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Autoinhibition of Bruton's tyrosine kinase (Btk) and activation by soluble inositol hexakisphosphate.

Wang Q, Vogan EM, Nocka LM, Rosen CE, Zorn JA, Harrison SC, Kuriyan J - Elife (2015)

Bottom Line: In addition to the expected activation of Btk by membranes containing phosphatidylinositol triphosphate (PIP3), we found that inositol hexakisphosphate (IP6), a soluble signaling molecule found in both animal and plant cells, also activates Btk.This activation is a consequence of a transient PH-TH dimerization induced by IP6, which promotes transphosphorylation of the kinase domains.Sequence comparisons with other Tec-family kinases suggest that activation by IP6 is unique to Btk.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States.

ABSTRACT
Bruton's tyrosine kinase (Btk), a Tec-family tyrosine kinase, is essential for B-cell function. We present crystallographic and biochemical analyses of Btk, which together reveal molecular details of its autoinhibition and activation. Autoinhibited Btk adopts a compact conformation like that of inactive c-Src and c-Abl. A lipid-binding PH-TH module, unique to Tec kinases, acts in conjunction with the SH2 and SH3 domains to stabilize the inactive conformation. In addition to the expected activation of Btk by membranes containing phosphatidylinositol triphosphate (PIP3), we found that inositol hexakisphosphate (IP6), a soluble signaling molecule found in both animal and plant cells, also activates Btk. This activation is a consequence of a transient PH-TH dimerization induced by IP6, which promotes transphosphorylation of the kinase domains. Sequence comparisons with other Tec-family kinases suggest that activation by IP6 is unique to Btk.

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Utilization of molecular dynamics in constructing a model for full-length Btk.(A) Overlay of the crystal structures of the Btk Src-like module and the PH-TH-kinase construct, using the kinase domain C lobe as the reference. Sidechain clashes (circled) are observed between helix α2 in the PH-TH module and the β3/β4 loop segment in the SH3 domain. (B) Fluctuations in the Btk Src-like module occurred during a 100 ns molecular dynamics simulation. An instantaneous structure (t = 89 ns) from the simulation is overlaid on the crystal structure of the Src-like module. (C) Fluctuations in the Btk PH-TH-kinase construct during a 100 ns molecular dynamics simulation. An instantaneous structure (t = 12 ns) from the simulation is overlaid on the crystal structure of the PH-TH-kinase construct using the kinase domain N lobe as the reference. The PH-TH module pivots (black arrow) about an apparent anchor point at the C terminus of helix α2. (D) Steps used to generate a composite model for full-length Btk. An instantaneous structure (t = 89 ns) of the Src-like module and an instantaneous structure (t = 12 ns) of the PH-TH-kinase construct from the molecular dynamics trajectory are overlaid, using the kinase domain C-lobe as the reference. In contrast to a corresponding overlay of the crystal structures, there are no sidechain clashes between helix α2 in the PH-TH module and the loop in the SH3 domain. A model for full-length Btk is obtained by combining the PH-TH module with the Src-like module in the two instantaneous structures, and is subject to further molecular dynamics simulation.DOI:http://dx.doi.org/10.7554/eLife.06074.011
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fig3s1: Utilization of molecular dynamics in constructing a model for full-length Btk.(A) Overlay of the crystal structures of the Btk Src-like module and the PH-TH-kinase construct, using the kinase domain C lobe as the reference. Sidechain clashes (circled) are observed between helix α2 in the PH-TH module and the β3/β4 loop segment in the SH3 domain. (B) Fluctuations in the Btk Src-like module occurred during a 100 ns molecular dynamics simulation. An instantaneous structure (t = 89 ns) from the simulation is overlaid on the crystal structure of the Src-like module. (C) Fluctuations in the Btk PH-TH-kinase construct during a 100 ns molecular dynamics simulation. An instantaneous structure (t = 12 ns) from the simulation is overlaid on the crystal structure of the PH-TH-kinase construct using the kinase domain N lobe as the reference. The PH-TH module pivots (black arrow) about an apparent anchor point at the C terminus of helix α2. (D) Steps used to generate a composite model for full-length Btk. An instantaneous structure (t = 89 ns) of the Src-like module and an instantaneous structure (t = 12 ns) of the PH-TH-kinase construct from the molecular dynamics trajectory are overlaid, using the kinase domain C-lobe as the reference. In contrast to a corresponding overlay of the crystal structures, there are no sidechain clashes between helix α2 in the PH-TH module and the loop in the SH3 domain. A model for full-length Btk is obtained by combining the PH-TH module with the Src-like module in the two instantaneous structures, and is subject to further molecular dynamics simulation.DOI:http://dx.doi.org/10.7554/eLife.06074.011

Mentions: We combined the two structures just described to build a model for full-length Btk (Figure 3A). Aligning the kinase domains in the two structures gives some local overlap between helix α2 in the PH-TH module and the β3/β4 loop in the SH3 domain, but otherwise compatible positions for the regulatory domains (Figure 3—figure supplement 1A). We therefore carried out molecular dynamics simulations of the two component structures and of the composite assembly, to determine whether energetically minor adjustments could resolve the remaining clashes. The trajectories ranged in length from 60 to 150 ns (see ‘Materials and methods’ for computational details).10.7554/eLife.06074.009Figure 3.A structural model for full-length Btk.


Autoinhibition of Bruton's tyrosine kinase (Btk) and activation by soluble inositol hexakisphosphate.

Wang Q, Vogan EM, Nocka LM, Rosen CE, Zorn JA, Harrison SC, Kuriyan J - Elife (2015)

Utilization of molecular dynamics in constructing a model for full-length Btk.(A) Overlay of the crystal structures of the Btk Src-like module and the PH-TH-kinase construct, using the kinase domain C lobe as the reference. Sidechain clashes (circled) are observed between helix α2 in the PH-TH module and the β3/β4 loop segment in the SH3 domain. (B) Fluctuations in the Btk Src-like module occurred during a 100 ns molecular dynamics simulation. An instantaneous structure (t = 89 ns) from the simulation is overlaid on the crystal structure of the Src-like module. (C) Fluctuations in the Btk PH-TH-kinase construct during a 100 ns molecular dynamics simulation. An instantaneous structure (t = 12 ns) from the simulation is overlaid on the crystal structure of the PH-TH-kinase construct using the kinase domain N lobe as the reference. The PH-TH module pivots (black arrow) about an apparent anchor point at the C terminus of helix α2. (D) Steps used to generate a composite model for full-length Btk. An instantaneous structure (t = 89 ns) of the Src-like module and an instantaneous structure (t = 12 ns) of the PH-TH-kinase construct from the molecular dynamics trajectory are overlaid, using the kinase domain C-lobe as the reference. In contrast to a corresponding overlay of the crystal structures, there are no sidechain clashes between helix α2 in the PH-TH module and the loop in the SH3 domain. A model for full-length Btk is obtained by combining the PH-TH module with the Src-like module in the two instantaneous structures, and is subject to further molecular dynamics simulation.DOI:http://dx.doi.org/10.7554/eLife.06074.011
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4384635&req=5

fig3s1: Utilization of molecular dynamics in constructing a model for full-length Btk.(A) Overlay of the crystal structures of the Btk Src-like module and the PH-TH-kinase construct, using the kinase domain C lobe as the reference. Sidechain clashes (circled) are observed between helix α2 in the PH-TH module and the β3/β4 loop segment in the SH3 domain. (B) Fluctuations in the Btk Src-like module occurred during a 100 ns molecular dynamics simulation. An instantaneous structure (t = 89 ns) from the simulation is overlaid on the crystal structure of the Src-like module. (C) Fluctuations in the Btk PH-TH-kinase construct during a 100 ns molecular dynamics simulation. An instantaneous structure (t = 12 ns) from the simulation is overlaid on the crystal structure of the PH-TH-kinase construct using the kinase domain N lobe as the reference. The PH-TH module pivots (black arrow) about an apparent anchor point at the C terminus of helix α2. (D) Steps used to generate a composite model for full-length Btk. An instantaneous structure (t = 89 ns) of the Src-like module and an instantaneous structure (t = 12 ns) of the PH-TH-kinase construct from the molecular dynamics trajectory are overlaid, using the kinase domain C-lobe as the reference. In contrast to a corresponding overlay of the crystal structures, there are no sidechain clashes between helix α2 in the PH-TH module and the loop in the SH3 domain. A model for full-length Btk is obtained by combining the PH-TH module with the Src-like module in the two instantaneous structures, and is subject to further molecular dynamics simulation.DOI:http://dx.doi.org/10.7554/eLife.06074.011
Mentions: We combined the two structures just described to build a model for full-length Btk (Figure 3A). Aligning the kinase domains in the two structures gives some local overlap between helix α2 in the PH-TH module and the β3/β4 loop in the SH3 domain, but otherwise compatible positions for the regulatory domains (Figure 3—figure supplement 1A). We therefore carried out molecular dynamics simulations of the two component structures and of the composite assembly, to determine whether energetically minor adjustments could resolve the remaining clashes. The trajectories ranged in length from 60 to 150 ns (see ‘Materials and methods’ for computational details).10.7554/eLife.06074.009Figure 3.A structural model for full-length Btk.

Bottom Line: In addition to the expected activation of Btk by membranes containing phosphatidylinositol triphosphate (PIP3), we found that inositol hexakisphosphate (IP6), a soluble signaling molecule found in both animal and plant cells, also activates Btk.This activation is a consequence of a transient PH-TH dimerization induced by IP6, which promotes transphosphorylation of the kinase domains.Sequence comparisons with other Tec-family kinases suggest that activation by IP6 is unique to Btk.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States.

ABSTRACT
Bruton's tyrosine kinase (Btk), a Tec-family tyrosine kinase, is essential for B-cell function. We present crystallographic and biochemical analyses of Btk, which together reveal molecular details of its autoinhibition and activation. Autoinhibited Btk adopts a compact conformation like that of inactive c-Src and c-Abl. A lipid-binding PH-TH module, unique to Tec kinases, acts in conjunction with the SH2 and SH3 domains to stabilize the inactive conformation. In addition to the expected activation of Btk by membranes containing phosphatidylinositol triphosphate (PIP3), we found that inositol hexakisphosphate (IP6), a soluble signaling molecule found in both animal and plant cells, also activates Btk. This activation is a consequence of a transient PH-TH dimerization induced by IP6, which promotes transphosphorylation of the kinase domains. Sequence comparisons with other Tec-family kinases suggest that activation by IP6 is unique to Btk.

Show MeSH
Related in: MedlinePlus