Mapping the Free Energy Landscape of PKA Inhibition and Activation: A Double-Conformational Selection Model for the Tandem cAMP-Binding Domains of PKA RIα

The holoenzyme complex of protein kinase A is in an inactive state; activation involves ordered cAMP binding to two tandem domains of the regulatory subunit and release of the catalytic subunit. Deactivation has been less studied, during which the two cAMPs unbind from the regulatory subunit to allow association of the catalytic subunit to reform the holoenzyme complex. Unbinding of the cAMPs appears ordered as indicated by a large difference in unbinding rates from the two sites, but the cause has remained elusive given the structural similarity of the two tandem domains. Even more intriguingly, NMR data show that allosteric communication between the two domains is unidirectional. Here, we present a mechanism for the unidirectionality, developed from extensive molecular dynamics simulations of the tandem domains in different cAMP-bound forms. Disparate responses to cAMP releases from the two sites (A and B) in conformational flexibility and chemical shift perturbation confirmed unidirectional allosteric communication. Community analysis revealed that the A-site cAMP, by forming across-domain interactions, bridges an essential pathway for interdomain communication. The pathway is impaired when this cAMP is removed but remains intact when only the B-site cAMP is removed. Specifically, removal of the A-site cAMP leads to the separation of the two domains, creating room for binding the catalytic subunit. Moreover, the A-site cAMP, by maintaining interdomain coupling, retards the unbinding of the B-site cAMP and stalls an unproductive pathway of cAMP release. Our work expands the perspective on allostery and implicates functional importance for the directionality of allostery.unidirectional allostery | cAMP | molecular dynamics | community analysis | NMR spectroscopy P rotein kinase A (PKA), also known as cAMP-dependent protein kinase, plays an important role in multiple cellular processes by catalyzing protein phosphorylation (1). Its dysregulation is involved in many diseases including cancer and inflammatory disorders (2). The holoenzyme complex, formed by two catalytic (C) subunits bound to a homodimer of regulatory (R) subunits, is inactive. Each regulatory subunit contains two tandem cAMP-binding domains (referred to as CBD-A and CBD-B) (3); in the holoenzyme complex, the A site is masked by the C subunit (4). Activation occurs when two cAMP molecules bind sequentially and cooperatively to the two sites on each R subunit (5). Possible structural changes in the activation process have been constructed from crystallographic studies (4). The first cAMP molecule binds to the B site and makes the A site accessible. Binding of the second cAMP molecule then leads to the release of the C subunit for catalyzing substrate phosphorylation. Deactivation, involving the unbinding of the two cAMP molecules from an R subunit and association of the R and C subunits to reform the holoenzyme complex, has been less studied, and a number of important questions remain open. In particular, kinetic studies have shown that the cAMP un...

Section: Nonreciprocal Responses In Conformational Flexibility To Campsupporting

confidence: 83%

Section: W260a Mutation Mimics Campmentioning

confidence: 83%

mentioning

confidence: 99%

See 1 more Smart Citation

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

Guo

Zhou

2016

“…R-subunit dynamics are important for holoenzyme activation, as highlighted by the extended αB/C/N-helix in the holoenzyme in contrast to the kinked helices in the cAMP-bound R-subunits. Several studies have characterized the dynamic features of the αB/C/N-helix, including a "flipped-back" model for PKA activation (38)(39)(40). The thermodynamic properties of the 2 CNB domains are critically intertwined and sensitive to ligand binding as well as mutation of the neighbor (41).…”

Section: Resultsmentioning

confidence: 99%

Two PKA RIα holoenzyme states define ATP as an isoform-specific orthosteric inhibitor that competes with the allosteric activator, cAMP

Aoto

et al. 2019

Self Cite

Protein kinase A (PKA) holoenzyme, comprised of a cAMP-binding regulatory (R)-subunit dimer and 2 catalytic (C)-subunits, is the master switch for cAMP-mediated signaling. Of the 4 R-subunits (RIα, RIβ, RIIα, RIIβ), RIα is most essential for regulating PKA activity in cells. Our 2 RIα2C2 holoenzyme states, which show different conformations with and without ATP, reveal how ATP/Mg2+ functions as a negative orthosteric modulator. Biochemical studies demonstrate how the removal of ATP primes the holoenzyme for cAMP-mediated activation. The opposing competition between ATP/cAMP is unique to RIα. In RIIβ, ATP serves as a substrate and facilitates cAMP-activation. The isoform-specific RI-holoenzyme dimer interface mediated by N3A–N3A′ motifs defines multidomain cross-talk and an allosteric network that creates competing roles for ATP and cAMP. Comparisons to the RIIβ holoenzyme demonstrate isoform-specific holoenzyme interfaces and highlights distinct allosteric mechanisms for activation in addition to the structural diversity of the isoforms.

“…Although this communication is straightforward in specific cases of two differing neighboring domains or heterodimers in which domains have distinct ligands (6)(7)(8), it is more elusive for the common case of symmetric homodimers. Homodimers present a challenge because it is difficult to either observe individual protomers or study states with a single ligand bound (referred to here as "lig 1 ") because of dynamic binding equilibria.…”

mentioning

confidence: 99%

Chemical shift imprint of intersubunit communication in a symmetric homodimer

Falk

Sapienza

Lee

2016

Allosteric communication is critical for protein function and cellular homeostasis, and it can be exploited as a strategy for drug design. However, unlike many protein-ligand interactions, the structural basis for the long-range communication that underlies allostery is not well understood. This lack of understanding is most evident in the case of classical allostery, in which a binding event in one protomer is sensed by a second symmetric protomer. A primary reason why study of interdomain signaling is challenging in oligomeric proteins is the difficulty in characterizing intermediate, singly bound species. Here, we use an NMR approach to isolate and characterize a singly ligated state ("lig 1 ") of a homodimeric enzyme that is otherwise obscured by rapid exchange with apo and saturated forms. Mixed labeled dimers were prepared that simultaneously permit full population of the lig 1 state and isotopic labeling of either protomer. Direct visualization of peaks from lig 1 yielded site-specific ligand-state multiplets that provide a convenient format for assessing mechanisms of intersubunit communication from a variety of NMR measurements. We demonstrate this approach on thymidylate synthase from Escherichia coli, a homodimeric enzyme known to be half-the-sites reactive. Resolving the dUMP 1 state shows that active site communication occurs not upon the first dUMP binding, but upon the second. Surprisingly, for many sites, dUMP 1 peaks are found beyond the limits set by apo and dUMP 2 peaks, indicating that binding the first dUMP pushes the enzyme ensemble to further conformational extremes than the apo or saturated forms. The approach used here should be generally applicable to homodimers.A llosteric regulation in proteins is a ubiquitous mechanism for controlling cellular behavior and an attractive strategy for therapeutic development. Even though broadly recognized, longrange communication is not well understood mechanistically (1-4). Although there have been numerous strategies to reveal the structural and dynamic underpinnings of allostery, oddly these strategies have largely focused on complex oligomeric or, alternatively, on small monomeric allosteric proteins. A likely more straightforward approach is to study allosteric mechanisms using simple symmetric homodimeric proteins, which would allow for answering the basic and general question of how the occurrence of an event in one subunit is communicated to another subunit, as occurs in classical multisubunit allosteric proteins. Given the large number of homodimeric proteins involved in cellular regulation (such as growth factors, cytokines, kinases, G protein-coupled receptors, transcription factors, and metabolic proteins), insights into intersubunit communication should be widely beneficial (5).As a key step toward a broad understanding of allosteric mechanisms, it will be important to observe how binding a ligand in one subunit is communicated to a second subunit, even in the absence of conformational change. Although this communication is straightforwar...