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. 2020 May 8;11(1):2319.
doi: 10.1038/s41467-020-16128-5.

Btk SH2-kinase Interface Is Critical for Allosteric Kinase Activation and Its Targeting Inhibits B-cell Neoplasms

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Free PMC article

Btk SH2-kinase Interface Is Critical for Allosteric Kinase Activation and Its Targeting Inhibits B-cell Neoplasms

Daniel P Duarte et al. Nat Commun. .
Free PMC article

Abstract

Bruton's tyrosine kinase (Btk) is critical for B-cell maturation and activation. Btk loss-of-function mutations cause human X-linked agammaglobulinemia (XLA). In contrast, Btk signaling sustains growth of several B-cell neoplasms which may be treated with tyrosine kinase inhibitors (TKIs). Here, we uncovered the structural mechanism by which certain XLA mutations in the SH2 domain strongly perturb Btk activation. Using a combination of molecular dynamics (MD) simulations and small-angle X-ray scattering (SAXS), we discovered an allosteric interface between the SH2 and kinase domain required for Btk activation and to which multiple XLA mutations map. As allosteric interactions provide unique targeting opportunities, we developed an engineered repebody protein binding to the SH2 domain and able to disrupt the SH2-kinase interaction. The repebody prevents activation of wild-type and TKI-resistant Btk, inhibiting Btk-dependent signaling and proliferation of malignant B-cells. Therefore, the SH2-kinase interface is critical for Btk activation and a targetable site for allosteric inhibition.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Mutations in Btk SH2 domain abrogates pY551 phosphorylation.
a Mapping of a subset of XLA-patient mutations (red sticks) onto the human Btk SH2 domain structure (PDB 2GE9). The residue R307 (orange sticks) is part of the pY-binding motif (FIVRD). Here and in all subsequent figures, the residue numbering refers to full-length human Btk. b Representative SDS-PAGE analysis of recombinant wild-type and mutant Btk SH2 domains purified from E. coli. c Averaged far-UV circular dichroism (CD) spectra of recombinant wild-type and XLA mutant Btk SH2 domains. Mean residue ellipticity (MRE) for each protein was calculated from three independent measurements. d Binding of a fluorescently labeled pY-peptide to recombinant Btk SH2 domains. Indicated Kd values were obtained from the fitting to a 1:1 binding model. Data are mean ± SD of two technical replicates. Non-binding (N.B). e HEK293 cells were transiently transfected with a construct containing an N-terminal 6xMyc-tagged human Btk SH2-KD wild-type or mutants. Immunoblotting of cell lysates was performed to assess Btk pY551 phosphorylation. f Quantification of pY551 immunoblot shown in e and normalized to total Btk (Myc) expression. Data are mean ± SD of three biological replicates (n = 4) and P-values were calculated using an unpaired t-test. **P ≤ 0.01 and ***P ≤ 0.001. Source data are provided as a Source Data file.
Fig. 2
Fig. 2. Btk SH2 domain is critical for kinase activation.
a Schematic representation of Btk constructs used in this study. Construct/domain boundaries and location of the key activating tyrosine phosphorylation sites (pY223 and pY551) are indicated. b Representative SDS-PAGE analysis of recombinant untagged Btk proteins purified from Sf9 cells. c In vitro autophosphorylation assay performed with recombinant Btk proteins at room temperature. The levels of pY551 (red channel) and total Btk (green channel) were assessed using immunoblotting in a dot blot apparatus. d Btk autophosphorylation kinetics shown in c normalized to total Btk signal and the calculated slopes of linear fits (relative velocities). Data are mean ± SD of at three independent experiments (n = 3). e Relative velocities of Btk autophosphorylation relative to KD. Data are mean ± SD of three independent experiments (n = 3) and P-values were calculated using an unpaired t-test. f HEK293 cells were transiently transfected with the indicated Btk constructs containing an N-terminal 6xMyc tag and kinase activation assessed by immunoblotting of cell lysates. g Quantification of pY551 and total pY shown in f normalized to total Btk (Myc) expression and relative to the KD. Data are mean ± SD of three biological replicates (n = 4) and P-values were calculated using an unpaired t-test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001. Source data are provided as a Source Data file.
Fig. 3
Fig. 3. SH2 domain interacts with the N-lobe of the KD in the active Btk.
a Scaled MD simulations were performed using a model including the Btk SH2 and KD crystal structures (PDB 6HTF and 1K2P, respectively). The most populated clusters of the SH2 positions (several colors) relative to the KD (white) are shown. The percentages indicate the population of the cluster with respect to the entire simulation time. b Detailed view of the SH2-KD interface of clusters 8, 11 and 15. SH2 residues mutated in XLA are indicated as sticks. c Comparative maximal particle dimension (Dmax) of recombinant Btk proteins and summary of structural paraments (Rg and Dmax ± error) obtained from SAXS. See Supplementary Table 2 for details. d Dimensionless Kratky plot of recombinant Btk proteins. The gray dashed line represents the theoretical peak assuming an ideal Guinier region for a globular particle. Ab initio envelope reconstructions obtained from SAXS (surface representation) superimposed on the crystal structures for Btk KD and SH3-SH2-KD (PDB 1K2P and 4XI2, respectively) are shown on top. For the SH2-KD protein, the structure of an elongated MD model with the best agreement with the experimental SAXS data is shown (model C15 shown in a and b). FL protein shows an extended conformation as observed in a previous SAXS reconstruction (SASDC52). Source data are provided as a Source Data file.
Fig. 4
Fig. 4. Mutation on the Btk SH2-KD interface perturbs its active conformation and decrease kinase activation.
a Representative SDS-PAGE analysis of recombinant untagged SH2-KD proteins purified from Sf9 cells. b In vitro autophosphorylation assay performed with recombinant Btk proteins at room temperature. The levels of pY551 (red channel) and total Btk (green channel) were assessed using immunoblotting in a dot blot apparatus. c Btk autophosphorylation kinetics shown in b normalized to total Btk signal and the calculated slopes of linear fits (relative velocities). Data shown are the mean ± SD of two independent experiments (n = 6). d Relative velocities of Btk autophosphorylation relative to wild-type. Data are mean ±  SD of two independent experiments (n = 6) and P-values were calculated using an unpaired t-test. **P ≤ 0.01 and ****P ≤ 0.0001. e Flexibility analysis of SH2-KD based on ensemble of optimization method (EOM) using the experimental SAXS data from recombinant Btk wild-type and mutant proteins. Data from a representative experiment shows the maximal particle dimension (Dmax) of selected conformers for each protein (lines) from a representative pool of theoretical conformations (dotted line). The table summarizes the obtained structural parameters (Rg and Dmax ± error). See Supplementary Table 2 for details. f Cartoon representation of the ensemble of SH2-KD conformations selected by EOM analysis (e, green line). The percentages represent the contribution of each conformer to a SAXS profile in good agreement with the experimental curve. The KD is represented in white and SH2 conformers are colored. SH2-linker distance estimated by the algorithm is represented as ribbon. Source data are provided as a Source Data file.
Fig. 5
Fig. 5. Development of a high-affinity protein binder to the human Btk SH2 domain.
a Representative SDS-PAGE analysis of recombinant repebodies rF10 and rNB purified from E. coli. b Representative ITC measurement of rF10 repebody to the SH2 domains of Btk, Abl and Lck kinases. The top panel show the raw signal and the bottom panel show the integrated calorimetric data of the area of each peak. The continuous line shows the best fit to the experimental data assuming a 1:1 binding model. Reported Kd value for Btk was calculated from three independent measurements. N.B. signifies non-binding. c Size-exclusion chromatogram (SEC) analysis of Btk SH2 and rF10 alone, and the SH2-rF10 complex formed by pre-incubation of SH2 and rF10 prior to column injection. d Peaks isolated from the SEC analysis shown in c resolved by SDS-PAGE and stained with Coomassie. e Binding-competition assay using fluorescently labeled pY-peptide to recombinant Btk SH2 domain in the presence of various concentrations of rF10 repebody. Data are mean ± SD from three technical replicates. f Cartoon representation of the crystal structure of human Btk SH2 (green) in complex with rF10 repebody (salmon), PDB 6HTF. Structural statistics are reported in Supplementary Table 3. The bottom panel shows the rF10 in surface representation, residue R307 (orange) indicates the position of the pY-binding site, and side chains of SH2 residues interacting with rF10 are shown as green sticks. g ITC measurement of rF10 repebody to Btk SH2 K374N performed as in b. The Kd value was calculated from two independent measurements. h Superimposition of the active Btk SH2-KD model (surface representation, SH2 in green and KD in blue) with the rF10-SH2 structure (cartoon representation, SH2 in green and rF10 in salmon). Source data are provided as a Source Data file.
Fig. 6
Fig. 6. rF10 repebody inhibits Btk activation in vitro and in cells.
a Representative SDS-PAGE analysis of in vitro autophosphorylation assay containing recombinant Btk proteins mixed with rF10 or non-binding (rNB) control repebodies. b In vitro autophosphorylation assay performed with recombinant Btk protein with 2-fold excess of rF10 or rNB at room temperature. The levels of pY551 (red channel) and total Btk (green channel) were assessed using immunoblotting in a dot blot apparatus. c Btk autophosphorylation kinetics in the presence of rF10 (dashed lines) or rNB (continuous lines) repebodies shown in b normalized to total Btk signal and the calculated slopes of linear fits (relative velocities). Data are mean ± SD of three independent experiments (n = 6). d Relative velocities of Btk autophosphorylation relative to control repebody. Data are mean ± SD of three independent experiments (n = 3) and P-values relative to rNB were calculated using an unpaired t-test. e HEK293 cells were transiently co-transfected with indicated Btk constructs and repebodies, and cell lysates subjected to immunoprecipitation using anti-Myc coated beads. A representative sample of cell lysate for each repebody is shown as expression control. f Immunoblot analysis of cell lysates from HEK293 cells transiently co-transfected with indicated Btk constructs and repebodies used to assess Btk pY551 phosphorylation. g Quantification of pY551 shown in f normalized to total Btk (Myc) expression and relative to rNB control. Data are mean ± SD of three biological replicates (n = 4) and P-values were calculated using an unpaired t-test. *P ≤ 0.05, ****P ≤ 0.0001, and non-significant (ns). Source data are provided as a Source Data file.
Fig. 7
Fig. 7. Targeting the Btk SH2-KD interface decreases the viability of B-cell lymphoma cells and inhibits BCR signaling.
a Cumulative cell number of HBL-1 cells lentivirally transduced with a doxycycline-inducible system for expression of rF10 or rNB control repebodies (n = 4). Parental cells are non-transduced cells. b Apoptosis analysis of transduced HBL-1 expressing the repebodies for 7 days. A representative gating of FACS staining is shown on top. The quantification of early (7AAD-/Annexin V+) and late (7AAD+/Annexin V+) apoptotic cells was obtained from two independent experiments (n = 2). Parental HBL-1 cells treated with 10 µM of ibrutinib for 48 h were used as positive control. c Immunoblot analysis from transduced HBL-1 cells expressing the repebodies (Flag-tagged) for 48 h. BCR signaling was stimulated with anti-human IgM or mock-treated for 2 min before cell lysis. Ibrutinib treatment (100 nM) was performed for 15 min prior to anti-IgM stimulation. Tubulin was used as loading control. d Quantification of Btk pY551 shown in c and normalized to total Btk expression. Data are mean ± SD from two biological replicates (n = 3) and P-values were calculated using an unpaired t-test. *P ≤ 0.05. e Immunoblotting from transduced DOHH2 cells expressing the repebodies as performed in c. Source data are provided as a Source Data file.
Fig. 8
Fig. 8. Targeting the Btk SH2-KD interface decreases activation of therapy-resistant Btk with mutation on C481.
a Immunoblot analysis of cell lysates from HEK293 cells transiently co-transfected with indicated Btk C481S constructs and repebodies used to assess Btk pY551 phosphorylation. b Quantification of pY551 shown in a normalized to total Btk (Myc) expression and relative to rNB control. Data are mean ± SD of two biological replicates (n = 3) and P-values were calculated using an unpaired t-test. *P ≤ 0.05. Source data are provided as a Source Data file.

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