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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

Proposed activation cascade of ZAP–70 kinase domain through the phosphorylation of Y493: upon phosphorylation of Y493 (green triangle), the salt bridge K369-E386 weakens and thus allows for movement of the αC helix (red) towards an active conformation.Concurrently, the activation loop (black) becomes more dynamic, thus allowing easier access to the active center.
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pcbi.1004560.g007: Proposed activation cascade of ZAP–70 kinase domain through the phosphorylation of Y493: upon phosphorylation of Y493 (green triangle), the salt bridge K369-E386 weakens and thus allows for movement of the αC helix (red) towards an active conformation.Concurrently, the activation loop (black) becomes more dynamic, thus allowing easier access to the active center.

Mentions: Phosphorylation was observed to lead to a global increase in dynamics, irrespective of the precise phosphorylation state. Double phosphorylation at Y492 and Y493 had a significantly stronger mobilizing effect than single phosphorylation of either residue. However, structural changes were distinctly different for the individual mono-phosphorylated complexes. Mutational studies suggest that Y492 is only weakly implicated in biological activation of ZAP–70. However, Y493 phosphorylation is of crucial importance to biological function as the Y493F mutation abolishes catalytic activity. Our simulations allow us to identify changes that are specific to Y493 phosphorylation and therefore let us trace the activation cascade: Y493 phosphorylation causes the salt bridge R369-E386 connecting the C-lobe with the αC helix to weaken. In contrast to Y492 phosphorylation, Y493 phosphorylation also promotes rearrangement of the αC helix towards an active conformation already observed for members of the Src kinase family. Concurrently, flexibility of the activation loop increases significantly, thus allowing for easier access to the catalytic center. A similar increase in activation loop exposure has been observed in the activation of focal adhesion kinase (FAK). However, the activation cascade of FAK is not directly analogous to ZAP–70, as FAK has an additional FERM domain involved in forming an auto-inhibited complex. [21] Our proposed activation cascade for ZAP–70 is summarized in Fig 7. Normal mode analysis further confirms these motions as characteristic for the biologically relevant Y493 phosphorylation.


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

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

Proposed activation cascade of ZAP–70 kinase domain through the phosphorylation of Y493: upon phosphorylation of Y493 (green triangle), the salt bridge K369-E386 weakens and thus allows for movement of the αC helix (red) towards an active conformation.Concurrently, the activation loop (black) becomes more dynamic, thus allowing easier access to the active center.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi.1004560.g007: Proposed activation cascade of ZAP–70 kinase domain through the phosphorylation of Y493: upon phosphorylation of Y493 (green triangle), the salt bridge K369-E386 weakens and thus allows for movement of the αC helix (red) towards an active conformation.Concurrently, the activation loop (black) becomes more dynamic, thus allowing easier access to the active center.
Mentions: Phosphorylation was observed to lead to a global increase in dynamics, irrespective of the precise phosphorylation state. Double phosphorylation at Y492 and Y493 had a significantly stronger mobilizing effect than single phosphorylation of either residue. However, structural changes were distinctly different for the individual mono-phosphorylated complexes. Mutational studies suggest that Y492 is only weakly implicated in biological activation of ZAP–70. However, Y493 phosphorylation is of crucial importance to biological function as the Y493F mutation abolishes catalytic activity. Our simulations allow us to identify changes that are specific to Y493 phosphorylation and therefore let us trace the activation cascade: Y493 phosphorylation causes the salt bridge R369-E386 connecting the C-lobe with the αC helix to weaken. In contrast to Y492 phosphorylation, Y493 phosphorylation also promotes rearrangement of the αC helix towards an active conformation already observed for members of the Src kinase family. Concurrently, flexibility of the activation loop increases significantly, thus allowing for easier access to the catalytic center. A similar increase in activation loop exposure has been observed in the activation of focal adhesion kinase (FAK). However, the activation cascade of FAK is not directly analogous to ZAP–70, as FAK has an additional FERM domain involved in forming an auto-inhibited complex. [21] Our proposed activation cascade for ZAP–70 is summarized in Fig 7. Normal mode analysis further confirms these motions as characteristic for the biologically relevant Y493 phosphorylation.

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