Solution structure of the cAMP-dependent protein kinase catalytic subunit and its contraction upon binding the protein kinase inhibitor peptide

Olah, Glenn A.; Mitchell, Ryan D.; Sosnick, Tobin R.; Walsh, Donal A.; Trewhella, Jill

doi:10.1021/bi00065a018

Cited by 81 publications

(82 citation statements)

References 21 publications

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“…Our results indicate that the linker must undergo a significant conformational change upon binding C subunits to allow simultaneous contact of the cAMPbinding domains of R with the C subunits and the pseudosubstrate inhibitor sequence with the catalytic cleft of C. The backbone conformation of the pseudosubstrate sequence bound to the active site of C is very likely to be similar to that of PKI bound to C (33,34). As the pseudosubstrate region adopts this conformation, an additional conformational change could be induced within other parts of the linker region that, in turn, affect the interdomain orientation of the homodimer.…”

Section: Discussionmentioning

confidence: 87%

C Subunits Binding to the Protein Kinase A RIα Dimer Induce a Large Conformational Change

Heller

Vigil

Brown

et al. 2004

Journal of Biological Chemistry

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We present structural data on the RI␣ isoform of the cAMP-dependent protein kinase A that reveal, for the first time, a large scale conformational change within the RI␣ homodimer upon catalytic subunit binding. This result infers that the inhibition of catalytic subunit activity is not the result of a simple docking process but rather is a multi-step process involving local conformational changes both in the cAMP-binding domains as well as in the linker region of the regulatory subunit that impact the global structure of the regulatory homodimer. The results were obtained using small-angle neutron scattering with contrast variation and deuterium labeling. From these experiments we derived information on the shapes and dispositions of the catalytic subunits and regulatory homodimer within a holoenzyme reconstituted with a deuterated regulatory subunit. The scattering data also show that, despite extensive sequence homology between the isoforms, the overall structure of the type I␣ holoenzyme is significantly more compact than the type II␣ isoform. We present a model of the type I␣ holoenzyme, built using available high-resolution structures of the component subunits and domains, which best fits the neutron-scattering data. In this model, the type I␣ holoenzyme forms a flattened V shape with the RI␣ dimerization domain at the point of the V and the cAMP-binding domains of the RI␣ subunits with their bound catalytic subunits at the ends.Many cellular signaling pathways in eukaryotes involve protein kinases, which phosphorylate Ser, Thr, and Tyr residues in a variety of target proteins. The cAMP-dependent protein kinase A (PKA 1 or protein kinase A) is one of the best-studied members of the Ser/Thr protein kinase family. PKA is known to be involved in the regulation of a large number of cellular processes including metabolism, contractile activity, growth, apoptosis, and ion flux (1). Mutations in PKA can lead to diseases such as Carney complex (2) and lupus (3). The catalytic (C) subunits of PKA are responsible for catalyzing the phospho-transfer reaction, whereas the regulatory (R) subunits serve both to confer cAMP dependence and to localize the holoenzyme to discrete subcellular locations via interactions with A-kinase anchoring proteins (AKAPs) (4). At low cAMP concentrations, PKA is maintained as an inactive tetrameric holoenzyme complex (R 2 C 2 ) consisting of a homodimeric R 2 subunit and two C subunits. When intracellular concentrations of cAMP increase in response to specific cellular stimuli, four cAMP molecules bind to each R 2 subunit. This event causes a release of inhibition of C by R, allowing the C subunits to phosphorylate their target proteins.There are four major isoforms of PKA, types I␣, I␤, II␣, and II␤, which differ with respect to their R subunits (RI␣, RI␤, RII␣, and RII␤, respectively). The isoforms have different biological functions, as determined by genetic studies using mice. For instance, mice lacking the RI␣ gene die in utero (5), whereas mice lacking the RII␤ gene are viable, lean,...

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Section: Discussionmentioning

confidence: 87%

C Subunits Binding to the Protein Kinase A RIα Dimer Induce a Large Conformational Change

Heller

Vigil

Brown

et al. 2004

Journal of Biological Chemistry

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“…Although the angle formed by the two domains opens by ca. 15" between the crystal structures of the closed and open forms, data from low-angle X-ray scattering experiments of cAPK alone in solution indicated an opening of as much as 39" (Olah et al, 1993). The conformational transition between the open and closed crystal structures has been described on a structural level in great detail by Cox et al (1994).…”

Section: Pki(5-24)mentioning

confidence: 99%

Kinase conformations: A computational study of the effect of ligand binding

Helms

McCammon

1997

Protein Science

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Protein function is often controlled by ligand-induced conformational transitions. Yet, in spite of the increasing number of three-dimensional crystal structures of proteins in different conformations, not much is known about the driving forces of these transitions. As an initial step toward exploring the conformational and energetic landscape of protein kinases by computational methods, intramolecular energies and hydration free energies were calculated for different conformations of the catalytic domain of CAMP-dependent protein kinase (cAPK) with a continuum (Poisson) model for the electrostatics. Three protein kinase crystal structures for ternary complexes of cAPK with the peptide inhibitor PKI(5-24) and ATP or AMP-PNP were modeled into idealized intermediate and open conformations. Concordant with experimental observation, we find that the binding of PKI(5-24) is more effective in stabilizing the closed and intermediate forms of cAPK than ATP. PKI(5-24) seems to drive the final closure of the active site cleft from intermediate to closed state because ATP does not distinguish between these two states. Binding of PKI(5-24) and ATP is energetically additive.

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“…This structural heterogeneity provides a more open conformation to allow the association of ATP even in the presence of inhibitors and substrates. Secondly, Olah et al (1993) showed by small angle X-ray scattering and Fourier transform infrared spectroscopy that a 9% decrease in the radius of gyration of the C-subunit accompanies inhibitor binding. The lack of any changes in secondary structure led these authors to propose that domain movement is the likely cause.…”

Section: Binary Atp Complex Have Equilibrated In Solutionmentioning

confidence: 99%

Divalent metal ions influence catalysis and active‐site accessibility in the camp‐dependent protein kinase

Adams

Taylor

1993

Protein Science

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Phosphorylation of the peptide LRRASLG by the catalytic subunit of CAMP-dependent protein kinase was measured in the presence of various divalent metals to establish the role of electrophiles in the kinetic mechanism. Under conditions of low or high metal concentrations, the apparent second-order rate constant, kc,,/Kpep,ide, and the maximal rate constant, kc,,, followed the trend Mg2+ > Co2+ > Mn2+. Competitive inhibition studies indicate that the former effect is not due to destabilization of the substrate complex, E-ATP.S. The effects of solvent viscosity on the steady-state kinetic parameters were interpreted according to a simple mechanism involving substrate binding, phosphotransfer, and product release steps and two metal chelation sites in the nucleotide pocket. Decreases in kc,, and kcol/Kpeplide result mostly from attenuations in the dissociation rate constant for ADP and the association rate constant for the substrate, respectively. Decreases in the phosphoryl transfer rate constant have only negligible to moderate effects on these parameters. The low observed values for the association rate constant of the substrate indicate that the metals control the concentration of the productive binary form, E,.ATP, and indirectly the accessibility of the active site. By comparison, Mg2+ is the best divalent metal catalyst because it uniformly lowers the transition state energies for all steps in the kinetic mechanism, permitting maximum flux of substrate to product. The data suggest that CAMP-dependent protein kinase uses metal ions to serve multiple roles in facilitating phosphotransfer and accelerating substrate association and product dissociation.Keywords: catalytic subunit; divalent metal ions; solvent viscosity; steady-state kinetics Many theories have been proposed to explain the large rate enhancements by metal catalysts at the active sites of enzymes. Data attained from enzymatic and model studies suggest that metal ions may accelerate reactions by lowering the chemical transition state energy through polarization of the electrophile, charge reduction, and stabilization of the leaving group (Jencks, 1987). In addition, ions may confer tight binding or proper orientation of the substrate's functional groups. An efficient enzyme recognizes substrate, easily modifies it, and then quickly releases the product without populating unproductive or very stable intermediate species. Enzymes accomplish this by lowering the transition state energies of the chemical and conformational steps and equalizing the ground states

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Solution structure of the cAMP-dependent protein kinase catalytic subunit and its contraction upon binding the protein kinase inhibitor peptide

Cited by 81 publications

References 21 publications

C Subunits Binding to the Protein Kinase A RIα Dimer Induce a Large Conformational Change

C Subunits Binding to the Protein Kinase A RIα Dimer Induce a Large Conformational Change

Kinase conformations: A computational study of the effect of ligand binding

Divalent metal ions influence catalysis and active‐site accessibility in the camp‐dependent protein kinase

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