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Structural insight into nucleotide recognition by human death-associated protein kinase.

McNamara LK, Watterson DM, Brunzelle JS - Acta Crystallogr. D Biol. Crystallogr. (2009)

Bottom Line: Death-associated protein kinase (DAPK) is a member of the Ca(2+)/calmodulin-regulated family of serine/threonine protein kinases.The structure of DAPK-ADP-Mg(2+) was compared with a newly determined DAPK-AMP-PNP-Mg(2+) structure and the previously determined apo DAPK structure (PDB code 1jks).The comparison shows that nucleotide-induced changes are localized to the glycine-rich loop region of DAPK.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Drug Discovery and Chemical Biology, Northwestern University, Chicago, Illinois 60611, USA.

ABSTRACT
Death-associated protein kinase (DAPK) is a member of the Ca(2+)/calmodulin-regulated family of serine/threonine protein kinases. The role of the kinase activity of DAPK in eukaryotic cell apoptosis and the ability of bioavailable DAPK inhibitors to rescue neuronal death after brain injury have made it a drug-discovery target for neurodegenerative disorders. In order to understand the recognition of nucleotides by DAPK and to gain insight into DAPK catalysis, the crystal structure of human DAPK was solved in complex with ADP and Mg(2+) at 1.85 A resolution. ADP is a product of the kinase reaction and product release is considered to be the rate-limiting step of protein kinase catalytic cycles. The structure of DAPK-ADP-Mg(2+) was compared with a newly determined DAPK-AMP-PNP-Mg(2+) structure and the previously determined apo DAPK structure (PDB code 1jks). The comparison shows that nucleotide-induced changes are localized to the glycine-rich loop region of DAPK.

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Comparisons of the glycine-rich loop. (a) Superposition of the glycine-rich loops of apo DAPK (beige), DAPK–ADP–Mg2+ (red) and DAPK–AMP-PNP–Mg2+ (blue) and their position relative to the nucleotide. The DAPK–AMP-PNP–Mg2+ loop has the most closed con­formation. Residues 17–28 are shown for simplicity. The calculated r.m.s. deviation over the Cα atoms of residues 20–25 between the apo DAPK structure and the DAPK–AMP-PNP–Mg2+ structure is 1.03 Å and that between the apo DAPK and DAPK–ADP–Mg2+ structures is 0.62 Å. (b) 2F                  o − F                  c electron-density map at 1.0σ of the glycine-rich loop of DAPK–ADP–Mg2+. One conformation of Gln23 is modeled such that the side chain is within proximity of the β-phosphate (conformation B). (c) 2F                  o − F                  c electron-density map of the glycine-rich loop of DAPK–AMP-PNP–Mg2+. The side chain of Phe24 in the DAPK–AMP-PNP structure can be modeled in the ‘open’ conformation (B) or a conformationally restricted position (A).
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fig5: Comparisons of the glycine-rich loop. (a) Superposition of the glycine-rich loops of apo DAPK (beige), DAPK–ADP–Mg2+ (red) and DAPK–AMP-PNP–Mg2+ (blue) and their position relative to the nucleotide. The DAPK–AMP-PNP–Mg2+ loop has the most closed con­formation. Residues 17–28 are shown for simplicity. The calculated r.m.s. deviation over the Cα atoms of residues 20–25 between the apo DAPK structure and the DAPK–AMP-PNP–Mg2+ structure is 1.03 Å and that between the apo DAPK and DAPK–ADP–Mg2+ structures is 0.62 Å. (b) 2F o − F c electron-density map at 1.0σ of the glycine-rich loop of DAPK–ADP–Mg2+. One conformation of Gln23 is modeled such that the side chain is within proximity of the β-phosphate (conformation B). (c) 2F o − F c electron-density map of the glycine-rich loop of DAPK–AMP-PNP–Mg2+. The side chain of Phe24 in the DAPK–AMP-PNP structure can be modeled in the ‘open’ conformation (B) or a conformationally restricted position (A).

Mentions: In a superposition of the ADP and AMP-PNP structures, the location of the magnesium ion differs by 1.2 Å (Fig. 5 ▶ a). The Mg2+ ion in the ADP structure is coordinated to six O atoms, which include the O1A atom of the α-phosphate, the O3B atom of the β-phosphate, a water molecule, Glu143 OE2, Asn144 OD1 and Asp161 OD1. In the AMP-PNP structure, the Mg2+ ion makes fewer interactions with the protein residues and only coordinates to Asp161 OD2, while other inter­actions are made with the O1A atom of the α-­phosphate, the O3G and O2G atoms of the γ-phosphate and a water molecule. An inspection of the crystal structure of an ADP complex obtained in the absence of Mg2+, DAPK–ADP (PDB code 3eh9), reveals an alternative conformation of the β-­phosphate. In this alternative conformation, the phosphate is in a similar location to the position where Mg2+ binds. This raises the possibility that the role of Mg2+ is to assist in the selection of a catalytically preferred conformation of the β-­phosphate. In the AMP-PNP structure obtained in the absence of Mg2+, DAPK–AMP-PNP (PDB code 3eha), only subtle changes in interaction distances are seen between the diaxial O atoms of the β- and γ-phosphates and the glycine-rich loop. For example, in the absence of Mg2+, the amide N interaction distances to the diaxial O atom of the β-phosphate are 3.3 Å to Ala25 and 2.9 Å to Asn24. In the Mg2+ structure, these distances are increased to 3.8 and 4.4 Å, respectively.


Structural insight into nucleotide recognition by human death-associated protein kinase.

McNamara LK, Watterson DM, Brunzelle JS - Acta Crystallogr. D Biol. Crystallogr. (2009)

Comparisons of the glycine-rich loop. (a) Superposition of the glycine-rich loops of apo DAPK (beige), DAPK–ADP–Mg2+ (red) and DAPK–AMP-PNP–Mg2+ (blue) and their position relative to the nucleotide. The DAPK–AMP-PNP–Mg2+ loop has the most closed con­formation. Residues 17–28 are shown for simplicity. The calculated r.m.s. deviation over the Cα atoms of residues 20–25 between the apo DAPK structure and the DAPK–AMP-PNP–Mg2+ structure is 1.03 Å and that between the apo DAPK and DAPK–ADP–Mg2+ structures is 0.62 Å. (b) 2F                  o − F                  c electron-density map at 1.0σ of the glycine-rich loop of DAPK–ADP–Mg2+. One conformation of Gln23 is modeled such that the side chain is within proximity of the β-phosphate (conformation B). (c) 2F                  o − F                  c electron-density map of the glycine-rich loop of DAPK–AMP-PNP–Mg2+. The side chain of Phe24 in the DAPK–AMP-PNP structure can be modeled in the ‘open’ conformation (B) or a conformationally restricted position (A).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2651756&req=5

fig5: Comparisons of the glycine-rich loop. (a) Superposition of the glycine-rich loops of apo DAPK (beige), DAPK–ADP–Mg2+ (red) and DAPK–AMP-PNP–Mg2+ (blue) and their position relative to the nucleotide. The DAPK–AMP-PNP–Mg2+ loop has the most closed con­formation. Residues 17–28 are shown for simplicity. The calculated r.m.s. deviation over the Cα atoms of residues 20–25 between the apo DAPK structure and the DAPK–AMP-PNP–Mg2+ structure is 1.03 Å and that between the apo DAPK and DAPK–ADP–Mg2+ structures is 0.62 Å. (b) 2F o − F c electron-density map at 1.0σ of the glycine-rich loop of DAPK–ADP–Mg2+. One conformation of Gln23 is modeled such that the side chain is within proximity of the β-phosphate (conformation B). (c) 2F o − F c electron-density map of the glycine-rich loop of DAPK–AMP-PNP–Mg2+. The side chain of Phe24 in the DAPK–AMP-PNP structure can be modeled in the ‘open’ conformation (B) or a conformationally restricted position (A).
Mentions: In a superposition of the ADP and AMP-PNP structures, the location of the magnesium ion differs by 1.2 Å (Fig. 5 ▶ a). The Mg2+ ion in the ADP structure is coordinated to six O atoms, which include the O1A atom of the α-phosphate, the O3B atom of the β-phosphate, a water molecule, Glu143 OE2, Asn144 OD1 and Asp161 OD1. In the AMP-PNP structure, the Mg2+ ion makes fewer interactions with the protein residues and only coordinates to Asp161 OD2, while other inter­actions are made with the O1A atom of the α-­phosphate, the O3G and O2G atoms of the γ-phosphate and a water molecule. An inspection of the crystal structure of an ADP complex obtained in the absence of Mg2+, DAPK–ADP (PDB code 3eh9), reveals an alternative conformation of the β-­phosphate. In this alternative conformation, the phosphate is in a similar location to the position where Mg2+ binds. This raises the possibility that the role of Mg2+ is to assist in the selection of a catalytically preferred conformation of the β-­phosphate. In the AMP-PNP structure obtained in the absence of Mg2+, DAPK–AMP-PNP (PDB code 3eha), only subtle changes in interaction distances are seen between the diaxial O atoms of the β- and γ-phosphates and the glycine-rich loop. For example, in the absence of Mg2+, the amide N interaction distances to the diaxial O atom of the β-phosphate are 3.3 Å to Ala25 and 2.9 Å to Asn24. In the Mg2+ structure, these distances are increased to 3.8 and 4.4 Å, respectively.

Bottom Line: Death-associated protein kinase (DAPK) is a member of the Ca(2+)/calmodulin-regulated family of serine/threonine protein kinases.The structure of DAPK-ADP-Mg(2+) was compared with a newly determined DAPK-AMP-PNP-Mg(2+) structure and the previously determined apo DAPK structure (PDB code 1jks).The comparison shows that nucleotide-induced changes are localized to the glycine-rich loop region of DAPK.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Drug Discovery and Chemical Biology, Northwestern University, Chicago, Illinois 60611, USA.

ABSTRACT
Death-associated protein kinase (DAPK) is a member of the Ca(2+)/calmodulin-regulated family of serine/threonine protein kinases. The role of the kinase activity of DAPK in eukaryotic cell apoptosis and the ability of bioavailable DAPK inhibitors to rescue neuronal death after brain injury have made it a drug-discovery target for neurodegenerative disorders. In order to understand the recognition of nucleotides by DAPK and to gain insight into DAPK catalysis, the crystal structure of human DAPK was solved in complex with ADP and Mg(2+) at 1.85 A resolution. ADP is a product of the kinase reaction and product release is considered to be the rate-limiting step of protein kinase catalytic cycles. The structure of DAPK-ADP-Mg(2+) was compared with a newly determined DAPK-AMP-PNP-Mg(2+) structure and the previously determined apo DAPK structure (PDB code 1jks). The comparison shows that nucleotide-induced changes are localized to the glycine-rich loop region of DAPK.

Show MeSH
Related in: MedlinePlus