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An autoinhibitory tyrosine motif in the cell-cycle-regulated Nek7 kinase is released through binding of Nek9.

Richards MW, O'Regan L, Mas-Droux C, Blot JM, Cheung J, Hoelder S, Fry AM, Bayliss R - Mol. Cell (2009)

Bottom Line: Tyrosine mutants of Nek7 and the related kinase Nek6 are constitutively active.The activity of Nek6 and Nek7, but not the tyrosine mutant, is increased by interaction with the Nek9 noncatalytic C-terminal domain, suggesting a mechanism in which the tyrosine is released from its autoinhibitory position.The autoinhibitory conformation is common to three Neks and provides a potential target for selective kinase inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cancer Research, London, UK.

ABSTRACT
Mitosis is controlled by multiple protein kinases, many of which are abnormally expressed in human cancers. Nek2, Nek6, Nek7, and Nek9 are NIMA-related kinases essential for proper mitotic progression. We determined the atomic structure of Nek7 and discovered an autoinhibited conformation that suggests a regulatory mechanism not previously described in kinases. Additionally, Nek2 adopts the same conformation when bound to a drug-like molecule. In both structures, a tyrosine side chain points into the active site, interacts with the activation loop, and blocks the alphaC helix. Tyrosine mutants of Nek7 and the related kinase Nek6 are constitutively active. The activity of Nek6 and Nek7, but not the tyrosine mutant, is increased by interaction with the Nek9 noncatalytic C-terminal domain, suggesting a mechanism in which the tyrosine is released from its autoinhibitory position. The autoinhibitory conformation is common to three Neks and provides a potential target for selective kinase inhibitors.

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Crystal Structure of Nek2 Bound to a Drug-like Molecule that Induces the Tyr-Down Conformation(A) Nek2-CCT241950 structure shown in cartoon representation in the equivalent view to that shown in Figure 1B. CCT241950 and Tyr70Nek2 are shown as spheres. The dashed box indicates the region magnified in (C).(B) Chemical structure of CCT241950 with potential H-bonds marked as dashed lines.(C) Cartoon and stick representation of Tyr70Nek2 and its surrounding environment. The putative H-bonds that form a network between CCT241950 and the protein are shown as dashed lines.
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fig4: Crystal Structure of Nek2 Bound to a Drug-like Molecule that Induces the Tyr-Down Conformation(A) Nek2-CCT241950 structure shown in cartoon representation in the equivalent view to that shown in Figure 1B. CCT241950 and Tyr70Nek2 are shown as spheres. The dashed box indicates the region magnified in (C).(B) Chemical structure of CCT241950 with potential H-bonds marked as dashed lines.(C) Cartoon and stick representation of Tyr70Nek2 and its surrounding environment. The putative H-bonds that form a network between CCT241950 and the protein are shown as dashed lines.

Mentions: We have previously described crystals of Nek2-ADP complex, and we have used this system to investigate the binding of small molecules to the ADP-binding pocket (Westwood et al., 2009). CCT241950 induces Tyr70Nek2 to jut into the ADP-binding pocket, similar to the Tyr-down conformation of Nek7 (Figure 4A). Tyr70Nek2 is the equivalent residue to Tyr97Nek7, and in the ADP-bound and other Nek2 structures in the PDB this residue is in the Tyr-up configuration (Figure 5A). CCT241950 is based on an aminopyridine scaffold, which has formed the core of c-Jun and Met inhibitors, although the pattern of H-bonds formed between the scaffold and the Nek2 hinge residues is different from what was observed in the crystal structures of these other two kinases (Figure 4B). The benzoic acid moiety of CCT241950 lies at the center of a network of putative H-bonds that connect the side chains of Lys37 Nek2 and Tyr70 Nek2 and the mainchain of Asp159 Nek2 (Figure 4C). By analogy with the H-bond formed between the DLG motif and Tyr97 of Nek7, this H-bond network is presumably a key determinant of the Tyr-down conformation in Nek2. In support of this assertion, the superposition of the Nek2-ADP and Nek2-CCT241950 structures shows that the conformational changes in the protein primarily involve side-chain movements of the residues that participate in the H-bond network (Figures 5A and 5B). The other major change involves the movement of Met86Nek2 and Leu59Nek2 side chains to create a pocket into which the side chain of Tyr70Nek2 inserts. In the Nek2-ADP structure, the closest contact between Met86Nek2 and Leu59Nek2 is 4.2 Å, whereas in the Nek2-CCT241950 structure it is 7.6 Å. The Met86Nek2 gatekeeper, which is flexible and adopts two rotamers in the ADP-bound structure, moves toward the compound, and the sulfur atom packs against the aromatic ring of the CCT241950 benzoic acid group. Overall there are no substantial changes in the backbone (0.36 Å Cα rmsd), although part of the Gly-rich loop and the activation loop are disordered in the CCT241950 structure, perhaps reflecting the loss of stabilizing interactions between the phosphate groups of ADP and the Gly-rich loop and the changes to the activation loop conformation induced through its interaction with CCT241950 and Tyr70Nek2. There are no substantial backbone changes (i.e., >1 Å C α rmsd) in the vicinity of Tyr70Nek2, which could be because the conformation is constrained by the crystal lattice. Nonetheless, the structure demonstrates that Nek2 can adopt the Tyr-down conformation, with relatively small changes in the overall structure.


An autoinhibitory tyrosine motif in the cell-cycle-regulated Nek7 kinase is released through binding of Nek9.

Richards MW, O'Regan L, Mas-Droux C, Blot JM, Cheung J, Hoelder S, Fry AM, Bayliss R - Mol. Cell (2009)

Crystal Structure of Nek2 Bound to a Drug-like Molecule that Induces the Tyr-Down Conformation(A) Nek2-CCT241950 structure shown in cartoon representation in the equivalent view to that shown in Figure 1B. CCT241950 and Tyr70Nek2 are shown as spheres. The dashed box indicates the region magnified in (C).(B) Chemical structure of CCT241950 with potential H-bonds marked as dashed lines.(C) Cartoon and stick representation of Tyr70Nek2 and its surrounding environment. The putative H-bonds that form a network between CCT241950 and the protein are shown as dashed lines.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2807034&req=5

fig4: Crystal Structure of Nek2 Bound to a Drug-like Molecule that Induces the Tyr-Down Conformation(A) Nek2-CCT241950 structure shown in cartoon representation in the equivalent view to that shown in Figure 1B. CCT241950 and Tyr70Nek2 are shown as spheres. The dashed box indicates the region magnified in (C).(B) Chemical structure of CCT241950 with potential H-bonds marked as dashed lines.(C) Cartoon and stick representation of Tyr70Nek2 and its surrounding environment. The putative H-bonds that form a network between CCT241950 and the protein are shown as dashed lines.
Mentions: We have previously described crystals of Nek2-ADP complex, and we have used this system to investigate the binding of small molecules to the ADP-binding pocket (Westwood et al., 2009). CCT241950 induces Tyr70Nek2 to jut into the ADP-binding pocket, similar to the Tyr-down conformation of Nek7 (Figure 4A). Tyr70Nek2 is the equivalent residue to Tyr97Nek7, and in the ADP-bound and other Nek2 structures in the PDB this residue is in the Tyr-up configuration (Figure 5A). CCT241950 is based on an aminopyridine scaffold, which has formed the core of c-Jun and Met inhibitors, although the pattern of H-bonds formed between the scaffold and the Nek2 hinge residues is different from what was observed in the crystal structures of these other two kinases (Figure 4B). The benzoic acid moiety of CCT241950 lies at the center of a network of putative H-bonds that connect the side chains of Lys37 Nek2 and Tyr70 Nek2 and the mainchain of Asp159 Nek2 (Figure 4C). By analogy with the H-bond formed between the DLG motif and Tyr97 of Nek7, this H-bond network is presumably a key determinant of the Tyr-down conformation in Nek2. In support of this assertion, the superposition of the Nek2-ADP and Nek2-CCT241950 structures shows that the conformational changes in the protein primarily involve side-chain movements of the residues that participate in the H-bond network (Figures 5A and 5B). The other major change involves the movement of Met86Nek2 and Leu59Nek2 side chains to create a pocket into which the side chain of Tyr70Nek2 inserts. In the Nek2-ADP structure, the closest contact between Met86Nek2 and Leu59Nek2 is 4.2 Å, whereas in the Nek2-CCT241950 structure it is 7.6 Å. The Met86Nek2 gatekeeper, which is flexible and adopts two rotamers in the ADP-bound structure, moves toward the compound, and the sulfur atom packs against the aromatic ring of the CCT241950 benzoic acid group. Overall there are no substantial changes in the backbone (0.36 Å Cα rmsd), although part of the Gly-rich loop and the activation loop are disordered in the CCT241950 structure, perhaps reflecting the loss of stabilizing interactions between the phosphate groups of ADP and the Gly-rich loop and the changes to the activation loop conformation induced through its interaction with CCT241950 and Tyr70Nek2. There are no substantial backbone changes (i.e., >1 Å C α rmsd) in the vicinity of Tyr70Nek2, which could be because the conformation is constrained by the crystal lattice. Nonetheless, the structure demonstrates that Nek2 can adopt the Tyr-down conformation, with relatively small changes in the overall structure.

Bottom Line: Tyrosine mutants of Nek7 and the related kinase Nek6 are constitutively active.The activity of Nek6 and Nek7, but not the tyrosine mutant, is increased by interaction with the Nek9 noncatalytic C-terminal domain, suggesting a mechanism in which the tyrosine is released from its autoinhibitory position.The autoinhibitory conformation is common to three Neks and provides a potential target for selective kinase inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cancer Research, London, UK.

ABSTRACT
Mitosis is controlled by multiple protein kinases, many of which are abnormally expressed in human cancers. Nek2, Nek6, Nek7, and Nek9 are NIMA-related kinases essential for proper mitotic progression. We determined the atomic structure of Nek7 and discovered an autoinhibited conformation that suggests a regulatory mechanism not previously described in kinases. Additionally, Nek2 adopts the same conformation when bound to a drug-like molecule. In both structures, a tyrosine side chain points into the active site, interacts with the activation loop, and blocks the alphaC helix. Tyrosine mutants of Nek7 and the related kinase Nek6 are constitutively active. The activity of Nek6 and Nek7, but not the tyrosine mutant, is increased by interaction with the Nek9 noncatalytic C-terminal domain, suggesting a mechanism in which the tyrosine is released from its autoinhibitory position. The autoinhibitory conformation is common to three Neks and provides a potential target for selective kinase inhibitors.

Show MeSH
Related in: MedlinePlus