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Molecular features of product release for the PKA catalytic cycle.

Bastidas AC, Wu J, Taylor SS - Biochemistry (2014)

Bottom Line: The ADP bound structure adopts a conformation that does not conform to the previously characterized open, closed, or intermediate states.These structures thus support a model where ADP release proceeds through release of the substrate and Mg1 followed by lifting of the Gly-rich loop and disengagement of the C-terminal tail.Coupling of these two structural elements with the release of the first metal ion fills in a key step in the catalytic cycle that has been missing and supports an ensemble of correlated conformational states that mediate the full catalytic cycle for a protein kinase.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of California, San Diego , San Diego, California 92093, United States.

ABSTRACT
Although ADP release is the rate limiting step in product turnover by protein kinase A, the steps and motions involved in this process are not well resolved. Here we report the apo and ADP bound structures of the myristylated catalytic subunit of PKA at 2.9 and 3.5 Å resolution, respectively. The ADP bound structure adopts a conformation that does not conform to the previously characterized open, closed, or intermediate states. In the ADP bound structure, the C-terminal tail and Gly-rich loop are more closed than in the open state adopted in the apo structure but are also much more open than the intermediate or closed conformations. Furthermore, ADP binds at the active site with only one magnesium ion, termed Mg2 from previous structures. These structures thus support a model where ADP release proceeds through release of the substrate and Mg1 followed by lifting of the Gly-rich loop and disengagement of the C-terminal tail. Coupling of these two structural elements with the release of the first metal ion fills in a key step in the catalytic cycle that has been missing and supports an ensemble of correlated conformational states that mediate the full catalytic cycle for a protein kinase.

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ADP bound structure ofthe C-subunit of PKA. The overall ADP boundstructure of the C-subunit is displayed in ribbon representation withchain A colored gray and chain B colored cyan, and the two moleculesfrom the asymmetric unit are aligned by the entire protein. ADP fromeach molecule is displayed in stick representation and colored byelement, Asn171 and Asp184 that bind to the magnesium are displayedin stick representation and colored by element, and the magnesiumion is colored by chain and displayed in sphere representation.
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fig2: ADP bound structure ofthe C-subunit of PKA. The overall ADP boundstructure of the C-subunit is displayed in ribbon representation withchain A colored gray and chain B colored cyan, and the two moleculesfrom the asymmetric unit are aligned by the entire protein. ADP fromeach molecule is displayed in stick representation and colored byelement, Asn171 and Asp184 that bind to the magnesium are displayedin stick representation and colored by element, and the magnesiumion is colored by chain and displayed in sphere representation.

Mentions: The ADP boundstructure of the C-subunit was obtained by soaking apo crystals withMg/ADP and was refined to 3.5 Å resolution (Table 1). The overall ADP bound structure is most similar to theapo structure with an RMSD of 0.74 Å between the apo and ADPbound structures aligned by the entire protein using both moleculesin the ASU. The two molecules in the ASU of the ADP bound structureare similar to each other with RMSD values between chain A and chainB for the overall protein of 0.61 Å. ADP and one magnesium ionare present in both molecules in the asymmetric unit (Figure 2). The main differences between chain A and chainB occur at the Gly-rich loop and C-terminal tail.


Molecular features of product release for the PKA catalytic cycle.

Bastidas AC, Wu J, Taylor SS - Biochemistry (2014)

ADP bound structure ofthe C-subunit of PKA. The overall ADP boundstructure of the C-subunit is displayed in ribbon representation withchain A colored gray and chain B colored cyan, and the two moleculesfrom the asymmetric unit are aligned by the entire protein. ADP fromeach molecule is displayed in stick representation and colored byelement, Asn171 and Asp184 that bind to the magnesium are displayedin stick representation and colored by element, and the magnesiumion is colored by chain and displayed in sphere representation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4295794&req=5

fig2: ADP bound structure ofthe C-subunit of PKA. The overall ADP boundstructure of the C-subunit is displayed in ribbon representation withchain A colored gray and chain B colored cyan, and the two moleculesfrom the asymmetric unit are aligned by the entire protein. ADP fromeach molecule is displayed in stick representation and colored byelement, Asn171 and Asp184 that bind to the magnesium are displayedin stick representation and colored by element, and the magnesiumion is colored by chain and displayed in sphere representation.
Mentions: The ADP boundstructure of the C-subunit was obtained by soaking apo crystals withMg/ADP and was refined to 3.5 Å resolution (Table 1). The overall ADP bound structure is most similar to theapo structure with an RMSD of 0.74 Å between the apo and ADPbound structures aligned by the entire protein using both moleculesin the ASU. The two molecules in the ASU of the ADP bound structureare similar to each other with RMSD values between chain A and chainB for the overall protein of 0.61 Å. ADP and one magnesium ionare present in both molecules in the asymmetric unit (Figure 2). The main differences between chain A and chainB occur at the Gly-rich loop and C-terminal tail.

Bottom Line: The ADP bound structure adopts a conformation that does not conform to the previously characterized open, closed, or intermediate states.These structures thus support a model where ADP release proceeds through release of the substrate and Mg1 followed by lifting of the Gly-rich loop and disengagement of the C-terminal tail.Coupling of these two structural elements with the release of the first metal ion fills in a key step in the catalytic cycle that has been missing and supports an ensemble of correlated conformational states that mediate the full catalytic cycle for a protein kinase.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of California, San Diego , San Diego, California 92093, United States.

ABSTRACT
Although ADP release is the rate limiting step in product turnover by protein kinase A, the steps and motions involved in this process are not well resolved. Here we report the apo and ADP bound structures of the myristylated catalytic subunit of PKA at 2.9 and 3.5 Å resolution, respectively. The ADP bound structure adopts a conformation that does not conform to the previously characterized open, closed, or intermediate states. In the ADP bound structure, the C-terminal tail and Gly-rich loop are more closed than in the open state adopted in the apo structure but are also much more open than the intermediate or closed conformations. Furthermore, ADP binds at the active site with only one magnesium ion, termed Mg2 from previous structures. These structures thus support a model where ADP release proceeds through release of the substrate and Mg1 followed by lifting of the Gly-rich loop and disengagement of the C-terminal tail. Coupling of these two structural elements with the release of the first metal ion fills in a key step in the catalytic cycle that has been missing and supports an ensemble of correlated conformational states that mediate the full catalytic cycle for a protein kinase.

Show MeSH
Related in: MedlinePlus