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Structural and functional characterization of the human protein kinase ASK1.

Bunkoczi G, Salah E, Filippakopoulos P, Fedorov O, Müller S, Sobott F, Parker SA, Zhang H, Min W, Turk BE, Knapp S - Structure (2007)

Bottom Line: Here, we present the structure of the human ASK1 catalytic domain in complex with staurosporine.Reporter gene assays showed that all three identified in vitro autophosphorylation sites (Thr813, Thr838, Thr842) regulate ASK1 signaling, but site-directed mutants showed catalytic activities similar to wild-type ASK1, suggesting a regulatory mechanism independent of ASK1 kinase activity.The determined high-resolution structure of ASK1 and identified ATP mimetic inhibitors will provide a first starting point for the further development of selective inhibitors.

View Article: PubMed Central - PubMed

Affiliation: University of Oxford, Structural Genomics Consortium, Botnar Research Centre, Oxford OX3 7LD, United Kingdom.

ABSTRACT
Apoptosis signal-regulating kinase 1 (ASK1) plays an essential role in stress and immune response and has been linked to the development of several diseases. Here, we present the structure of the human ASK1 catalytic domain in complex with staurosporine. Analytical ultracentrifugation (AUC) and crystallographic analysis showed that ASK1 forms a tight dimer (K(d) approximately 0.2 microM) interacting in a head-to-tail fashion. We found that the ASK1 phosphorylation motifs differ from known ASK1 phosphorylation sites but correspond well to autophosphorylation sites identified by mass spectrometry. Reporter gene assays showed that all three identified in vitro autophosphorylation sites (Thr813, Thr838, Thr842) regulate ASK1 signaling, but site-directed mutants showed catalytic activities similar to wild-type ASK1, suggesting a regulatory mechanism independent of ASK1 kinase activity. The determined high-resolution structure of ASK1 and identified ATP mimetic inhibitors will provide a first starting point for the further development of selective inhibitors.

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Binding of StauroporineMain interacting active site residues with the ATP competitive inhibitor staurosporine are shown in ball-and-stick representation. Hydrogen bonds formed between the ligand and the protein are shown as dotted lines.
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fig2: Binding of StauroporineMain interacting active site residues with the ATP competitive inhibitor staurosporine are shown in ball-and-stick representation. Hydrogen bonds formed between the ligand and the protein are shown as dotted lines.

Mentions: Staurosporine binds to the ATP binding site of ASK1 in a mode that has been described for a number of kinase-staurosporine complexes (Underwood et al., 2003; Bertrand et al., 2003) (Figure 2). Two hydrogen bonds anchor the lactam moiety to the hinge region and mimic the hydrogen-bonding pattern of the adenine base. The stauroporine ligand forms a third hydrogen bond with the backbone oxygen of the catalytic loop residue Asn808, an interaction that has been described previously (Lamers et al., 1999; Lawrie et al., 1997; Prade et al., 1997; Zhao et al., 2002; Zhu et al., 1999). The heterocyclic condensed five-ring system packs against the hydrophobic side chains of the ASK1 residues Leu686, Val694, and Ala707 on the N-terminal lobe side as well as Val738 and Leu810 on the C-terminal lobe side of the binding pocket. In addition to staurosporine, we identified a number of diverse inhibitors in the melting temperature screen performed on a library of 158 known kinase inhibitors (Table S1 and Figure S1 in the Supplemental Data available with this article online). The inhibitors have been used in cocrystallization experiments, but well-diffracting crystals were only obtained in the presence of staurosporine.


Structural and functional characterization of the human protein kinase ASK1.

Bunkoczi G, Salah E, Filippakopoulos P, Fedorov O, Müller S, Sobott F, Parker SA, Zhang H, Min W, Turk BE, Knapp S - Structure (2007)

Binding of StauroporineMain interacting active site residues with the ATP competitive inhibitor staurosporine are shown in ball-and-stick representation. Hydrogen bonds formed between the ligand and the protein are shown as dotted lines.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2100151&req=5

fig2: Binding of StauroporineMain interacting active site residues with the ATP competitive inhibitor staurosporine are shown in ball-and-stick representation. Hydrogen bonds formed between the ligand and the protein are shown as dotted lines.
Mentions: Staurosporine binds to the ATP binding site of ASK1 in a mode that has been described for a number of kinase-staurosporine complexes (Underwood et al., 2003; Bertrand et al., 2003) (Figure 2). Two hydrogen bonds anchor the lactam moiety to the hinge region and mimic the hydrogen-bonding pattern of the adenine base. The stauroporine ligand forms a third hydrogen bond with the backbone oxygen of the catalytic loop residue Asn808, an interaction that has been described previously (Lamers et al., 1999; Lawrie et al., 1997; Prade et al., 1997; Zhao et al., 2002; Zhu et al., 1999). The heterocyclic condensed five-ring system packs against the hydrophobic side chains of the ASK1 residues Leu686, Val694, and Ala707 on the N-terminal lobe side as well as Val738 and Leu810 on the C-terminal lobe side of the binding pocket. In addition to staurosporine, we identified a number of diverse inhibitors in the melting temperature screen performed on a library of 158 known kinase inhibitors (Table S1 and Figure S1 in the Supplemental Data available with this article online). The inhibitors have been used in cocrystallization experiments, but well-diffracting crystals were only obtained in the presence of staurosporine.

Bottom Line: Here, we present the structure of the human ASK1 catalytic domain in complex with staurosporine.Reporter gene assays showed that all three identified in vitro autophosphorylation sites (Thr813, Thr838, Thr842) regulate ASK1 signaling, but site-directed mutants showed catalytic activities similar to wild-type ASK1, suggesting a regulatory mechanism independent of ASK1 kinase activity.The determined high-resolution structure of ASK1 and identified ATP mimetic inhibitors will provide a first starting point for the further development of selective inhibitors.

View Article: PubMed Central - PubMed

Affiliation: University of Oxford, Structural Genomics Consortium, Botnar Research Centre, Oxford OX3 7LD, United Kingdom.

ABSTRACT
Apoptosis signal-regulating kinase 1 (ASK1) plays an essential role in stress and immune response and has been linked to the development of several diseases. Here, we present the structure of the human ASK1 catalytic domain in complex with staurosporine. Analytical ultracentrifugation (AUC) and crystallographic analysis showed that ASK1 forms a tight dimer (K(d) approximately 0.2 microM) interacting in a head-to-tail fashion. We found that the ASK1 phosphorylation motifs differ from known ASK1 phosphorylation sites but correspond well to autophosphorylation sites identified by mass spectrometry. Reporter gene assays showed that all three identified in vitro autophosphorylation sites (Thr813, Thr838, Thr842) regulate ASK1 signaling, but site-directed mutants showed catalytic activities similar to wild-type ASK1, suggesting a regulatory mechanism independent of ASK1 kinase activity. The determined high-resolution structure of ASK1 and identified ATP mimetic inhibitors will provide a first starting point for the further development of selective inhibitors.

Show MeSH