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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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Aspects of the binding of IP6 to Btk.(A) An open-ended chain of PH-TH dimers in the crystal lattice, mediated by IP6 at the pheripheral site. IP6 is bound at the peripheral site in such a way that only one IP6 molecule occupies the two sites at each interface. (B) Structure superposition of the IP6-bound PH-TH module with apo- and the IP4-bound PH-TH module. The β1/β2 loop, which coordinates IP4 binding, adopts a different conformation in the apo-structure, and is partially disordered in the IP6-bound structure. (C) IP6 coordination in the peripheral binding site by a crystal symmetry mate of the PH-TH module in the crystal structure of IP6-bound PH-TH module. IP6 and the residues involved in coordination are shown in sticks. The hydrogen bonds are shown in dotted lines. This is a detailed view of the interfacial sites shown in the schematic diagram in Panel A. (D) Isothermal titration calorimetry data for Btk PH-TH mutant R52E/K49E/R28C/N24D titrated with IP6. Arg 52 and Lys 49 are critical residues coordinating IP6 binding in the peripheral site. Arg 28 and Asn 24 are critical residues coordinating IP6 binding in the canonical site. Note that IP6 does not bind to this mutant form of the PH-TH module. (E) Activation of full-length Btk, Btk R52S/K49S mutant, and Btk R28C/N24D mutant in the presence of IP6 (100 μM).DOI:http://dx.doi.org/10.7554/eLife.06074.019
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fig7s1: Aspects of the binding of IP6 to Btk.(A) An open-ended chain of PH-TH dimers in the crystal lattice, mediated by IP6 at the pheripheral site. IP6 is bound at the peripheral site in such a way that only one IP6 molecule occupies the two sites at each interface. (B) Structure superposition of the IP6-bound PH-TH module with apo- and the IP4-bound PH-TH module. The β1/β2 loop, which coordinates IP4 binding, adopts a different conformation in the apo-structure, and is partially disordered in the IP6-bound structure. (C) IP6 coordination in the peripheral binding site by a crystal symmetry mate of the PH-TH module in the crystal structure of IP6-bound PH-TH module. IP6 and the residues involved in coordination are shown in sticks. The hydrogen bonds are shown in dotted lines. This is a detailed view of the interfacial sites shown in the schematic diagram in Panel A. (D) Isothermal titration calorimetry data for Btk PH-TH mutant R52E/K49E/R28C/N24D titrated with IP6. Arg 52 and Lys 49 are critical residues coordinating IP6 binding in the peripheral site. Arg 28 and Asn 24 are critical residues coordinating IP6 binding in the canonical site. Note that IP6 does not bind to this mutant form of the PH-TH module. (E) Activation of full-length Btk, Btk R52S/K49S mutant, and Btk R28C/N24D mutant in the presence of IP6 (100 μM).DOI:http://dx.doi.org/10.7554/eLife.06074.019

Mentions: We determined a crystal structure of the Btk PH-TH module bound to IP6 at 2.3 Å (Figure 7). There are four PH-TH modules in the asymmetric unit of the crystal, which form two dimers, similar to those seen in other structures of the Btk PH-TH module (Figure 7D) (Hyvönen and Saraste, 1997; Baraldi et al., 1999). The only structural changes in the IP6-bound PH-TH module, relative to the apo- or IP4-bound PH-TH module, are in the loop regions (Figure 7—figure supplement 1B).10.7554/eLife.06074.018Figure 7.Crystal structure of the PH-TH module bound to IP6.


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)

Aspects of the binding of IP6 to Btk.(A) An open-ended chain of PH-TH dimers in the crystal lattice, mediated by IP6 at the pheripheral site. IP6 is bound at the peripheral site in such a way that only one IP6 molecule occupies the two sites at each interface. (B) Structure superposition of the IP6-bound PH-TH module with apo- and the IP4-bound PH-TH module. The β1/β2 loop, which coordinates IP4 binding, adopts a different conformation in the apo-structure, and is partially disordered in the IP6-bound structure. (C) IP6 coordination in the peripheral binding site by a crystal symmetry mate of the PH-TH module in the crystal structure of IP6-bound PH-TH module. IP6 and the residues involved in coordination are shown in sticks. The hydrogen bonds are shown in dotted lines. This is a detailed view of the interfacial sites shown in the schematic diagram in Panel A. (D) Isothermal titration calorimetry data for Btk PH-TH mutant R52E/K49E/R28C/N24D titrated with IP6. Arg 52 and Lys 49 are critical residues coordinating IP6 binding in the peripheral site. Arg 28 and Asn 24 are critical residues coordinating IP6 binding in the canonical site. Note that IP6 does not bind to this mutant form of the PH-TH module. (E) Activation of full-length Btk, Btk R52S/K49S mutant, and Btk R28C/N24D mutant in the presence of IP6 (100 μM).DOI:http://dx.doi.org/10.7554/eLife.06074.019
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fig7s1: Aspects of the binding of IP6 to Btk.(A) An open-ended chain of PH-TH dimers in the crystal lattice, mediated by IP6 at the pheripheral site. IP6 is bound at the peripheral site in such a way that only one IP6 molecule occupies the two sites at each interface. (B) Structure superposition of the IP6-bound PH-TH module with apo- and the IP4-bound PH-TH module. The β1/β2 loop, which coordinates IP4 binding, adopts a different conformation in the apo-structure, and is partially disordered in the IP6-bound structure. (C) IP6 coordination in the peripheral binding site by a crystal symmetry mate of the PH-TH module in the crystal structure of IP6-bound PH-TH module. IP6 and the residues involved in coordination are shown in sticks. The hydrogen bonds are shown in dotted lines. This is a detailed view of the interfacial sites shown in the schematic diagram in Panel A. (D) Isothermal titration calorimetry data for Btk PH-TH mutant R52E/K49E/R28C/N24D titrated with IP6. Arg 52 and Lys 49 are critical residues coordinating IP6 binding in the peripheral site. Arg 28 and Asn 24 are critical residues coordinating IP6 binding in the canonical site. Note that IP6 does not bind to this mutant form of the PH-TH module. (E) Activation of full-length Btk, Btk R52S/K49S mutant, and Btk R28C/N24D mutant in the presence of IP6 (100 μM).DOI:http://dx.doi.org/10.7554/eLife.06074.019
Mentions: We determined a crystal structure of the Btk PH-TH module bound to IP6 at 2.3 Å (Figure 7). There are four PH-TH modules in the asymmetric unit of the crystal, which form two dimers, similar to those seen in other structures of the Btk PH-TH module (Figure 7D) (Hyvönen and Saraste, 1997; Baraldi et al., 1999). The only structural changes in the IP6-bound PH-TH module, relative to the apo- or IP4-bound PH-TH module, are in the loop regions (Figure 7—figure supplement 1B).10.7554/eLife.06074.018Figure 7.Crystal structure of the PH-TH module bound to IP6.

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