Electrostatic Interactions as Mediators in the Allosteric Activation of Protein Kinase A RIα

Barros, Emília P.; Malmstrom, Robert D.; Nourbakhsh, Kimya; Rio, Jason C. Del; Kornev, Alexandr P.; Taylor, Susan S.; Amaro, Rommie E.

doi:10.1021/acs.biochem.6b01152

Cited by 17 publications

(23 citation statements)

References 34 publications

(68 reference statements)

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“…Finally, disruption of the salt bridge, especially one that involved two negative charges, may induce protein remodeling through partial unfolding due to the colocalization of two negatively charged side chains. Similar reorganizations of electrostatic networks and allosteric effects resulting from disrupted charge clusters have also been observed in other systems, including PKA and VraR (40,41). These examples as well as others (42) suggest that neither mono-nor divalent cation binding would completely recover the energy invested in the colocalization of D134 and E135 in RKIP, and, thermodynamically, the partial unfolding of the loop is favored.…”

supporting

confidence: 66%

Conserved salt-bridge competition triggered by phosphorylation regulates the protein interactome

Skinner

Wang

Lee

et al. 2017

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

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Phosphorylation is a major regulator of protein interactions; however, the mechanisms by which regulation occurs are not well understood. Here we identify a salt-bridge competition or "theft" mechanism that enables a phospho-triggered swap of protein partners by Raf Kinase Inhibitory Protein (RKIP). RKIP transitions from inhibiting Raf-1 to inhibiting G-protein-coupled receptor kinase 2 upon phosphorylation, thereby bridging MAP kinase and G-Protein-Coupled Receptor signaling. NMR and crystallography indicate that a phosphoserine, but not a phosphomimetic, competes for a lysine from a preexisting salt bridge, initiating a partial unfolding event and promoting new protein interactions. Structural elements underlying the theft occurred early in evolution and are found in 10% of homo-oligomers and 30% of hetero-oligomers including Bax, Troponin C, and Early Endosome Antigen 1. In contrast to a direct recognition of phosphorylated residues by binding partners, the salt-bridge theft mechanism represents a facile strategy for promoting or disrupting protein interactions using solvent-accessible residues, and it can provide additional specificity at protein interfaces through local unfolding or conformational change.

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supporting

confidence: 66%

Conserved salt-bridge competition triggered by phosphorylation regulates the protein interactome

Skinner

Wang

Lee

et al. 2017

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

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“…The thermodynamic properties of the 2 CNB domains are critically intertwined and sensitive to ligand binding as well as mutation of the neighbor (41). Both mutagenesis and simulations have shown that the helical propensity of the αB/C/N-helix correlates with R-C dissociation in the (Δ1-91) RIα:C complex (42). The "flip-back" model of the R-C complex, which has been observed in long MD simulations, also highlights the dynamic properties of the αB/C/N-helix and the CNB domains for PKA activation (43).…”

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

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

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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.

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“…Such mobility of the CNB-B domain is consistent with previously obtained SAXS data of the RIα(91-379):PKAcα heterodimer and led to a suggestion that the CNB-B domain of RIα is mobile and moves away from PKAcα with Gly235 serving as a hinge point (Cheng et al, 2009). Recent studies have shown that CNB-B domain flexibility is linked to cAMP activation in the RIα(91-379):PKAcα truncated heterodimer (Hirakis et al, 2017; Barros et al, 2017). However, this view of CNB-B movement in the holoenzyme is different from the packing observed here in the full-length chimeric and wt RIα holoenzymes (Figures.…”

Section: Resultsmentioning

confidence: 99%

Structures of the PKA RIα holoenzyme with the FLHCC driver J-PKAcα or wild type PKAcα

Cao

Fiesco

et al. 2018

Preprint

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1Fibrolamellar hepatocellular carcinoma (FLHCC) is driven by J-PKAcα, a kinase fusion chimera 2 2 of the J-domain of DnaJB1 with PKAcα, the catalytic subunit of Protein Kinase A (PKA). Here 2 3 we report the crystal structures of the chimeric fusion RIα 2 :J-PKAcα 2 holoenzyme formed by J-2 4 PKAcα and the PKA regulatory (R) subunit RIα, and the wild type (wt) RIα 2 :PKAcα 2 2 5 holoenzyme. The chimeric and wt RIα holoenzymes have quaternary structures different from 2 6 the previously solved wt RIβ and RIIβ holoenzymes. The chimeric holoenzyme shows an 2 7isoform-specific interface dominated by antiparallel interactions between the N3A-N3A' motifs 2 8of RIα that serves as an anchor for RIα structural rearrangements during cAMP activation. The 2 9wt RIα holoenzyme showed the same configuration as well as a distinct second conformation. In 3 0 the structure of the chimeric fusion RIα 2 :J-PKAcα 2 holoenzyme, the presence of the J-domain 3 1 does not prevent formation of the holoenzymes, and is positioned away from the symmetrical 3 2 interface between the two RIα:J-PKAcα heterodimers in the holoenzyme. The J-domains have 3 3 significantly higher temperature factors than the rest of the holoenzyme, implying a large degree 3 4 of conformational flexibility. Furthermore molecular dynamics simulations were applied to 3 5 analyze the conformational states of chimeric fusion and wt RIα holoenzymes, and showed an 3 6 ensemble of conformations in the majority of which the J-domain was dynamic and rotated away 3 7 from the R:J-PKAcα interface. Thus, rather than affecting the interactions with the regulatory 3 8 subunits, the fusion of the J-domain to the PKAcα alters the conformational landscape of the 3 9 chimeric fusion holoenzymes and potentially, as result, the interactions with other molecules.4 0The structural and dynamic features of these holoenzymes enhance our understanding of the 4 1 fusion chimera protein J-PKAcα that drives FLHCC as well as the isoform specificity of PKA. 4 2 4 3

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Electrostatic Interactions as Mediators in the Allosteric Activation of Protein Kinase A RIα

Cited by 17 publications

References 34 publications

Conserved salt-bridge competition triggered by phosphorylation regulates the protein interactome

Conserved salt-bridge competition triggered by phosphorylation regulates the protein interactome

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

Structures of the PKA RIα holoenzyme with the FLHCC driver J-PKAcα or wild type PKAcα

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