Switch-like activation of Bruton’s tyrosine kinase by membrane-mediated dimerization

Abstract

The transformation of molecular binding events into cellular decisions is the basis of most biological signal transduction. A fundamental challenge faced by these systems is that reliance on protein-ligand chemical affinities alone generally results in poor sensitivity to ligand concentration, endangering the system to error. Here, we examine the lipid-binding pleckstrin homology and Tec homology (PH-TH) module of Bruton's tyrosine kinase (Btk). Using fluorescence correlation spectroscopy (FCS) and membranebinding kinetic measurements, we identify a phosphatidylinositol (3-5)-trisphosphate (PIP 3 ) sensing mechanism that achieves switchlike sensitivity to PIP 3 levels, surpassing the intrinsic affinity discrimination of PIP 3 :PH binding. This mechanism employs multiple PIP 3 binding as well as dimerization of Btk on the membrane surface. Studies in live cells confirm that mutations at the dimer interface and peripheral site produce effects comparable to that of the kinase-dead Btk in vivo. These results demonstrate how a single protein module can institute an allosteric counting mechanism to achieve high-precision discrimination of ligand concentration. Furthermore, this activation mechanism distinguishes Btk from other Tec family member kinases, Tec and Itk, which we show are not capable of dimerization through their PH-TH modules. This suggests that Btk plays a critical role in the stringency of the B cell response, whereas T cells rely on other mechanisms to achieve stringency.Bruton's tyrosine kinase | PIP 3 | ultrasensitivity | signaling

A mechanism for localized dynamics-driven activation in Bruton's tyrosine kinase

“…There are five structural domains in BTK (figure 1a), each of which has its own activation mechanism, and then jointly form the overall activation process of BTK. To date, many studies have explored to some extent the activation mechanism of different BTK domains [15,23,[32][33][34][35][36][37]. For example, some studies have shown PH dimerization [34,38], which could mediate the dimerization of the catalytic region of BTK [23], and that the A-loop may be associated with kinase domain dimerization [39].…”

Reining in BTK: Interdomain Interactions and Their Importance in the Regulatory Control of BTK

“…Pose 1 ( Figure 4 ) involves specific contacts between R133 and Y134 in the PHTH domain and the N-lobe of the kinase domain. Importantly, this crystallographically determined structure shows the PHTH domain adopting the “Saraste dimer” ( Hyvönen and Saraste, 1997 ), a structure that has been associated with binding of the BTK PHTH domain to the plasma membrane following production of PIP 3 from PIP 2 by PI3K ( Chung et al, 2019 ). Molecular dynamics simulations suggest that the BTK PHTH Saraste dimer is stabilized at the membrane by binding multiple PIP 3 lipids and mutations that are known to have disease relevance destabilized the dimer interface ( Wang et al, 2019 ).…”

“…Molecular dynamics simulations suggest that the BTK PHTH Saraste dimer is stabilized at the membrane by binding multiple PIP 3 lipids and mutations that are known to have disease relevance destabilized the dimer interface ( Wang et al, 2019 ). Furthermore, elegant studies using supported lipid bilayers provide evidence that the peripheral site originally identified in the crystal structure of the BTK PH domain bound to IP 6 ( Wang et al, 2015 ) stabilizes membrane association ( Chung et al, 2019 ). This requirement for the occupancy of both PIP 3 sites for activation of BTK suggests that the PH domain is sensitive to the concentration of PIP 3 in the membrane.…”

“…This transient opening of the BTK autoinhibited structure might favor phospholipid association once PIP 3 is present at the membrane ( Figure 5C ). Once membrane associated, BTK will dimerize via the Saraste dimer and membrane association will be further stabilized by PIP 3 binding to both the canonical and peripheral sites on the PH domain ( Figure 5D ; Chung et al, 2019 ). Upon membrane dimerization it is likely that further rearrangement of the SH2 domain to create contacts with the kinase domain N-lobe will stabilize the kinase domain in its active state ( Figure 5D ).…”

Diffusion-based determination of protein homodimerization on reconstituted membrane surfaces

“…Thus, SLBs have been demonstrated in its usefulness in many works examining molecular interactions on membrane surfaces. Two such examples are K-Ras4B GTPase ( 25 ) and the pleckstrin homology (PH) domain of Bruton’s tyrosine kinase (Btk) in their dimerization behavior ( 26 ). Homodimerization is perhaps the simplest possible protein-protein interaction and is ubiquitous in signal transduction.…”