Autoinhibition of Bruton's tyrosine kinase (Btk) and activation by soluble inositol hexakisphosphate
- Howard Hughes Medical Institute, University of California, Berkeley, United States
- University of California, Berkeley, United States
- Beryllium Inc, United States
- Harvard Medical School, Howard Hughes Medical Institute, United States
- Lawrence Berkeley National Laboratory, United States
Figures
Figure 1 with 1 supplement
Crystal structure of the Src-like module of Btk.
(A) Domain architectures of Btk, c-Abl, and c-Src. (B) Model for the Src-like module of Btk, based on the crystal structure of the domain-swapped dimer. (C) Comparison of the Src-like modules of Btk …
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Figure 1—source data 1
Structures of fragments of Tec family kinases in the Protein Data Bank.
- https://doi.org/10.7554/eLife.06074.017
Figure 1—figure supplement 1
Structural details of the Src-like module of Btk.
(A) The Src-like module of Btk forms a domain-swapped dimer in the crystal lattice, with one molecule per asymmetric unit. The SH3 domain and the kinase domain are intact, but the SH2 domain forms a …
Figure 2 with 1 supplement
Crystal structure of the PH-TH-kinase construct of Btk.
(A) Crystal structure of the PH-TH-kinase construct. The PH-TH module is connected to the kinase domain via a 13-residue linker from the SH2-kinase linker of Btk. The kinase domain is bound to the …
Figure 2—figure supplement 1
Structure of the kinase domain of Btk and its interaction with the PH domain.
(A) Crystal structure of the isolated Btk kinase domain with mutations in the activation loop. These mutations (L542M, S543T, V555T, R562K, S564A, and P565S) are based on a previous study (Joseph et …
Figure 3 with 1 supplement
A structural model for full-length Btk.
(A) A composite model for full-length Btk. The PH-TH domain sits on top of the kinase domain and the SH3 domain, serving as a ‘latch’ that presumably stabilizes the Src-like module in the …
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Figure 3—source data 1
Structures from several time points in the simulation of the composite model for full-length Btk.
The structures are taken every 20 ns from a 360 ns molecule dynamic simulation of the composite model. See ‘Materials and methods’ for the details of simulations.
- https://doi.org/10.7554/eLife.06074.010
Figure 3—figure supplement 1
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 …
Figure 4
Autoinhibition of Btk.
(A) Activation of full-length bovine Btk (residues 1 to 659, 2 μM). Reactions are carried out in the presence of 10 mM Mg2+, 150 mM NaCl, 1 mM ATP, 25 mM Tris-HCl pH 8.0. The level of …
Figure 5 with 1 supplement
Activation of Btk.
(A) Activation of full-length Btk on membranes. Left panel: Btk (1 μM) autophosphorylates rapidly on lipid vesicles (total lipid concentration, 1 mM) containing 75% DOPC, 20% DOPS and 5% PIP3. Right …
Figure 5—figure supplement 1
Activation of various deletion constructs of Btk.
(A) End-point autophosphorylation assay for full-length Btk, the Src-like module, the SH2-kinase construct and the kinase domain in the presence/absence of IP6. The measurements are carried out …
Figure 6
Binding of IP4 and IP6 to the Btk PH-TH module.
(A) Representative isothermal titration calorimetry data for the Btk PH-TH module and its R28C/N24D variant binding to IP4 or IP6. The protein concentration is 20 μM and that of the inositol …
Figure 7 with 1 supplement
Crystal structure of the PH-TH module bound to IP6.
(A) Structure of the PH-TH module bound to two IP6 molecules. One IP6 molecule is in the canonical lipid-binding site and the other IP6 molecule is in the peripheral binding site formed by strands …
Figure 7—figure supplement 1
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 …
Figure 8
The Saraste dimer of the PH-TH module is critical for Btk activation by IP6.
(A) Molecular details of the Saraste dimer interface in the crystal structure of the IP6-bound PH-TH module. (B) End-point autophosphorylation assays for wild-type Btk, Btk L29R/I9R mutant, Btk …
Figure 9 with 1 supplement
Possible allosteric and electrostatic effects of binding IP6.
(A) Fluctuations of the PH-TH module during a molecular dynamic simulation. An instantaneous structure (t = 100 ns) from a 100 ns simulation of the PH-TH module shows a conformational change that …
Figure 9—figure supplement 1
Flexibility in the PH domain of Btk and simulation of the kinetics of Btk autophosphorylation.
(A) Fluctuations in the β3/β4 loop of the wild-type PH-TH module and its I9A/Y42A/F44A/I95A variant. Each trajectory was sampled every 1 ns. The instantaneous structures are aligned using the …
Figure 10 with 1 supplement
Models of Btk autoinhibition and activation.
(A) The PH-TH module stabilizes the assembled conformation of the Src-like module of Btk in its inactive conformation. The activation of Btk involves formation of a transient dimer through the PH-TH …
Figure 10—figure supplement 1
Sequence alignment of Btk and Itk.
The alignment was performed using ClustalW2 (Larkin et al., 2007) and visualized by Espript3 (Robert and Gouet, 2014). The alignment shows that the adoption of the compact autoinhibited conformation …
Tables
Table 1
Data collection and refinement statistics
| Src-like module of mouse Btk (217-659) | PH-TH-kinase unit of bovine Btk | Btk PH-TH module bound to IP6 | Btk kinase domain with mutations in the activation loop | |
|---|---|---|---|---|
| PDB ID | 4XI2 | 4Y93 | 4Y94 | 4Y95 |
| Data collection | ||||
| KAu(CN)2 | Native | Native | Native | |
| Wavelength (Å) | 0.9474 | 1.000 | 1.000 | 1.000 |
| Space group | P 31 2 1 | P 2 21 21 | P 1 | P 1 |
| a,b,c (Å) | 132.2, 132.2, 107.6 | 78.6, 38.3, 157.6 | 37.2, 64.0, 80.0 | 50.9, 79.0, 79.2 |
| α,β,γ (°) | 90.0, 90.0, 120.0 | 90.0, 90.0, 90.0 | 82.0, 88.8, 89.8 | 90.7, 89.9, 90.0 |
| Resolution (Å) | 43.2–2.6 | 50–1.7 | 43.9–2.3 | 47.9–1.6 |
| Rsym (%) | 8.7 (>100) | 12.2 (79.7) | 6.0 (53.8) | 5.1 (71.7) |
| I/σ(I) | 10.6 (1.1) | 17.0 (3.5) | 10.8 (1.2) | 13.2 (3.2) |
| Completeness (%) | 91.9 (69.6) | 99.2 (98.5) | 94.9 (71.8) | 96.9 (94.7) |
| Redundancy | 4.8 (3.5) | 7.8 (7.4) | 2.0 (1.7) | 24.4 (3.1) |
| Wilson B factor | 72.8 | 15.7 | 52.3 | 18.1 |
| Refinement | ||||
| Resolution | 43.2–2.6 | 50–1.7 | 46.5–2.3 | 47.9–1.6 |
| Reflections | 57,643(30,933) | 53,357 | 30,516 | 157,562 |
| Rfree reflections | 1525 | 2000 | 2025 | 2009 |
| Rwork/Rfree | 0.237/0.252 | 0.167/0.195 | 0.236/0.254 | 0.155/0.187 |
| No. atoms | ||||
| Protein | 3459 | 6970 | 5053 | 8911 |
| Ligands | 2 | 80 | 216 | 961 |
| Water | 0 | 525 | 39 | 737 |
| Average B factors | ||||
| Protein | 114.4 | 27.7 | 62.1 | 22.5 |
| Solvent | N/A | 33.0 | 66.2 | 30.1 |
| Root mean square deviation from ideality | ||||
| Bonds (Å) | 0.006 | 0.005 | 0.003 | 0.014 |
| Angles (°) | 1.11 | 0.977 | 0.764 | 1.571 |
| Ramachandran statistics | ||||
| Favored (%) | 91 | 98.58 | 98.2 | 97.11 |
| Disallowed (%) | 2.4 | 0.0 | 0.0 | 0.18 |
| MolProbity clash score | 9.6 | 2.8 | 5.16 | 2.66 |
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The CC1/2 values for the PH-TH-kinase dataset, IP6-bound PH-TH dataset and the kinase domain with mutations in the activation loop dataset are 99.9 (86.5), 99.9 (55.4) and 99.9 (90.7), respectively.
Table 2
Data statistics for the Src-like module of Btk
| Src-like module of mouse Btk (217-659) | ||||
|---|---|---|---|---|
| Data collection | ||||
| KAu(CN)2 | DMA | Au2(NO3)3 | native | |
| Wavelength (Å) | 0.9474 | 0.9474 | 0.9474 | 0.9474 |
| Space group | P31 2 1 | P31 2 1 | P31 2 1 | P31 2 1 |
| a,b,c (Å) | 132.2, 132.2, 107.6 | 132.5, 132.5, 107.3 | 131.9, 131.9, 107.6 | 131.8, 131.8, 107.0 |
| α,β,γ (°) | 90.0, 90.0, 120.0 | 90.0, 90.0, 120.0 | 90.0, 90.0, 120.0 | 90.0, 90.0, 120.0 |
| Resolution (Å) | 43.2–2.6 | 41.7–3.5 | 41.7–3.4 | 40–4.0 |
| Rsym (%) | 8.7(>100) | 8.7 (32.7) | 7.5 (32.5) | 5.1 (71.7) |
| I/σ(I) | 10.6 (1.1) | 9.7 (3.0) | 12.1 (3.6) | 18.4 (12.3) |
| Completeness (%) | 91.9 (69.6) | 92.4 (94.2) | 94.5 (95.9) | 92.3 (100) |
| Redundancy | 4.8 (3.5) | 4.3 | 4.1 | N/A |
| Wilson B factor | 72.8 | 58.8 | 70.8 | 67.3 |
Table 3
Thermodynamic parameters for Btk PH-TH module binding to IP6 and IP4
| Protein | Ligand | N | Ka (×106) | Kd (nM) | ΔH (Kcal/mol) | ΔS (cal/mol/deg) |
|---|---|---|---|---|---|---|
| Wild-type PH-TH | IP6 | 1.1 ± 0.1 | 4.2 ± 0.3 | 238 ± 32 | −1.1 ± 0.2 | 26.7 |
| Wild-type PHTH | IP4 | 0.9 ± 0.1 | 39 ± 11 | 26 ± 6 | 4.4 ± 0.1 | 49.5 |
| PH-TH R28C/D24N | IP6 | 0.7 ± 0.2 | 0.21 ± 0.03 | 4760 ± 700 | −1.7 ± 0.6 | 17.5 |
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The integrated heat from representative isothermal titration calorimetry experiments was fit with a one-binding site model. See methods for the details of data analysis.
