Communication between Tandem cAMP Binding Domains in the Regulatory Subunit of Protein Kinase A-Iα as Revealed by Domain-silencing Mutations

McNicholl, Eric Tyler; Das, Rahul; SilDas, Soumita; Taylor, Susan S.; Melacini, Giuseppe

doi:10.1074/jbc.m110.105783

Cited by 50 publications

(61 citation statements)

References 37 publications

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“…Below, we elaborate on the calculated CSPs and highlight pertinent experimental results from ref. 7.…”

Section: Calculated Chemical Shift Perturbations Confirm Unidirectionalmentioning

confidence: 99%

“…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.…”

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confidence: 99%

“…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).…”

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confidence: 99%

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

Guo

Zhou

2016

Proc. Natl. Acad. Sci. U.S.A.

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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...

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“…Below, we elaborate on the calculated CSPs and highlight pertinent experimental results from ref. 7.…”

Section: Calculated Chemical Shift Perturbations Confirm Unidirectionalmentioning

confidence: 99%

mentioning

confidence: 99%

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

Guo

Zhou

2016

Proc. Natl. Acad. Sci. U.S.A.

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“…However, what remains experimentally challenging is often defining the networks of residues that mediate the cross-talk between distal sites. Such clusters of coupled residues are particularly elusive in allosteric processes with a significant dynamically driven component (11)(12)(13)(14)(15)(16)(17), as in this case the allosteric signal propagation relies on subtle, but critical, conformational and side-chain packing rearrangements that often fall below the resolution of common X-ray or NMR structure determination methods (2,7,24).…”

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confidence: 99%

Mapping allostery through the covariance analysis of NMR chemical shifts

Selvaratnam¹,

Chowdhury²,

VanSchouwen³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Allostery is a fundamental mechanism of regulation in biology. The residues at the end points of long-range allosteric perturbations are commonly identified by the comparative analyses of structures and dynamics in apo and effector-bound states. However, the networks of interactions mediating the propagation of allosteric signals between the end points often remain elusive. Here we show that the covariance analysis of NMR chemical shift changes caused by a set of covalently modified analogs of the allosteric effector (i.e., agonists and antagonists) reveals extended networks of coupled residues. Unexpectedly, such networks reach not only sites subject to effector-dependent structural variations, but also regions that are controlled by dynamically driven allostery. In these regions the allosteric signal is propagated mainly by dynamic rather than structural modulations, which result in subtle but highly correlated chemical shift variations. The proposed chemical shift covariance analysis (CHESCA) identifies interresidue correlations based on the combination of agglomerative clustering (AC) and singular value decomposition (SVD). AC results in dendrograms that define functional clusters of coupled residues, while SVD generates score plots that provide a residue-specific dissection of the contributions to binding and allostery. The CHESCA approach was validated by applying it to the cAMP-binding domain of the exchange protein directly activated by cAMP (EPAC) and the CHESCA results are in full agreement with independent mutational data on EPAC activation. Overall, CHESCA is a generally applicable method that utilizes a selected chemical library of effector analogs to quantitatively decode the binding and allosteric information content embedded in chemical shift changes. L ong-range allosteric perturbations are propagated not only by structural changes but also by effector-dependent modulations in dynamics (1-23). The end points of these long-range allosteric signal propagations are effectively characterized by the comparative analysis of the structural and dynamic profiles of apo and effector-bound states (2, 7). However, what remains experimentally challenging is often defining the networks of residues that mediate the cross-talk between distal sites. Such clusters of coupled residues are particularly elusive in allosteric processes with a significant dynamically driven component (11-17), as in this case the allosteric signal propagation relies on subtle, but critical, conformational and side-chain packing rearrangements that often fall below the resolution of common X-ray or NMR structure determination methods (2, 7, 24).Here we introduce a general experimental method to map allosteric networks based on the covariance analysis of NMR chemical shifts. The chemical shift covariance analysis (CHESCA) is based on two simple but general notions. The first assumption is that the subtle but functionally relevant structural changes that underlie the allosteric modulations of dynamics are effectively probed by accurately ...

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“…CBD-A and CBD-B (Johnson et al, 2001). cAMP is the essential second messenger that activates PKA (Berman et al, 2005;McNicholl et al). In the absence of cAMP, the R-subunit and the C-subunit create a complex that blocks substrate access and thus prevents the kinase activity.…”

Section: Pkamentioning

confidence: 99%

Role of Protein Kinase Network in Excitation-Contraction Coupling in Smooth Muscle Cell

Roux¹,

Mbikou²,

Fajmut³

2012

Protein Kinases

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Communication between Tandem cAMP Binding Domains in the Regulatory Subunit of Protein Kinase A-Iα as Revealed by Domain-silencing Mutations

Cited by 50 publications

References 37 publications

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

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

Mapping allostery through the covariance analysis of NMR chemical shifts

Role of Protein Kinase Network in Excitation-Contraction Coupling in Smooth Muscle Cell

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