Structural mechanism for Bruton's tyrosine kinase activation at the cell membrane

Abstract

Bruton's tyrosine kinase (Btk) is critical for B cell proliferation and activation, and the development of Btk inhibitors is a vigorously pursued strategy for the treatment of various B cell malignancies. A detailed mechanistic understanding of Btk activation has, however, been lacking. Here, inspired by a previous suggestion that Btk activation might depend on dimerization of its lipid-binding PH-TH module on the cell membrane, we performed long-timescale molecular dynamics simulations of membrane-bound PH-TH modules and observed that they dimerized into a single predominant conformation. We found that the phospholipid PIP₃ stabilized the dimer allosterically by binding at multiple sites, and that the effects of PH-TH mutations on dimer stability were consistent with their known effects on Btk activity. Taken together, our simulation results strongly suggest that PIP₃-mediated dimerization of Btk at the cell membrane is a critical step in Btk activation.

Keywords: Bruton’s tyrosine kinase; PIP3; allosteric activation; dimerization; enhanced sampling.

Fig. 1.

Spontaneous binding of the PH–TH module to the membrane. (A) Cartoon illustration of a membrane-binding simulation setup (see SI Appendix, Methods for details). (B, Right) Minimum distance between atoms in PIP₃ lipids and in the canonical binding site for three independent membrane-binding simulations. (B, Left) A structure taken from one of these simulations (t = 10 μs) showing a PIP₃ molecule bound at the canonical site. (C, Right) Minimum distance between atoms in PIP₃ lipids and in the peripheral binding site for three independent membrane-binding simulations. (C, Left) A structure taken from one of these simulations (t = 10 μs) showing a PIP₃ molecule bound at the peripheral site.

Fig. 2.

Dimerization of the PH–TH module on the membrane. (A) Cartoon illustration of an encounter simulation setup (see SI Appendix, Methods for details). (B) Conformation clusters of PH–TH dimers formed in the simulations (see SI Appendix, Methods for details). A representative structure of the most populated dimer conformation is shown (see Inset). (The PIP₃ lipids bound in the canonical binding sites are also shown.) (C) Reversible dimerization of two membrane-bound PH–TH modules on a membrane that contains 1.5% PIP₃ and 98.5% POPC in tempered binding simulations. The rmsd at the Saraste interface is calculated for all interface residues (residues 9, 11, 42, 44, 92, and 95) of the two modules with respect to the Saraste dimer interface conformation seen in a crystal structure of the PH–TH module (PDB ID code 1BTK). (This same interface rmsd calculation is used for the rmsd plots in all other figures, unless stated otherwise.)

Fig. 3.

Structural analysis of Saraste dimers formed on membranes. (A) Structural comparison of Saraste dimers formed in the encounter simulations and in the Saraste dimer crystal structure (PDB ID code 1BTK). (B) An instantaneous structure of the pre-Saraste interface formed in the encounter simulations.

Fig. 4.

Stability analysis of Saraste dimer variants on a membrane. (A) Representative tempered binding simulation trajectories showing dissociation of the tightly packed Saraste conformation in two individual trajectories. The tempered binding simulations started from a Saraste dimer on a membrane containing 6% PIP₃ and 94% POPC. (B) Stability analysis of the F98V mutant, starting from the Saraste dimer conformation, on a membrane. An instantaneous structure from a simulation (t = 20 μs) shows the local conformational rearrangement that occurred at the Saraste interface before the dimer dissociated. Representative tempered binding simulation trajectories show the dissociation of the F98V dimer in two individual trajectories. (C) Stability analysis of the E41K mutant, starting from the Saraste dimer conformation, on a membrane. An instantaneous structure from a simulation (t = 100 μs) shows PIP₃ bound to residue Lys-41 at the bridging site before the dimer dissociated. Representative tempered binding simulation trajectories show the dissociation of the E41K dimer in two individual trajectories.

Fig. 5.

Fluctuations at the pre-Saraste dimer interface. (A) Representative tempered binding simulation trajectories of two PH–TH modules on a membrane containing eight PIP₃ lipids. (B) Fluctuations at the pre-Saraste dimer interface that formed on membranes with two and eight PIP₃ lipids (1.5% and 6% membrane PIP₃ content, respectively).

Fig. 6.

Sensitivity of fluctuations at the pre-Saraste dimer interface to membrane PIP₃ concentration. Fluctuations at the pre-Saraste dimer interface are shown, measured under various membrane PIP₃ concentrations. The histograms of the rmsd of the interface residues are calculated from 100-μs simulation trajectories initiated from Saraste dimers that originally formed in tempered binding simulations; only data from the portion of the trajectories where the interactions were unscaled are used.

Fig. 7.

Speculation on the mechanisms by which binding multiple PIP₃ molecules stabilizes the Saraste dimer on the membrane. (A) Fluctuations at the Saraste dimer interface region in an individual PH–TH module in conventional MD simulations in solution and on the membrane. (B) An instantaneous structure from a tempered binding simulation showing that a PIP₃ lipid can simultaneously interact with both modules in the Saraste dimer at the bridging site, which formed near the dimer interface.