Allostery through the computational microscope: cAMP activation of a canonical signalling domain

Malmstrom, Robert D.; Kornev, Alexandr P.; Taylor, Susan S.; Amaro, Rommie E.

doi:10.1038/ncomms8588

Cited by 95 publications

(97 citation statements)

References 60 publications

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“…We simulated, WT Holo R in five 200ns replicates using the AMBER 17 force field in NTP ensemble at 310K. MD of WT-R Holo reveals a very flexible B/C helix as observed in other simulations 18, 19 . Mutagenesis of B/C helix residues recently showed pronounced effects on PKA activation.…”

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confidence: 68%

Molecular Simulations Reveal an Unresolved Conformation of the Type IA Protein Kinase A Regulatory Subunit and Suggest Its Role in the cAMP Regulatory Mechanism

2017

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We identify a previously unresolved, unrecognized, and highly stable conformation of the Protein Kinase A (PKA) regulatory subunit RIα. This conformation, which we refer to as the “Flipback” structure, bridges conflicting characteristics in the crystallographic structures and solution experiments of the PKA RIα heterotetramer. Our simulations reveal a hinge residue in the B/C helix that is conserved through all isoforms of RI. Brownian dynamics simulations suggest that the Flipback conformation plays a role in cAMP association to the A domain of R subunit.

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confidence: 68%

Molecular Simulations Reveal an Unresolved Conformation of the Type IA Protein Kinase A Regulatory Subunit and Suggest Its Role in the cAMP Regulatory Mechanism

2017

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“…30–31,69 In these models, microstates are obtained by clustering snapshots according to structural similarity, and transition probabilities between microstates are estimated from molecular dynamics trajectories.…”

Section: Approaches For Characterizing Allosteric Communicationmentioning

confidence: 99%

Protein Allostery and Conformational Dynamics

2016

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The functions of many proteins are regulated through allostery, whereby effector binding at a distal site changes the functional activity (e.g., substrate binding affinity or catalytic efficiency) at the active site. Most allosteric studies have focused on thermodynamic properties, in particular, substrate binding affinity. Changes in substrate binding affinity by allosteric effectors have generally been thought to be mediated by conformational transitions of the proteins, or alternatively, by changes in the broadness of the free energy basin of the protein conformational state without shifting the basin minimum position. When effector binding changes the free energy landscape of a protein in conformational space, the change not only affects thermodynamic properties but also dynamic properties, including the amplitudes of motions on different timescales and rates of conformational transitions. Here we assess the roles of conformational dynamics in allosteric regulation. Two cases are highlighted where NMR spectroscopy and molecular dynamics simulation have been used as complementary approaches to identify residues possibly involved in allosteric communication. Perspectives on contentious issues, e.g., the relation between picosecond-nanosecond local and microsecond-millisecond conformational exchange dynamics, are presented.

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“…Previous simulation studies have revealed how unidirectional allosteric communication was achieved in Pin1 (19) and sortase A (20). Several simulation studies have already enriched our knowledge on the conformational properties of RIα, including the conformational space sampled by the tandem CBDs in the apo form (21,22) and cAMP-induced conformational changes of the isolated CBD-A (23,24). However, the mechanism of unidirectional allosteric communication between the two cAMP-binding sites has not been addressed and its potential role in PKA deactivation has yet to be explored.…”

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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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Allostery through the computational microscope: cAMP activation of a canonical signalling domain

Cited by 95 publications

References 60 publications

Molecular Simulations Reveal an Unresolved Conformation of the Type IA Protein Kinase A Regulatory Subunit and Suggest Its Role in the cAMP Regulatory Mechanism

Molecular Simulations Reveal an Unresolved Conformation of the Type IA Protein Kinase A Regulatory Subunit and Suggest Its Role in the cAMP Regulatory Mechanism

Protein Allostery and Conformational Dynamics

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

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