Btk SH2-kinase interface is critical for allosteric kinase activation and its targeting inhibits B-cell neoplasms

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. Constitutive Btk signaling drives several B-cell cancers. Here the authors demonstrate key allosteric intramolecular interactions between the SH2 domain and the kinase domain of Btk, and propose an alternative approach for inhibition of both wild-type and tyrosine kinase inhibitor-resistant Btk.

Allosteric regulation and targeting of Bruton's tyrosine kinase (Btk) in B-cell lymphoma

Daniel Pereira Duarte

Bruton's tyrosine kinase (Btk) is a key component of BCR signaling required for B-cell maturation and activation. Loss-of-function mutations throughout the Btk protein cause human X-linked agammaglobulinemia (XLA), a heritable genetic disorder characterized by the absence of mature B-cells and lack of adaptive immune response. In contrast, Btk signaling sustains the growth of several B-cell neoplasms, which may be treated with small-molecule tyrosine kinase inhibitors (TKIs) targeting the ATP-site of the kinase. No alternative targeted therapy is available for patients who acquire resistance to current Btk inhibitors. Whereas a number of intramolecular interactions amongst the PH, SH3, SH2, and kinase domain (KD) are well resolved and stabilize a compact autoinhibited conformation, the molecular mechanisms governing Btk activation are not fully understood. Using a combination of in silico, structural, cellular, and molecular approaches, we discovered an allosteric interface between the SH2 and the N-lobe of the KD that is critical for kinase activation in vitro and cells. In contrast to the low Btk activity of the KD alone, the presence of the SH2 domain is sufficient to increase Btk phosphorylation at Y551 and multiple other sites. This provides the structural mechanism by which a subset of XLA mutations mapped to the SH2 domain near the interface region strongly perturbs Btk activation, even though the SH2 canonical function is preserved. Furthermore, we demonstrated that the active Btk SH2-kinase adopts an elongated conformation with a dynamic contact interface structurally distinct than to the ones observed for other tyrosine kinases, such as Abl, Fes, and Csk. As allosteric interactions provide unique targeting opportunities far from the ATP-site, we developed an engineered repebody protein binding to the human Btk SH2 domain. The crystal structure of the SH2-repebody complex combined with other structural approaches demonstrated that the repebody disrupts the SH2-kinase interaction. The repebody prevented activation of wild-type and TKI-resistant Btk in vitro and cells seen by a significant decrease in Btk phosphorylation. In parallel, sole allosteric targeting inhibited Btk-dependent signaling and reduced proliferation of malignant B-cells dependent on the BCR signaling. Therefore, the SH2-kinase interface is critical for Btk activation and is the first targetable site able to trigger allosteric inhibition. As Btk targeted therapy has a great impact on several conditions, including B-cell malignancies and autoimmune diseases, this study provides a rationale to support the development of alternative Btk inhibitors. This approach for targeted inhibition could particularly benefit patients with current therapy-resistant Btk variants.

EPFL2020

HRD Motif as the Central Hub of the Signaling Network for Activation Loop Autophosphorylation in Abl Kinase

Oliver Hantschel, Grégory La Sala

A number of structural factors modulate the activity of Abelson (Abl) tyrosine kinase, whose deregulation is often related to oncogenic processes. First, only the open conformation of the Abl kinase domain's activation loop (A-loop) favors ATP binding to the catalytic cleft. In this regard, the trans-autophosphorylation of the Y412 residue, which is located along the A-loop, favors the stability of the open conformation, in turn enhancing Abl activity. Another key factor for full Abl activity is the formation of active conformations of the catalytic DFG motif in the Abl kinase domain. Furthermore, binding of the SH2 domain to the N-lobe of the Abl kinase was recently demonstrated to have a long-range allosteric effect on the stabilization of the A-loop open state. Intriguingly, these distinct structural factors imply a complex signal transmission network for controlling the A-loop's flexibility and conformational preference for optimal Abl function. However, the exact dynamical features of this signal transmission network structure remain unclear. Here, we report on microsecond-long molecular dynamics coupled with enhanced sampling simulations of multiple Abl model systems, in the presence or absence of the SH2 domain and with the DFG motif flipped in two ways (in or out conformation). Through comparative analysis, our simulations augment the interpretation of the existing Abl experimental data, revealing a dynamical network of interactions that interconnect SH2 domain binding with A-loop plasticity and Y412 autophosphorylation in Abl. This signaling network engages the DFG motif and, importantly, other conserved structural elements of the kinase domain, namely, the EPK-ELK H-bond network and the HRD motif. Our results show that the signal propagation for modulating the A-loop spatial localization is highly dependent on the HRD motif conformation, which thus acts as the central hub of this (allosteric) signaling network controlling Abl activation and function.

Amer Chemical Soc2016

Computational Investigation of Intracellular Signalling Cascades

Siri Camee van Keulen

In order to adjust to changes in the environment, cells can communicate via signalling cascades. A common pathway for intercellular communication is the G-protein-coupled- receptor (GPCR) signal transduction cascade which is triggered by a (chemical or physical) extracellular signal, such as light or small molecules. This external interaction induces conformational changes that can lead either to GPCR activation or deactivation. In the active form, GPCRs can induce the activation of trimeric G proteins, which results in the dissociation of the Galpha from the beta and gamma subunits. Subsequently, active Galpha can interact with other cytosolic proteins, such as adenylyl cyclase (AC), which converts adenosine triphosphate to cyclic adenosine monophosphate (cAMP) upon stimulation. AC activation can be induced via stimulatory Galpha subunit (Galpha_s) interactions, while inhibition is related to inhibitory Galpha (Galpha_i) association. X-ray crystallography is crucial for the computational study of the GPCR signal transduction pathway. However, sometimes important molecules or moieties can become lost or cannot be incorporated during crystallisation procedures, which can have a significant effect on a protein’s conformation and function. In this thesis, three steps in the GPCR signal transduction cascade have been studied, rhodopsin activation (i), the conformation of active Galpha_i (ii) and the interaction between Galpha_i and AC (iii). In all three cases some groups or molecules that are absent in the X-ray structures play a vital role in the function of the proteins. In the first project, rhodopsin’s early intermediates after photon exposure are investigated together with a deprotonation reaction that takes place in the later stages of the activation pathway. The classical molecular dynamics (MD) and mixed quantum mechanics/molecular mechanics MD results suggest that photon exposure induces active site rearrangements which increase the stability of rhodopsin’s deprotonated Schiff base state. A bridging water molecule in the active site that is absent in the X-ray structure appears to play an important role during the deprotonation mechanism, which demonstrates the dependence of the protein’s function on its environment. The second study investigates the conformation of the active soluble form of Galpha_i, which has not yet been fully resolved via experimental studies due to crystallisation difficulties of the lipidated N-terminus. To investigate the effect of lipidation via classical MD simulations, a model of soluble lipid-bound Galpha_i was constructed. The simulations show that Galpha_i can form a hydrophobic pocket for the lipid on the protein surface. Other regions of the protein, important for protein-protein interaction, are adjusted as well. Hence, the post-translational modification appears to have a significant impact on Galpha_i’s active conformation and function, which was not captured via the unlipidated X-ray structures. The last project is devoted to a complex of active soluble Galpha_i and AC (Galpha_i :AC). While the interaction site for Galpha_s on AC is known, the interaction site for Galpha_i has not yet been identified. Classical MD simulations were performed on a docked Galpha_i:AC complex to understand the inhibition mechanisms as well as the differentiation between AC stimulation/inhibition. The results show that Galpha_i binding not only leads to AC inhibition via impeding ATP binding but also by preventing re-activation via Galpha_s through closure of the subunit’s interaction site on AC. Keywords: GPCR signal transduction pathway, G-protein-coupled recepter, rhodopsin, G protein, complex formation, cis-trans isomerisation, adenylyl cyclase.

EPFL2017