dissociation under physiologically relevant conditions (for reviews, see Refs. 2 and 6). Newly transcribed R subunit (apoR) in the cell can complex with either cAMP or the C subunit of PKA. Binding of cAMP leads to a dramatically decreased affinity for the C subunit, whereas binding of the C subunit lowers the cAMP affinity by about 3 orders of magnitude (7), allowing the holoenzyme to respond to fluctuations in physiological cAMP concentrations (8, 9).Comparing the crystal structures of RI␣-(103-376) (numbering for bovine RI␣) with 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 CNB domains, in particular with respect to their relative positioning (Fig. 1). However, little is known about the structure of the ligand-free (apo) state of the R subunits. A truncated RI␣ (residues 119 -244), comprising most of the A domain, has been investigated by NMR (11-13). Note, however, that this truncated form lacks not only the B domain but also the C-terminal end of the A domain, in particular the ␣C:A and ␣CЈ:A helices. These helices are at the junction between domains A and B and are important elements for interaction with the C subunit (4). Moreover, this region is conserved in all CAP-related eukaryotic cAMP binding proteins, as...

Using Markov State Models to Develop a Mechanistic Understanding of Protein Kinase A Regulatory Subunit RIα Activation in Response to cAMP Binding

“…Also, the interactions between the CBD-As both in the free R-subunit and in the R-C complex were necessary for the formation and fitting of the best model. This is consistent with recent publications (26,28). However, the diverse R 2 C 2 structures that various PKA isoforms exhibit suggest that although this model is consistent with current PKA-RI␣ experiments it would be expected that RI␤, RII␣, and RII␤ will exhibit significantly different mechanistic activations possibly with less interaction between the R-C heterodimers (13,29,30).…”

Unidirectional allostery in the regulatory subunit RIα facilitates efficient deactivation of protein kinase A

“…Although the masking of the A site by a C subunit provides a simple explanation for why the first cAMP molecule binds to the B site, it is not clear why unbinding should first occur from the A site, given the structural similarities between the two CBDs. Even more intriguingly, NMR studies have presented evidence that allosteric communication between the two cAMP-binding sites is unidirectional (6,7), but the mechanism is uncertain. The present study aimed to address these questions on deactivation through extensive moleculardynamics simulations.…”

“…Molecular insight into the allosteric communication between the two cAMP-binding sites in RIα N-terminal deletion constructs was provided by recent NMR and hydrogen/deuterium (H/D) exchange studies (6,7). Gronenborn and coworkers carried out NMR titrations of cAMP and two site-selective analogs (6).…”

“…Gronenborn and coworkers carried out NMR titrations of cAMP and two site-selective analogs (6). Upon ligand binding to the A site alone, some CBD-B residues experienced chemical shift changes that corresponded to conformations intermediate between apo and cAMP-bound forms.…”

The cAMP-Dependent Protein Kinase Pathway as Therapeutic Target – Possibilities and Pitfalls

“…Recent progress in the structural understanding of PKA activation has come from comparison of cAMP-bound Rsubunit and the resolved structures of PKAI and II holoenzymes [1,61,62,63,64,65]. The structures of the holoenzymes show large surface interactions between the R and C subunits which are greatly diminished upon binding of cAMP (reviewed in [66]).…”