Biology subjects

Tsutsui, Y.

Publications and source records attributed to Tsutsui, Y..

4 recordsLinked to original sources

bioRxiv2024

EGFR and tyrosine kinase inhibitor interactions probed by hydrogen-deuterium exchange and mass spectrometry (HDX-MS)

EGFR is one of the primary drug targets for treating non-small cell lung cancer (NSCLC) patients carrying EGFR oncogenic mutations in the tyrosine kinase domain (TKD). Such patients typically receive tyrosine kinase inhibitors (TKIs) to inhibit aberrant activation of EGFR; however, together with the appearance of the TKI-resistant mutations, TKIs severe side effects often limit their clinical usage. To develop TKIs with the wild-type sparing effect, the wild-type structures bound to various TKIs ought to be characterized, though comparisons of such crystal structures do not show clear differences. To characterize subtle EGFR TKD structural changes upon TKI binding that cannot be gleaned from crystal structure comparisons, we employed HDX-MS. We show inhibitor-dependent EGFR dynamics that are displayed even among the TKD bound to chemically similar inhibitors. Such inhibitor-dependent structural changes appear to underlie TKI side effects and the selectivity of covalent inhibitors. HighlightsO_LIEGFR shows TKI-dependent dynamics even if TKIs are structurally similar. C_LIO_LIThe stability of the TKI-encounter complexes correlates with their side effects. C_LIO_LICovalent TKIs disrupt the binding pocket of wild-type EGFR. C_LIO_LIThe structure of the osimertinib-L858R/T790M complex is extremely rigid. C_LI

biophysics↗

bioRxiv2024

Coupled Solvent Dynamics and Protein Dynamics Help Drive Functional Differences in Exon-19 Deletion Mutants in the Epidermal Growth Factor Receptor (EGFR/ErbB1/HER1) Kinase Domain

Deletions in Exon-19 of the epidermal growth factor receptor (EGFR) play a pivotal role in the pathogenesis of non-small cell lung cancer (NSCLC), influencing patient response to tyrosine kinase inhibitors (TKIs). Although these mutations are known to affect treatment efficacy, the precise molecular mechanisms have been unclear. Building upon recent insights from the study [DOI: 10.1038/s41467-022-34398-z], which identified two distinct mutation profiles associated with differential drug sensitivity and clinical outcomes, our research delves into the molecular dynamics that drive these variances. We employed molecular dynamics simulations, enhanced sampling methods, and machine learning to classify Exon-19 deletion mutations into two profiles based on their conformational dynamics. Profile 1 mutations display only localized motions in key subdomains in their fluctuations about the equilibrium state, and a high affinity for ATP and consequent resistance to TKIs, while profile 2 mutations show reduced ATP binding affinity due to delocalized motion characterized by an increased flexibility between the N- and C-lobes of the EGFR kinase domain. This structural flexibility perturbs the ATP binding site, leading to decreased affinity and, heightened sensitivity to TKIs. Our use of the INDirect Umbrella Sampling (INDUS) technique has shed light on the collective solvent dynamics, further elucidating the coupling between long timescale solvent fluctuations and protein conformational dynamics, that likely contributes to the observations in HDX-MS studies. Our free energy analysis, covering timescales relevant to both HDX-MS and ligand interaction, provides a deeper understanding of the relationship between protein and solvent dynamics and their collective impact on drug efficacy in NSCLC with EGFR Exon-19 deletions. Significance StatementEGFR Exon 19 deletion mutations are key drivers in non-small cell lung cancer (NSCLC), yet their drug sensitivity to tyrosine kinase inhibitors (TKIs) varies significantly. This study identifies two mutation profiles: mutations that exhibit high ATP binding affinity and localized conformational motion, driving TKI resistance, and mutations that show reduced ATP affinity due to delocalized structural flexibility, enhancing TKI sensitivity. Using molecular dynamics simulations and free energy sampling techniques, we reveal how solvent fluctuations and protein dynamics collectively affect drug binding and efficacy. These findings provide a mechanistic basis for differential drug sensitivity, informing precision medicine strategies for NSCLC patients with EGFR Exon-19 mutations.

biophysics↗

bioRxiv2024

Physical basis for the interaction between Drosophila ROS1 and the GPCR BOSS

Abstract/SummaryDrosophila ROS1 (dROS1, Sevenless) is a receptor tyrosine kinase (RTK) essential for the differentiation of Drosophila R7 photoreceptor cells1, 2. Activation of dROS1 is mediated by binding to the extracellular region (ECR) of the GPCR (G protein coupled receptor) BOSS (Bride Of Sevenless) on adjacent cells1, 3, 4. Genetic evidence together with in vitro activity assays confirmed the activation of dROS1 by BOSS and identified subsequent downstream signaling pathways including SOS (Son of Sevenless)1, 5. However, the physical basis for how dROS1 interacts with the GPCR BOSS has long remained unknown. Here we provide the first structure, using Cryo-Electron Microscopy (CryoEM), of dROS1s extracellular region, which mediates ligand binding. We show that the N-terminal region of dROS1 adopts a folded-over conformation harboring a novel structural domain. We further narrowed down the interacting binding epitopes on both dROS1 and BOSS. This includes a beta-strand in dROS1s third Fibronectin type III (FNIII) domain and the C-terminal portion of BOSS ECR. Our mutagenesis studies, coupled with AlphaFold complex predictions, support a binding interaction mediated by a hydrophobic interaction and beta-strand augmentation between these regions. Our findings provide a fundamental understanding of the regulatory function of dROS1 and further provide mechanistic insight into the human ortholog and oncogene ROS1.

biophysics↗

bioRxiv2023

Allosteric activation of the co-receptor BAK1 by the EFR receptor kinase initiates immune signaling

Transmembrane signaling by plant receptor kinases (RKs) has long been thought to involve reciprocal trans-phosphorylation of their intracellular kinase domains. The fact that many of these are pseudokinase domains, however, suggests that additional mechanisms must govern RK signaling activation. Non-catalytic signaling mechanisms of protein kinase domainshave been described in metazoans, but information is scarce for plants. Recently, a non-catalytic function was reported for the leucine-rich repeat (LRR)-RK subfamily XIIa member EFR (ELONGATION FACTOR TU RECEPTOR) and phosphorylation-dependent conformational changes were proposed to regulate signaling of RKs with non-RD kinase domains. Here, using EFR as a model, we describe a non-catalytic activation mechanism for LRR-RKs with non-RD kinase domains. EFR is an active kinase, but a kinase-dead variant retains the ability to enhance catalytic activity of its co-receptor kinase BAK1/SERK3 (BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1/SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3). Applying hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis and designing homology-based intragenic suppressor mutations, we provide evidence that the EFR kinase domain must adopt its active conformation in order to activate BAK1 allosterically, likely by supporting C-helix positioning in BAK1. Our results suggest a conformational toggle model for signaling, in which BAK1 first phosphorylates EFR in the activation loop to stabilize its active conformation, allowing EFR in turn to allosterically activate BAK1.

biochemistry↗