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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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Apo structure of the catalytic subunit of PKA. (A) Theoverallapo structure is displayed in ribbon representation with chain A coloredgray and chain B colored olive and with chains A and B aligned bythe entire protein. (B) (left) The myristic acid binding pocket forchains A and B, which are colored as in panel A, is displayed alongwith the 2Fo – Fc electron density at 1σ shown in blue for the myristicacid group from chain A. There is no electron density for myristicacid in chain B. (right) In contrast, the 2Fo – Fc electron densityat 1σ is visible for the entire myristic acid group in the closedstate (4DFX).25 (C) The 2Fo – Fc electron density at 1σ is shown in bluefor the C-terminal tail of chains A and B.
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fig1: Apo structure of the catalytic subunit of PKA. (A) Theoverallapo structure is displayed in ribbon representation with chain A coloredgray and chain B colored olive and with chains A and B aligned bythe entire protein. (B) (left) The myristic acid binding pocket forchains A and B, which are colored as in panel A, is displayed alongwith the 2Fo – Fc electron density at 1σ shown in blue for the myristicacid group from chain A. There is no electron density for myristicacid in chain B. (right) In contrast, the 2Fo – Fc electron densityat 1σ is visible for the entire myristic acid group in the closedstate (4DFX).25 (C) The 2Fo – Fc electron density at 1σ is shown in bluefor the C-terminal tail of chains A and B.

Mentions: The myristylated C-subunitof PKA was crystallized in an apo state to determine any effects ofmyristylation in the absence of ligands. A K7C mutant was crystallizedbecause it increases the yield of the myristylated protein.25 The apo crystal structure was refined at 2.9Å resolution (Table 1, Figure 1A). There is electron density for myristic acidin one of the two molecules in the asymmetric unit (ASU); however,there is density for only 4 of the 14 carbon atoms of myristic acid(Figure 1B). The lack of electron density formyristic acid implies that the myristic acid group binding withinthe hydrophobic pocket could be involved in forming a closed statesince there is more electron density for the myristic acid group instructures that adopt a closed conformation, particularly in the binarycomplex that contains only peptide, compared with the open conformationadopted here in an apo state.25 The lackof density likely reflects high flexibility or disorder of the myristylgroup, and this flexibility may correlate with different steps ofthe catalytic cycle.


Molecular features of product release for the PKA catalytic cycle.

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

Apo structure of the catalytic subunit of PKA. (A) Theoverallapo structure is displayed in ribbon representation with chain A coloredgray and chain B colored olive and with chains A and B aligned bythe entire protein. (B) (left) The myristic acid binding pocket forchains A and B, which are colored as in panel A, is displayed alongwith the 2Fo – Fc electron density at 1σ shown in blue for the myristicacid group from chain A. There is no electron density for myristicacid in chain B. (right) In contrast, the 2Fo – Fc electron densityat 1σ is visible for the entire myristic acid group in the closedstate (4DFX).25 (C) The 2Fo – Fc electron density at 1σ is shown in bluefor the C-terminal tail of chains A and B.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4295794&req=5

fig1: Apo structure of the catalytic subunit of PKA. (A) Theoverallapo structure is displayed in ribbon representation with chain A coloredgray and chain B colored olive and with chains A and B aligned bythe entire protein. (B) (left) The myristic acid binding pocket forchains A and B, which are colored as in panel A, is displayed alongwith the 2Fo – Fc electron density at 1σ shown in blue for the myristicacid group from chain A. There is no electron density for myristicacid in chain B. (right) In contrast, the 2Fo – Fc electron densityat 1σ is visible for the entire myristic acid group in the closedstate (4DFX).25 (C) The 2Fo – Fc electron density at 1σ is shown in bluefor the C-terminal tail of chains A and B.
Mentions: The myristylated C-subunitof PKA was crystallized in an apo state to determine any effects ofmyristylation in the absence of ligands. A K7C mutant was crystallizedbecause it increases the yield of the myristylated protein.25 The apo crystal structure was refined at 2.9Å resolution (Table 1, Figure 1A). There is electron density for myristic acidin one of the two molecules in the asymmetric unit (ASU); however,there is density for only 4 of the 14 carbon atoms of myristic acid(Figure 1B). The lack of electron density formyristic acid implies that the myristic acid group binding withinthe hydrophobic pocket could be involved in forming a closed statesince there is more electron density for the myristic acid group instructures that adopt a closed conformation, particularly in the binarycomplex that contains only peptide, compared with the open conformationadopted here in an apo state.25 The lackof density likely reflects high flexibility or disorder of the myristylgroup, and this flexibility may correlate with different steps ofthe catalytic cycle.

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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