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The Structural Basis for Activation and Inhibition of ZAP-70 Kinase Domain.

Huber RG, Fan H, Bond PJ - PLoS Comput. Biol. (2015)

Bottom Line: Furthermore, we rationalize previously observed staurosporine-bound crystal structures, suggesting that whilst the KD superficially resembles an "active-like" conformation, the inhibitor modulates the underlying protein dynamics and restricts it in a compact, rigid state inaccessible to ligands or cofactors.Finally, our analysis reveals a novel, potentially druggable pocket in close proximity to the activation loop of the kinase, and we subsequently use its structure in fragment-based virtual screening to develop a pharmacophore model.The pocket is distinct from classical type I or type II kinase pockets, and its discovery offers promise in future design of specific kinase inhibitors, whilst mutations in residues associated with this pocket are implicated in immunodeficiency in humans.

View Article: PubMed Central - PubMed

Affiliation: Bioinformatics Institute (BII), Agency for Science, Technology and Research (A*STAR), Singapore.

ABSTRACT
ZAP-70 (Zeta-chain-associated protein kinase 70) is a tyrosine kinase that interacts directly with the activated T-cell receptor to transduce downstream signals, and is hence a major player in the regulation of the adaptive immune response. Dysfunction of ZAP-70 causes selective T cell deficiency that in turn results in persistent infections. ZAP-70 is activated by a variety of signals including phosphorylation of the kinase domain (KD), and binding of its regulatory tandem Src homology 2 (SH2) domains to the T cell receptor. The present study investigates molecular mechanisms of activation and inhibition of ZAP-70 via atomically detailed molecular dynamics simulation approaches. We report microsecond timescale simulations of five distinct states of the ZAP-70 KD, comprising apo, inhibited and three phosphorylated variants. Extensive analysis of local flexibility and correlated motions reveal crucial transitions between the states, thus elucidating crucial steps in the activation mechanism of the ZAP-70 KD. Furthermore, we rationalize previously observed staurosporine-bound crystal structures, suggesting that whilst the KD superficially resembles an "active-like" conformation, the inhibitor modulates the underlying protein dynamics and restricts it in a compact, rigid state inaccessible to ligands or cofactors. Finally, our analysis reveals a novel, potentially druggable pocket in close proximity to the activation loop of the kinase, and we subsequently use its structure in fragment-based virtual screening to develop a pharmacophore model. The pocket is distinct from classical type I or type II kinase pockets, and its discovery offers promise in future design of specific kinase inhibitors, whilst mutations in residues associated with this pocket are implicated in immunodeficiency in humans.

No MeSH data available.


Related in: MedlinePlus

Overview of ZAP–70 kinase domain (PDB 1U59 [11]): Ligands staurosporine or ATP are located in the hinge region between the C-lobe (top) and N-lobe (bottom) of the protein (cartoons format) and are depicted in gray/CPK wireframe format.The αC helix, depicted in red, contains the salt bridge K369-E386 indicated in magenta/CPK wireframe. Phosphorylation sites Y492 and Y493 are indicated in cyan/CPK wireframe. The DFG motif, D479, F480 and G481, is indicated in blue/CPK wireframe. The activation loop comprises all residues from the DFG motif to seven residues beyond the phosphorylation sites. The N-lobal region of residues 537–569 (green) exhibits significant changes in flexibility depending upon phosphorylation state.
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pcbi.1004560.g001: Overview of ZAP–70 kinase domain (PDB 1U59 [11]): Ligands staurosporine or ATP are located in the hinge region between the C-lobe (top) and N-lobe (bottom) of the protein (cartoons format) and are depicted in gray/CPK wireframe format.The αC helix, depicted in red, contains the salt bridge K369-E386 indicated in magenta/CPK wireframe. Phosphorylation sites Y492 and Y493 are indicated in cyan/CPK wireframe. The DFG motif, D479, F480 and G481, is indicated in blue/CPK wireframe. The activation loop comprises all residues from the DFG motif to seven residues beyond the phosphorylation sites. The N-lobal region of residues 537–569 (green) exhibits significant changes in flexibility depending upon phosphorylation state.

Mentions: Available three-dimensional structures for the ZAP–70 KD include the isolated domain in complex with staurosporine (PDB 1U59 [11]), a well-characterized ATP-competitive inhibitor of kinases [11–12]. Moreover, structures of the full-length complex of ZAP–70 are available, with the KD bound to ANP, but auto-inhibited by its tandem of SH2 domains. The first such structure (PDB 2OZO [13]) included mutations which masked an inhibitory interface between regulatory domain and KD resolved in a subsequent, otherwise similar wild-type structure (PDB 4K2R [14]). Fig 1 illustrates the architecture of the isolated, inhibited KD and highlights significant functional regions. The domain exhibits the distinct bilobal architecture common to other protein kinases, with the activation loop containing Y492 and Y493 located between the two lobes. Staurosporine occupies the ATP binding pocket, which is located at the linkage region between lobes. Despite being bound to inhibitor, Jin et al. reported that the KD is in an active-like state, due to the conformation of the activation loop resembling the geometry of active states observed in the Syk kinase family. However, they noted that the activation loop forms a crystal contact in their structure. Hence, it is unclear whether the loop in the isolated staurosporine complex would likewise adopt an active conformation. Similar conformations of the non-phosphorylated activation loop have been observed for Chk1-staurosporine complexes, although these also involved crystal contacts (PDB 1NVR [15]). The salt bridge formed between K369 and E386 (residue numbers for ZAP–70), located in the αC helix comprising residues D379 to Q392, is a conserved motif of active kinase conformations [16] that is normally broken in inactive kinase states, but was formed in the staurosporine ZAP–70 complex. Deindl et al. observed striking similarities between auto-inhibited ZAP–70 and inhibited Hck [17] and c-Src kinase [18] structures. In comparison to the staurosporine complex, the auto-inhibited, ANP-bound crystal structures of ZAP–70 revealed that the αC helix is displaced outwards leading to a loss of this key salt bridge.


The Structural Basis for Activation and Inhibition of ZAP-70 Kinase Domain.

Huber RG, Fan H, Bond PJ - PLoS Comput. Biol. (2015)

Overview of ZAP–70 kinase domain (PDB 1U59 [11]): Ligands staurosporine or ATP are located in the hinge region between the C-lobe (top) and N-lobe (bottom) of the protein (cartoons format) and are depicted in gray/CPK wireframe format.The αC helix, depicted in red, contains the salt bridge K369-E386 indicated in magenta/CPK wireframe. Phosphorylation sites Y492 and Y493 are indicated in cyan/CPK wireframe. The DFG motif, D479, F480 and G481, is indicated in blue/CPK wireframe. The activation loop comprises all residues from the DFG motif to seven residues beyond the phosphorylation sites. The N-lobal region of residues 537–569 (green) exhibits significant changes in flexibility depending upon phosphorylation state.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4608720&req=5

pcbi.1004560.g001: Overview of ZAP–70 kinase domain (PDB 1U59 [11]): Ligands staurosporine or ATP are located in the hinge region between the C-lobe (top) and N-lobe (bottom) of the protein (cartoons format) and are depicted in gray/CPK wireframe format.The αC helix, depicted in red, contains the salt bridge K369-E386 indicated in magenta/CPK wireframe. Phosphorylation sites Y492 and Y493 are indicated in cyan/CPK wireframe. The DFG motif, D479, F480 and G481, is indicated in blue/CPK wireframe. The activation loop comprises all residues from the DFG motif to seven residues beyond the phosphorylation sites. The N-lobal region of residues 537–569 (green) exhibits significant changes in flexibility depending upon phosphorylation state.
Mentions: Available three-dimensional structures for the ZAP–70 KD include the isolated domain in complex with staurosporine (PDB 1U59 [11]), a well-characterized ATP-competitive inhibitor of kinases [11–12]. Moreover, structures of the full-length complex of ZAP–70 are available, with the KD bound to ANP, but auto-inhibited by its tandem of SH2 domains. The first such structure (PDB 2OZO [13]) included mutations which masked an inhibitory interface between regulatory domain and KD resolved in a subsequent, otherwise similar wild-type structure (PDB 4K2R [14]). Fig 1 illustrates the architecture of the isolated, inhibited KD and highlights significant functional regions. The domain exhibits the distinct bilobal architecture common to other protein kinases, with the activation loop containing Y492 and Y493 located between the two lobes. Staurosporine occupies the ATP binding pocket, which is located at the linkage region between lobes. Despite being bound to inhibitor, Jin et al. reported that the KD is in an active-like state, due to the conformation of the activation loop resembling the geometry of active states observed in the Syk kinase family. However, they noted that the activation loop forms a crystal contact in their structure. Hence, it is unclear whether the loop in the isolated staurosporine complex would likewise adopt an active conformation. Similar conformations of the non-phosphorylated activation loop have been observed for Chk1-staurosporine complexes, although these also involved crystal contacts (PDB 1NVR [15]). The salt bridge formed between K369 and E386 (residue numbers for ZAP–70), located in the αC helix comprising residues D379 to Q392, is a conserved motif of active kinase conformations [16] that is normally broken in inactive kinase states, but was formed in the staurosporine ZAP–70 complex. Deindl et al. observed striking similarities between auto-inhibited ZAP–70 and inhibited Hck [17] and c-Src kinase [18] structures. In comparison to the staurosporine complex, the auto-inhibited, ANP-bound crystal structures of ZAP–70 revealed that the αC helix is displaced outwards leading to a loss of this key salt bridge.

Bottom Line: Furthermore, we rationalize previously observed staurosporine-bound crystal structures, suggesting that whilst the KD superficially resembles an "active-like" conformation, the inhibitor modulates the underlying protein dynamics and restricts it in a compact, rigid state inaccessible to ligands or cofactors.Finally, our analysis reveals a novel, potentially druggable pocket in close proximity to the activation loop of the kinase, and we subsequently use its structure in fragment-based virtual screening to develop a pharmacophore model.The pocket is distinct from classical type I or type II kinase pockets, and its discovery offers promise in future design of specific kinase inhibitors, whilst mutations in residues associated with this pocket are implicated in immunodeficiency in humans.

View Article: PubMed Central - PubMed

Affiliation: Bioinformatics Institute (BII), Agency for Science, Technology and Research (A*STAR), Singapore.

ABSTRACT
ZAP-70 (Zeta-chain-associated protein kinase 70) is a tyrosine kinase that interacts directly with the activated T-cell receptor to transduce downstream signals, and is hence a major player in the regulation of the adaptive immune response. Dysfunction of ZAP-70 causes selective T cell deficiency that in turn results in persistent infections. ZAP-70 is activated by a variety of signals including phosphorylation of the kinase domain (KD), and binding of its regulatory tandem Src homology 2 (SH2) domains to the T cell receptor. The present study investigates molecular mechanisms of activation and inhibition of ZAP-70 via atomically detailed molecular dynamics simulation approaches. We report microsecond timescale simulations of five distinct states of the ZAP-70 KD, comprising apo, inhibited and three phosphorylated variants. Extensive analysis of local flexibility and correlated motions reveal crucial transitions between the states, thus elucidating crucial steps in the activation mechanism of the ZAP-70 KD. Furthermore, we rationalize previously observed staurosporine-bound crystal structures, suggesting that whilst the KD superficially resembles an "active-like" conformation, the inhibitor modulates the underlying protein dynamics and restricts it in a compact, rigid state inaccessible to ligands or cofactors. Finally, our analysis reveals a novel, potentially druggable pocket in close proximity to the activation loop of the kinase, and we subsequently use its structure in fragment-based virtual screening to develop a pharmacophore model. The pocket is distinct from classical type I or type II kinase pockets, and its discovery offers promise in future design of specific kinase inhibitors, whilst mutations in residues associated with this pocket are implicated in immunodeficiency in humans.

No MeSH data available.


Related in: MedlinePlus