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

Boras, Britton; Kornev, Alexandr P.; Taylor, Susan S.; McCulloch, Andrew D.

doi:10.1074/jbc.m114.568907

Cited by 29 publications

(39 citation statements)

References 33 publications

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“…Previous kinetic studies as well as mutations of the essential PBC Arg in CNB-A (Arg209) and CNB-B (Arg333) led to a model for cooperative activation of the RIα holoenzyme where cAMP binds first to CNB-B [27-29]. This mechanism is consistent with the R:C heterodimer structure [1, 2], is supported by urea denaturation studies [30], and was quantitatively confirmed recently by MD simulations [31]. Although the molecular mechanisms are likely different, all ACRDYS1 mutations lead to a desensitized enzyme that is resistant to activation by cAMP, providing further evidence for the “gatekeeper” role of the CNB-B domain.…”

Section: Discussionsupporting

confidence: 53%

Structure of a PKA RIα Recurrent Acrodysostosis Mutant Explains Defective cAMP-Dependent Activation

Bruystens

Fortezzo

et al. 2016

Journal of Molecular Biology

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Most disease related mutations that impair PKA signaling are present within the regulatory PKA RI alpha-subunit (RIα). Although mutations in the PRKAR1A gene are linked to Carney complex disease (CNC) and more recently to acrodysostosis-1 (ACRDYS1), the two diseases show contrasting phenotypes. While CNC mutations cause increased PKA activity, ACRDYS1 mutations result in decreased PKA activity and cAMP resistant holoenzymes. Mapping the ACRDYS1 disease mutations reveals their localization to the second of two tandem cAMP binding domains (CNB-B) and here we characterize a recurrent deletion mutant where the last 14 residues are missing. The crystal structure of a monomeric form of this mutant (RIα92-365) bound to the C subunit reveals the dysfunctional regions of the RIα-subunit. Beyond the missing residues, the entire capping motif is disordered (residues 357-379) and explains the disrupted cAMP binding. Moreover, the effects of the mutation extend far beyond the CNB-B domain and include the active site and N-lobe of the C-subunit, which is in a partially open conformation with the C-tail disordered. A key residue that contributes to this crosstalk, D267, is altered in our structure and we confirmed its functional importance by mutagenesis. In particular, the D267 interaction with Arg241, a residue shown earlier to be important for allosteric regulation, is disrupted thereby strengthening the interaction of D267 with the C-subunit residue Arg194 at the R:C interface. We see here how the switch between active (cAMP-bound) and inactive (holoenzyme) conformations is perturbed and how the dynamically controlled crosstalk between the helical domains of the two CNB-domains is necessary for functional regulation of PKA activity.

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Section: Discussionsupporting

confidence: 53%

Structure of a PKA RIα Recurrent Acrodysostosis Mutant Explains Defective cAMP-Dependent Activation

Bruystens

Fortezzo

et al. 2016

Journal of Molecular Biology

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“…Domain-B preference in Holo validates the standing “gate-keeper” theory of PKA RI α , which holds that cAMP binds to CBD-B first. 27 Domain B preference is neutralized in R 2 C 2 Flipback , where both CBD-A and CBD-B bind on the order of ~10 6 M −1 s −1 (Table 1 and Scheme 1). …”

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confidence: 99%

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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“…MSMs use discrete-time Markov chain to describe the conformational dynamics of proteins in terms of jumps between the microstates extracted from the simulation data, providing structural, thermodynamic, and long-timescale kinetic information (16,17). For example, this approach was used to examine the activation of PKA regulatory subunit RIα in response to cAMP binding (18). Yang et al (19) carried out a MSM analysis of a simplified coarse-grained model of the catalytic domain of Hck, a member of the Src family of kinases.…”

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confidence: 99%

Transition path theory analysis of c-Src kinase activation

Meng

Shukla

Pande

et al. 2016

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

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Nonreceptor tyrosine kinases of the Src family are large multidomain allosteric proteins that are crucial to cellular signaling pathways. In a previous study, we generated a Markov state model (MSM) to simulate the activation of c-Src catalytic domain, used as a prototypical tyrosine kinase. The long-time kinetics of transition predicted by the MSM was in agreement with experimental observations. In the present study, we apply the framework of transition path theory (TPT) to the previously constructed MSM to characterize the main features of the activation pathway. The analysis indicates that the activating transition, in which the activation loop first opens up followed by an inward rotation of the αC-helix, takes place via a dense set of intermediate microstates distributed within a fairly broad "transition tube" in a multidimensional conformational subspace connecting the two end-point conformations. Multiple microstates with negligible equilibrium probabilities carry a large transition flux associated with the activating transition, which explains why extensive conformational sampling is necessary to accurately determine the kinetics of activation. Our results suggest that the combination of MSM with TPT provides an effective framework to represent conformational transitions in complex biomolecular systems.transition path theory | conformational transition | Markov state models P roteins, rather than being static molecular structures, often exhibit large-scale collective motions that are biologically essential for their function (1, 2). Dynamics and flexibility form the foundation of both the conformational selection and induced-fit mechanisms of protein-protein or protein-ligand binding (3). An energy landscape theory was proposed in the early 1990s to conceptualize protein dynamics (4) and was extensively used to characterize the protein folding problem (5) and allostery (6). According to the energy landscape theory, the complex topography of the landscape that underlies the dynamics can give rise to multiple and significantly populated conformational states (metastable states). The study of protein dynamics is not only interested in obtaining the thermodynamic properties of those metastable states but also pays significant attention to the transition pathways linking them and the kinetic information. A noteworthy example of biological significance is presented by the nonreceptor tyrosine kinases of the Src family. A well-characterized prototypical tyrosine kinase is c-Src, which plays vital roles in cellular signaling pathways (7); overactivation is key to tumorgenesis and metastasis and obesity (8). Although the configurations of the regulatory domains are important in controlling kinase activity, the conformational changes occurring within the catalytic domain itself (Fig.1A and SI Methods for more details) is of special interest. Intramolecular motions, both at short and large length scales, control the activation of c-Src. Therefore, a better understanding of the internal motions displayed within the ...

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

Cited by 29 publications

References 33 publications

Structure of a PKA RIα Recurrent Acrodysostosis Mutant Explains Defective cAMP-Dependent Activation

Structure of a PKA RIα Recurrent Acrodysostosis Mutant Explains Defective cAMP-Dependent Activation

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

Transition path theory analysis of c-Src kinase activation

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