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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

Specific structural features of the various states of ZAP70 kinase domain: (a) The salt bridge D379-R496 and the intermittent hydrogen bonding between N348-S497 (yellow) connect the C- and N-lobes and restrict access to the catalytic cleft.Additionally these bonds restrict activation loop positioning. (b) The cryptic pocket adjacent to the activation loop spontaneously opened during simulation and was unique to the non-phosphorylated state. Key residues lining the pocket are indicated in gray. Four consensus pharmacophore features were identified by fragment screening. These comprise the acceptor A, donor D, ring R, and a hydrophobic feature H, indicated inset. (c) The cryptic pocket repeatedly opened and closed over the final ~1μs of simulation time, reaching a maximum volume of ~1400 Å 3
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pcbi.1004560.g004: Specific structural features of the various states of ZAP70 kinase domain: (a) The salt bridge D379-R496 and the intermittent hydrogen bonding between N348-S497 (yellow) connect the C- and N-lobes and restrict access to the catalytic cleft.Additionally these bonds restrict activation loop positioning. (b) The cryptic pocket adjacent to the activation loop spontaneously opened during simulation and was unique to the non-phosphorylated state. Key residues lining the pocket are indicated in gray. Four consensus pharmacophore features were identified by fragment screening. These comprise the acceptor A, donor D, ring R, and a hydrophobic feature H, indicated inset. (c) The cryptic pocket repeatedly opened and closed over the final ~1μs of simulation time, reaching a maximum volume of ~1400 Å 3

Mentions: Structural features specific for the staurosporine complex during our simulations comprise an additional salt bridge (D379-R496) formed across the catalytic cleft, and an adjacent intermittent hydrogen bond between the S497 hydroxyl and the N348 side chain amide group. This interaction is not observed in any X-ray structures of ZAP–70. Moreover, this behavior was not observed in any other complex. These interactions span the catalytic cleft and connect the N- and C-lobes, thereby significantly restricting substrate access. Moreover, these stabilize the position of the activation loop. These structural features are illustrated in Fig 4a. Additionally, an intra-strand interaction hydrogen bond between the side chains of R514 and Y493 could be discerned for significant parts of the trajectories even in the absence of Y493 phosphorylation.


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

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

Specific structural features of the various states of ZAP70 kinase domain: (a) The salt bridge D379-R496 and the intermittent hydrogen bonding between N348-S497 (yellow) connect the C- and N-lobes and restrict access to the catalytic cleft.Additionally these bonds restrict activation loop positioning. (b) The cryptic pocket adjacent to the activation loop spontaneously opened during simulation and was unique to the non-phosphorylated state. Key residues lining the pocket are indicated in gray. Four consensus pharmacophore features were identified by fragment screening. These comprise the acceptor A, donor D, ring R, and a hydrophobic feature H, indicated inset. (c) The cryptic pocket repeatedly opened and closed over the final ~1μs of simulation time, reaching a maximum volume of ~1400 Å 3
© Copyright Policy
Related In: Results  -  Collection

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

pcbi.1004560.g004: Specific structural features of the various states of ZAP70 kinase domain: (a) The salt bridge D379-R496 and the intermittent hydrogen bonding between N348-S497 (yellow) connect the C- and N-lobes and restrict access to the catalytic cleft.Additionally these bonds restrict activation loop positioning. (b) The cryptic pocket adjacent to the activation loop spontaneously opened during simulation and was unique to the non-phosphorylated state. Key residues lining the pocket are indicated in gray. Four consensus pharmacophore features were identified by fragment screening. These comprise the acceptor A, donor D, ring R, and a hydrophobic feature H, indicated inset. (c) The cryptic pocket repeatedly opened and closed over the final ~1μs of simulation time, reaching a maximum volume of ~1400 Å 3
Mentions: Structural features specific for the staurosporine complex during our simulations comprise an additional salt bridge (D379-R496) formed across the catalytic cleft, and an adjacent intermittent hydrogen bond between the S497 hydroxyl and the N348 side chain amide group. This interaction is not observed in any X-ray structures of ZAP–70. Moreover, this behavior was not observed in any other complex. These interactions span the catalytic cleft and connect the N- and C-lobes, thereby significantly restricting substrate access. Moreover, these stabilize the position of the activation loop. These structural features are illustrated in Fig 4a. Additionally, an intra-strand interaction hydrogen bond between the side chains of R514 and Y493 could be discerned for significant parts of the trajectories even in the absence of 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