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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

Conformations of the DFG motif: Closed (gray), semi-closed (green) and open conformation (cyan). Staurosporine causes the DFG motif in ZAP–70 to adopt the closed conformation exclusively, presumably because D479 is unable to interact with the Mg2+ ion coordinated by ATP. The Y0Y0 and Y0YP variants predominantly adopt the semi-closed form. The open extended geometry is only observed in the YPY0 and YPYP states. Labels within the figure indicate measured distances, in Angstroms.
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pcbi.1004560.g006: Conformations of the DFG motif: Closed (gray), semi-closed (green) and open conformation (cyan). Staurosporine causes the DFG motif in ZAP–70 to adopt the closed conformation exclusively, presumably because D479 is unable to interact with the Mg2+ ion coordinated by ATP. The Y0Y0 and Y0YP variants predominantly adopt the semi-closed form. The open extended geometry is only observed in the YPY0 and YPYP states. Labels within the figure indicate measured distances, in Angstroms.

Mentions: Common patterns observed in all simulated states were characterized by two distinct, comparatively rigid cores of the C-lobe and N-lobe, as well as flexible segments of the activation loop and the region formed by residues 537–569. Baseline flexibility in the non-phosphorylated and inhibited states was significantly lower than in either mono- or di-phosphorylated states, as indicated by the calculated B-factors. In order to differentiate between the alternative phosphorylated states, it should be noted that experimentally Y492F does not adversely affect ZAP–70 activity, whereas Y493F abolishes ZAP–70 function. [8] Therefore, we can use the effects of Y492 phosphorylation as a baseline for dynamic changes induced by monophosphorylation at the C-terminal end of the activation loop and contrast its effects with those observed in states containing phosphorylated Y493. The differences in dynamics between the two states offer some indication of the functional relevance of the observed changes across systems. Whereas phosphorylation caused a global increase in protein flexibility, specific changes were localized around the activation loop as well as the αC helix region. Strikingly, the conserved DFG motif at the N-terminal side of the activation loop adopted three distinct states across the different simulation systems, characterized by a cyclization through hydrogen bonding. By considering the distance between the carboxylate carbon of D479 and the backbone amide hydrogen of G481, we could identify a cyclic, closed state at approximately 3 Å, a semi-closed state at a distance of ~6 Å, and an open state at a distance of ~8 Å (Fig 6). We surmise that the semi-closed state of the DFG motif is relevant for the catalytic activity in ZAP–70 as it occurs in the un-phosphorylated state as well as in the Y0YP state. YPYP and YPY0 bias the DFG conformation towards the open state whereas staurosporine inhibition results in stabilization of the closed state. We postulate that YPY0 causes a repulsive charge-charge interaction with D479, thereby promoting the open state. As staurosporine is bereft of a negative charge proximal to the DFG motif and misses an Mg2+ ion, D479 is free to position itself in a hydrogen bonding orientation towards the backbone N-H of G481. This behavior of the DFG motif is consistent with differing orientations of D479 observed in the staurosporine-bound X-ray structure 1U59 versus the ANP-bound complexes 2OZO and 4K2R. Generally, our observations signify that residue D479 located close to the catalytic center is highly sensitive to its local electrostatic environment. It should be noted that all simulations started from the staurosporine-bound protein conformation represented by 1U59, as it is the only structure in which all residues of the activation loop are resolved. While the choice of this starting state may introduce a bias towards active-like states in the remaining simulations, the reorientation of the DFG motif is consistent with the 2OZO and 4K2R structures. Thus we assume that the simulation times are sufficient to sample at a minimum conformational transitions between the active and intermediate states [19]. Despite the length of our trajectories, we were unable to observe DFG-in/DFG-out transitions as indicated by the interaction of F480 with M390 in the αC helix (S1 Fig).


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

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

Conformations of the DFG motif: Closed (gray), semi-closed (green) and open conformation (cyan). Staurosporine causes the DFG motif in ZAP–70 to adopt the closed conformation exclusively, presumably because D479 is unable to interact with the Mg2+ ion coordinated by ATP. The Y0Y0 and Y0YP variants predominantly adopt the semi-closed form. The open extended geometry is only observed in the YPY0 and YPYP states. Labels within the figure indicate measured distances, in Angstroms.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi.1004560.g006: Conformations of the DFG motif: Closed (gray), semi-closed (green) and open conformation (cyan). Staurosporine causes the DFG motif in ZAP–70 to adopt the closed conformation exclusively, presumably because D479 is unable to interact with the Mg2+ ion coordinated by ATP. The Y0Y0 and Y0YP variants predominantly adopt the semi-closed form. The open extended geometry is only observed in the YPY0 and YPYP states. Labels within the figure indicate measured distances, in Angstroms.
Mentions: Common patterns observed in all simulated states were characterized by two distinct, comparatively rigid cores of the C-lobe and N-lobe, as well as flexible segments of the activation loop and the region formed by residues 537–569. Baseline flexibility in the non-phosphorylated and inhibited states was significantly lower than in either mono- or di-phosphorylated states, as indicated by the calculated B-factors. In order to differentiate between the alternative phosphorylated states, it should be noted that experimentally Y492F does not adversely affect ZAP–70 activity, whereas Y493F abolishes ZAP–70 function. [8] Therefore, we can use the effects of Y492 phosphorylation as a baseline for dynamic changes induced by monophosphorylation at the C-terminal end of the activation loop and contrast its effects with those observed in states containing phosphorylated Y493. The differences in dynamics between the two states offer some indication of the functional relevance of the observed changes across systems. Whereas phosphorylation caused a global increase in protein flexibility, specific changes were localized around the activation loop as well as the αC helix region. Strikingly, the conserved DFG motif at the N-terminal side of the activation loop adopted three distinct states across the different simulation systems, characterized by a cyclization through hydrogen bonding. By considering the distance between the carboxylate carbon of D479 and the backbone amide hydrogen of G481, we could identify a cyclic, closed state at approximately 3 Å, a semi-closed state at a distance of ~6 Å, and an open state at a distance of ~8 Å (Fig 6). We surmise that the semi-closed state of the DFG motif is relevant for the catalytic activity in ZAP–70 as it occurs in the un-phosphorylated state as well as in the Y0YP state. YPYP and YPY0 bias the DFG conformation towards the open state whereas staurosporine inhibition results in stabilization of the closed state. We postulate that YPY0 causes a repulsive charge-charge interaction with D479, thereby promoting the open state. As staurosporine is bereft of a negative charge proximal to the DFG motif and misses an Mg2+ ion, D479 is free to position itself in a hydrogen bonding orientation towards the backbone N-H of G481. This behavior of the DFG motif is consistent with differing orientations of D479 observed in the staurosporine-bound X-ray structure 1U59 versus the ANP-bound complexes 2OZO and 4K2R. Generally, our observations signify that residue D479 located close to the catalytic center is highly sensitive to its local electrostatic environment. It should be noted that all simulations started from the staurosporine-bound protein conformation represented by 1U59, as it is the only structure in which all residues of the activation loop are resolved. While the choice of this starting state may introduce a bias towards active-like states in the remaining simulations, the reorientation of the DFG motif is consistent with the 2OZO and 4K2R structures. Thus we assume that the simulation times are sufficient to sample at a minimum conformational transitions between the active and intermediate states [19]. Despite the length of our trajectories, we were unable to observe DFG-in/DFG-out transitions as indicated by the interaction of F480 with M390 in the αC helix (S1 Fig).

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