Quaternary Structures of a Catalytic Subunit-Regulatory Subunit Dimeric Complex and the Holoenzyme of the cAMP-dependent Protein Kinase by Neutron Contrast Variation

Zhao, Jinkui; Hoye, Elaine; Boylan, S A; Walsh, Donal A.; Trewhella, Jill

doi:10.1074/jbc.273.46.30448

Cited by 58 publications

(69 citation statements)

References 37 publications

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“…The template R subunits used in both cases here were the cAMP-bound crystal structures (19,20). Our previous x-ray scattering data show that there is no change in the overall shape of R II␣ upon cAMP binding (1), suggesting that the cAMP-bound protein is a suitable template structure to determine the target R subunit structure.…”

Section: Resultsmentioning

confidence: 99%

“…Amino acids were represented as 4-Å radius spheres centered at their respective C-␣ coordinate positions. The docking procedure was divided into two steps, the first being the coarse docking of the two subunits into the ellipsoid model for the complex derived from the neutron scattering study (1), while the second step is a detailed search in the conformational space around the structures derived from the coarse docking procedure.…”

Section: Methodsmentioning

confidence: 99%

“…The cAMP-dependent protein kinase (PKA) 1 is a multifunctional kinase that serves as a prototype for understanding second messenger signaling and protein phosphorylation (1)(2)(3). In the absence of a cAMP signal, the enzyme is inactive and exists as a dimer of dimers, having two identical regulatory (R) and two identical catalytic (C) subunits.…”

mentioning

confidence: 99%

“…The crystal structure of C ␣ shows that it has the now classical protein kinase bilobal structure, with the catalytic cleft located between the two lobes (14 -16). Both small angle solution scattering (1,17) and crystallographic studies (18) have shown that binding of peptides that model either substrate or pseudosubstrate sequences results in closure of the catalytic cleft. This closure is achieved via a hinge movement about a conserved Gly residue (Gly 125 ) located in the sequence that joins the two lobes.…”

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confidence: 99%

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A Structural Model of the Catalytic Subunit-regulatory Subunit Dimeric Complex of the cAMP-dependent Protein Kinase

Tung

Walsh

Trewhella

2002

Journal of Biological Chemistry

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Previous neutron scattering studies elaborated the topographical relationship of the regulatory (R II␣ ) and catalytic (C ␣ ) subunits of the cAMP-dependent protein kinase. We present here the results of a set of computations that lead to an atomic model of the cAMP-dependent protein kinase heterodimer, ⌬ 1-91 R II␣ -C ␣ . The first step in the modeling utilized the crystal structures for the porcine C ␣ and bovine ⌬ 1-90 R I␣ or rat ⌬ 1-111 R II␤ , to homology-model structures of the species and isoforms that had been used in the neutron scattering experiments (bovine C ␣ subunit and murine ⌬ 1-91 R II␣ subunit, respectively). A docking procedure, constrained by the dimensions and positions of the ellipsoids in the neutron-derived R-C model as well as mutagenesis data, was used to develop "best fit" models for the heterodimer. Simulated annealing, molecular dynamics, and energy minimization were then used to refine the side chain packing at the heterodimer interface. For comparison, the calculations were done using the homology models derived from both the R I␣ and R II␤ crystal structures. Both resultant models had many similarities. Each predicted similar interfaces. The R I␣ -based model has 25% more hydrogen bonds than that based on R II␤ , with seven of these potential bonds in common. The distribution of hydrophobic, polar, and charged residues at the interface was similar for both models, with a distribution more characteristic of the exposed surface residues than those in the protein interior. The calculated interface area in each is relatively small (<2000 Å 2 ). The R I␣ -based model, however, has a significantly better fit with the scattering data and is therefore the one of distinctly higher probability. With its small interface area that has a high proportion of charged and polar residues, the complex appears poised for dissociation, and each subunit existing as a stable entity. This result is consistent with the known physiological events required for cAMP-dependent activation of the kinase.The cAMP-dependent protein kinase (PKA) 1 is a multifunctional kinase that serves as a prototype for understanding second messenger signaling and protein phosphorylation (1-3). In the absence of a cAMP signal, the enzyme is inactive and exists as a dimer of dimers, having two identical regulatory (R) and two identical catalytic (C) subunits. The C subunit has the conserved catalytic core structure that is common to the majority of protein kinases. The R subunit has a dimerization domain, followed by a pseudosubstrate sequence that inhibits C subunit activity and two in tandem cAMP-binding domains (in order from the N to C termini). When two cAMP molecules bind to each of the R subunits, the C subunit is activated, presumably via some sort of release of the pseudosubstrate sequence. The activation of the C subunit has been assumed to involve dissociation of the C subunits from the R 2 homodimer (4 -6). The strongest evidence for the dissociation of the C subunit is the fact that the C subunit, but not R 2 , is...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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A Structural Model of the Catalytic Subunit-regulatory Subunit Dimeric Complex of the cAMP-dependent Protein Kinase

Tung

Walsh

Trewhella

2002

Journal of Biological Chemistry

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“…Therefore, our results imply that RII⅐EYFP associates to form dimers in the CHO cells. Because Ht31 can bind to the N termini of dimerized RII in vitro (23,24), the ECFP tag at the C terminus of RII should not interfere with AKAP (or Ht31) binding to RII. We tested this hypothesis using surface plasmon resonance.…”

Section: Expression Of Fluorescent Fusion Proteins In Transfectedmentioning

confidence: 99%

Cyclic AMP-dependent Protein Kinase Binding to A-kinase Anchoring Proteins in Living Cells by Fluorescence Resonance Energy Transfer of Green Fluorescent Protein Fusion Proteins

Ruehr

Zakhary

Damron

et al. 1999

Journal of Biological Chemistry

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There is now increasing evidence for clustering and compartmentalization of signaling enzymes with their activators and target proteins. This targeting of enzymes with their substrates may promote specificity of signaling events (1-3). To better understand the physiological role of scaffolding proteins, such as A-kinase anchoring proteins (AKAPs), 1 which target cyclic AMP-dependent protein kinase (PKA) (2, 4), it is essential to monitor in real time the interactions between the components of this signaling complex in living cells. However, to date such measurements have not been reported for the PKA pathway. AKAPs and PKA co-localize in cells. This was first shown by the redistribution of the type II regulatory (RII) and the catalytic subunits of PKA from the cytosolic to the particulate fraction following overexpression of AKAP75 in HEK293 cells (5). In studies in HEK293 cells, S-AKAP84 targeted RII to mitochondria (6). In rat cardiac myocytes, endogenous AKAP100 co-localized with RII in the region of the junctional sarcoplasmic reticulum/transverse tubule (7). A functional role of AKAP targeting of PKA is further indicated by studies in which Ht31 peptide, a competitive inhibitor of AKAP/PKA binding, was introduced into AKAP-overexpressing cells. For example, transfection of RINm5F pancreatic beta cells with Ht31 caused redistribution of RII from a perinuclear to a diffuse cytoplasmic localization and prevented cAMP-dependent insulin secretion (8). Similarly, in cardiac myocytes transfected with AKAP79, microinjection of Ht31 impaired the PKA-dependent increase in L-type Ca 2ϩ current (9). These studies demonstrate a role for AKAPs in the regulation of PKA distribution and in the specificity of PKA function.To date, the binding of RII with full-length AKAPs or with the peptide Ht31 has been demonstrated in vitro by RII overlay assay (10 -12), by band shift assays on nondenaturing gels (12, 13), by equilibrium dialysis measurements (12, 13), and recently by solution NMR measurements of the N-terminal dimerization domain of RII with Ht31 (14). Equilibrium dialysis measurements (12) and surface plasmon resonance 2 showed that Ht31 binds RII with nanomolar affinity. Although these in vitro approaches provide evidence for high affinity RII/AKAP or RII/Ht31 interactions in vitro, the results presented here extend these studies by measuring real-time interactions between these molecules in living cells. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.ʈ To whom correspondence should be addressed: Dept. EXPERIMENTAL PROCEDURES Construction of Vectors for the Expression of GFP-tagged Proteins-The cDNAs for RII␣, Ht31, and Ht31P were gifts from John Scott (Vollum Institute, Howard Hughes Medical Institute, Portland, OR). The construct for Ht31, used in the FRET experiments, encodes residues 418 -718 of the human thyroid RII anchoring protein (...

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Neutron Scattering Techniques and Applications in Structural Biology

et al. 2013

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Neutron scattering is exquisitely sensitive to the position, concentration, and dynamics of hydrogen atoms in materials and is a powerful tool for the characterization of structure-function and interfacial relationships in biological systems. Modern neutron scattering facilities offer access to a sophisticated, nondestructive suite of instruments for biophysical characterization that provides spatial and dynamic information spanning from Ångstroms to microns and from picoseconds to microseconds, respectively. Applications in structural biology range from the atomic-resolution analysis of individual hydrogen atoms in enzymes through to meso- and macro-scale analysis of complex biological structures, membranes, and assemblies. The large difference in neutron scattering length between hydrogen and deuterium allows contrast variation experiments to be performed and enables H/D isotopic labeling to be used for selective and systematic analysis of the local structure, dynamics, and interactions of multi-component systems. This overview describes the available techniques and summarizes their practical application to the study of biomolecular systems.

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Quaternary Structures of a Catalytic Subunit-Regulatory Subunit Dimeric Complex and the Holoenzyme of the cAMP-dependent Protein Kinase by Neutron Contrast Variation

Cited by 58 publications

References 37 publications

A Structural Model of the Catalytic Subunit-regulatory Subunit Dimeric Complex of the cAMP-dependent Protein Kinase

A Structural Model of the Catalytic Subunit-regulatory Subunit Dimeric Complex of the cAMP-dependent Protein Kinase

Cyclic AMP-dependent Protein Kinase Binding to A-kinase Anchoring Proteins in Living Cells by Fluorescence Resonance Energy Transfer of Green Fluorescent Protein Fusion Proteins

Neutron Scattering Techniques and Applications in Structural Biology

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