Limits...
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

Normal mode projection amplitudes for the four lowest-frequency normal modes correlated with state of the complex.Normal mode (a) is characteristic for mono-phosphorylation as it is concurrent in the YPY0 and the Y0YP state. Normal mode (b) is associated with Y493 phosphorylation Y0YP. Characteristic for this motion is the movement of the αC helix with concurrent extension of the activation loop. The tertiary mode (c) is characteristic for YPY0. It consists of an extension of the activation loop. However, no motion of the αC helix is associated with this mode. Normal mode (d) characterizes the non-phosphorylated state. Predominant motions increase compactness, close the catalytic cleft and bury the αC helix.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4608720&req=5

pcbi.1004560.g005: Normal mode projection amplitudes for the four lowest-frequency normal modes correlated with state of the complex.Normal mode (a) is characteristic for mono-phosphorylation as it is concurrent in the YPY0 and the Y0YP state. Normal mode (b) is associated with Y493 phosphorylation Y0YP. Characteristic for this motion is the movement of the αC helix with concurrent extension of the activation loop. The tertiary mode (c) is characteristic for YPY0. It consists of an extension of the activation loop. However, no motion of the αC helix is associated with this mode. Normal mode (d) characterizes the non-phosphorylated state. Predominant motions increase compactness, close the catalytic cleft and bury the αC helix.

Mentions: Fig 5 shows Normal modes and projections of the four lowest-frequency modes during the entire trajectory. Mode number 4 was specific for the non-phosphorylated complex, and is associated with a movement of the αC helix towards the remainder of the C-lobe. Concomitantly, the section of the activation loop containing phosphorylation targets Y492 and Y493 moves towards the catalytic cleft, thereby restricting access. (Fig 5d) Notably, scanning the surfaces of the KD in the non-phosphorylated complex over 1.5 μs of simulation revealed the spontaneous formation of a cryptic pocket adjacent to the activation loop (Fig 4b). This cryptic pocket repeatedly opened and closed from ~700 ns, and reached a maximum volume of ~1400 Å3 (Fig 4c). The protein backbone geometry of the maximum open states encountered at 1050 ns, 1257 ns and 1378 ns is identical. The formation of the pocket primarily arose from the sidechain movement of a single residue, W505, which is highly conserved across kinase domains. In the initial structure, W505 forms the core of a hydrophobic cluster; thus, its aromatic ring is wedged between P539 and P502, and is in van der Waal’s contact with V527, A463, and the alkyl groups of K504 and R465. Coupled to the motion of the activation loop, the W505 ring underwent conformational switching within the hydrophobic core (S2 and S3 Figs), with a gradual shift in its position relative to the nearby ATP site, increasing the distance of separation by up to 8 Å over the final ~700 ns (S3 Fig). In its final sidechain orientation (S3 Fig) which resulted in the formation of the fully open cryptic pocket, W505 came to rest on the surface of Y506, W523, I552, and W576, encompassing residues in or nearby to the mobile N-lobal region, whilst remaining in contact with V527 and P502.


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

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

Normal mode projection amplitudes for the four lowest-frequency normal modes correlated with state of the complex.Normal mode (a) is characteristic for mono-phosphorylation as it is concurrent in the YPY0 and the Y0YP state. Normal mode (b) is associated with Y493 phosphorylation Y0YP. Characteristic for this motion is the movement of the αC helix with concurrent extension of the activation loop. The tertiary mode (c) is characteristic for YPY0. It consists of an extension of the activation loop. However, no motion of the αC helix is associated with this mode. Normal mode (d) characterizes the non-phosphorylated state. Predominant motions increase compactness, close the catalytic cleft and bury the αC helix.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi.1004560.g005: Normal mode projection amplitudes for the four lowest-frequency normal modes correlated with state of the complex.Normal mode (a) is characteristic for mono-phosphorylation as it is concurrent in the YPY0 and the Y0YP state. Normal mode (b) is associated with Y493 phosphorylation Y0YP. Characteristic for this motion is the movement of the αC helix with concurrent extension of the activation loop. The tertiary mode (c) is characteristic for YPY0. It consists of an extension of the activation loop. However, no motion of the αC helix is associated with this mode. Normal mode (d) characterizes the non-phosphorylated state. Predominant motions increase compactness, close the catalytic cleft and bury the αC helix.
Mentions: Fig 5 shows Normal modes and projections of the four lowest-frequency modes during the entire trajectory. Mode number 4 was specific for the non-phosphorylated complex, and is associated with a movement of the αC helix towards the remainder of the C-lobe. Concomitantly, the section of the activation loop containing phosphorylation targets Y492 and Y493 moves towards the catalytic cleft, thereby restricting access. (Fig 5d) Notably, scanning the surfaces of the KD in the non-phosphorylated complex over 1.5 μs of simulation revealed the spontaneous formation of a cryptic pocket adjacent to the activation loop (Fig 4b). This cryptic pocket repeatedly opened and closed from ~700 ns, and reached a maximum volume of ~1400 Å3 (Fig 4c). The protein backbone geometry of the maximum open states encountered at 1050 ns, 1257 ns and 1378 ns is identical. The formation of the pocket primarily arose from the sidechain movement of a single residue, W505, which is highly conserved across kinase domains. In the initial structure, W505 forms the core of a hydrophobic cluster; thus, its aromatic ring is wedged between P539 and P502, and is in van der Waal’s contact with V527, A463, and the alkyl groups of K504 and R465. Coupled to the motion of the activation loop, the W505 ring underwent conformational switching within the hydrophobic core (S2 and S3 Figs), with a gradual shift in its position relative to the nearby ATP site, increasing the distance of separation by up to 8 Å over the final ~700 ns (S3 Fig). In its final sidechain orientation (S3 Fig) which resulted in the formation of the fully open cryptic pocket, W505 came to rest on the surface of Y506, W523, I552, and W576, encompassing residues in or nearby to the mobile N-lobal region, whilst remaining in contact with V527 and P502.

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