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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 binds at the activesite with Mg2. (A) The active site of chainB from the ADP bound structure is displayed in cartoon representationwith ADP displayed in stick representation and magnesium displayedin sphere representation. The side chain of Asn171 and Asp184, whichchelate the magnesium ions, are displayed in stick representation.(B) The resulting Fo – Fc electron density map obtained by omittingthe magnesium ion from the final ADP bound structure is displayedcontoured to 3σ showing strong positive electron density atthe Mg2 site with no density for the Mg1 site. The ternary structure, 1RDQ,7 colored olive was aligned by the entire protein to theADP bound structure to illustrate the location of Asn171, Asp184,ATP, and the magnesium ions from the ternary structure.
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fig6: ADP binds at the activesite with Mg2. (A) The active site of chainB from the ADP bound structure is displayed in cartoon representationwith ADP displayed in stick representation and magnesium displayedin sphere representation. The side chain of Asn171 and Asp184, whichchelate the magnesium ions, are displayed in stick representation.(B) The resulting Fo – Fc electron density map obtained by omittingthe magnesium ion from the final ADP bound structure is displayedcontoured to 3σ showing strong positive electron density atthe Mg2 site with no density for the Mg1 site. The ternary structure, 1RDQ,7 colored olive was aligned by the entire protein to theADP bound structure to illustrate the location of Asn171, Asp184,ATP, and the magnesium ions from the ternary structure.

Mentions: There is only one magnesium ion present in theADP bound structureunlike typical PKA ternary structures, which display two magnesiumions at the active site.7 The magnesiumion at the active site in the ADP bound structure corresponds to Mg2.Although the location of the magnesium ion does not exactly matchthe typical position of Mg2, the magnesium ion is clearly situatedbetween Asp184 and Asn171, which would correspond to the Mg2 ion (Figure 6A). To further verify that the magnesium ion ismodeled correctly and corresponds to Mg2, the magnesium ion was removedfrom the final model of the structure, which was then refined. Thecorresponding Fo – Fc map shows strong positive electron density at the sitewhere the magnesium ion was modeled, and again, the site correspondsto Mg2 based on its location between Asp184 and Asn171 (Figure 6B). Therefore, Mg2 is the ion that binds more stronglywith ADP and verifies previous studies suggesting that Mg1 is lostfollowing phosphoryl transfer.22,23


Molecular features of product release for the PKA catalytic cycle.

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

ADP binds at the activesite with Mg2. (A) The active site of chainB from the ADP bound structure is displayed in cartoon representationwith ADP displayed in stick representation and magnesium displayedin sphere representation. The side chain of Asn171 and Asp184, whichchelate the magnesium ions, are displayed in stick representation.(B) The resulting Fo – Fc electron density map obtained by omittingthe magnesium ion from the final ADP bound structure is displayedcontoured to 3σ showing strong positive electron density atthe Mg2 site with no density for the Mg1 site. The ternary structure, 1RDQ,7 colored olive was aligned by the entire protein to theADP bound structure to illustrate the location of Asn171, Asp184,ATP, and the magnesium ions from the ternary structure.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: ADP binds at the activesite with Mg2. (A) The active site of chainB from the ADP bound structure is displayed in cartoon representationwith ADP displayed in stick representation and magnesium displayedin sphere representation. The side chain of Asn171 and Asp184, whichchelate the magnesium ions, are displayed in stick representation.(B) The resulting Fo – Fc electron density map obtained by omittingthe magnesium ion from the final ADP bound structure is displayedcontoured to 3σ showing strong positive electron density atthe Mg2 site with no density for the Mg1 site. The ternary structure, 1RDQ,7 colored olive was aligned by the entire protein to theADP bound structure to illustrate the location of Asn171, Asp184,ATP, and the magnesium ions from the ternary structure.
Mentions: There is only one magnesium ion present in theADP bound structureunlike typical PKA ternary structures, which display two magnesiumions at the active site.7 The magnesiumion at the active site in the ADP bound structure corresponds to Mg2.Although the location of the magnesium ion does not exactly matchthe typical position of Mg2, the magnesium ion is clearly situatedbetween Asp184 and Asn171, which would correspond to the Mg2 ion (Figure 6A). To further verify that the magnesium ion ismodeled correctly and corresponds to Mg2, the magnesium ion was removedfrom the final model of the structure, which was then refined. Thecorresponding Fo – Fc map shows strong positive electron density at the sitewhere the magnesium ion was modeled, and again, the site correspondsto Mg2 based on its location between Asp184 and Asn171 (Figure 6B). Therefore, Mg2 is the ion that binds more stronglywith ADP and verifies previous studies suggesting that Mg1 is lostfollowing phosphoryl transfer.22,23

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