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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

Conformational dynamics of ZAP–70 kinase domain.(a) Cα RMSD, (b) B-factors over final 500 ns, and (c) B-factor variance over 10 sequential independent trajectory segments of all investigated systems. All phosphorylated states exhibit elevated flexibility compared to the staurosporine-inhibited and the ATP-bound non-phosphorylated complexes. Regions of interest comprise the N-lobal residues 500–519 and the “flap-like” residues 537–569.
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pcbi.1004560.g002: Conformational dynamics of ZAP–70 kinase domain.(a) Cα RMSD, (b) B-factors over final 500 ns, and (c) B-factor variance over 10 sequential independent trajectory segments of all investigated systems. All phosphorylated states exhibit elevated flexibility compared to the staurosporine-inhibited and the ATP-bound non-phosphorylated complexes. Regions of interest comprise the N-lobal residues 500–519 and the “flap-like” residues 537–569.

Mentions: The staurosporine-bound complex remained stable throughout the course of the simulation. This is illustrated by the constant Cα-RMSD (Fig 2a), which rapidly plateaued at ~2–3 Å, as well as the low B-factors (Fig 2b) across the entire domain and associated variance throughout the trajectory (Fig 2c). Residual flexibility was observed within the DFG motif, the activation loop downstream of the unmodified phosphorylation sites Y492 and Y493 and the solvent-exposed regions of the αC helix. The DFG motif was observed to form a stable, closed loop structure through a hydrogen bond from the side chain carboxylate-oxygen on D479 through the backbone amide hydrogen on G481 (Fig 3a). This closed loop structure is also present in the staurosporine X-ray structure, 1U59. The distance of the αC helix from the center of mass of the C-lobe initially increased from a value of 14 Å (in the crystallographic state) to 15 Å within ~150 ns and remained stable at this level for the remainder of the trajectory (Fig 3b). Moreover, the phenyl ring of F480 of the DFG motif forms a close contact with the backbone amide of M390 within the αC helix (S1 Fig). The salt bridge K369-E386 was nevertheless present during the entire course of the simulation (Fig 3c). It is present in the staurosporine complex structure 1U59 but absent in the ANP complexes 2OZO and 4K2R.


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

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

Conformational dynamics of ZAP–70 kinase domain.(a) Cα RMSD, (b) B-factors over final 500 ns, and (c) B-factor variance over 10 sequential independent trajectory segments of all investigated systems. All phosphorylated states exhibit elevated flexibility compared to the staurosporine-inhibited and the ATP-bound non-phosphorylated complexes. Regions of interest comprise the N-lobal residues 500–519 and the “flap-like” residues 537–569.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi.1004560.g002: Conformational dynamics of ZAP–70 kinase domain.(a) Cα RMSD, (b) B-factors over final 500 ns, and (c) B-factor variance over 10 sequential independent trajectory segments of all investigated systems. All phosphorylated states exhibit elevated flexibility compared to the staurosporine-inhibited and the ATP-bound non-phosphorylated complexes. Regions of interest comprise the N-lobal residues 500–519 and the “flap-like” residues 537–569.
Mentions: The staurosporine-bound complex remained stable throughout the course of the simulation. This is illustrated by the constant Cα-RMSD (Fig 2a), which rapidly plateaued at ~2–3 Å, as well as the low B-factors (Fig 2b) across the entire domain and associated variance throughout the trajectory (Fig 2c). Residual flexibility was observed within the DFG motif, the activation loop downstream of the unmodified phosphorylation sites Y492 and Y493 and the solvent-exposed regions of the αC helix. The DFG motif was observed to form a stable, closed loop structure through a hydrogen bond from the side chain carboxylate-oxygen on D479 through the backbone amide hydrogen on G481 (Fig 3a). This closed loop structure is also present in the staurosporine X-ray structure, 1U59. The distance of the αC helix from the center of mass of the C-lobe initially increased from a value of 14 Å (in the crystallographic state) to 15 Å within ~150 ns and remained stable at this level for the remainder of the trajectory (Fig 3b). Moreover, the phenyl ring of F480 of the DFG motif forms a close contact with the backbone amide of M390 within the αC helix (S1 Fig). The salt bridge K369-E386 was nevertheless present during the entire course of the simulation (Fig 3c). It is present in the staurosporine complex structure 1U59 but absent in the ANP complexes 2OZO and 4K2R.

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