Biology subjects

Lemmon, M. A.

Publications and source records attributed to Lemmon, M. A..

7 recordsLinked to original sources

bioRxiv2026

Dual recognition of negative charge by pleckstrin homology (PH) domains

Pleckstrin homology (PH) domains are typically assumed to be phosphoinositide-binding modules, although most lack strong lipid specificity and their broader ligand repertoire remains poorly defined. We find that many yeast PH domains bind Nsr1p, the ortholog of nucleolin, also identified as a PH domain ligand, suggesting widespread recognition of negatively charged protein regions. The phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-binding PLC{delta}1 PH domain also binds a highly phosphorylated region of IRBIT through the same site that recognizes PtdIns(4,5)P2. Using high-throughput integrated phosphopeptide (Hi-P) screening, we surveyed 38,624 mammalian sequences containing three documented phosphoserines. PLC{delta}1-PH bound numerous phosphorylated and unphosphorylated acidic peptides, and phosphorylation generally strengthened pre-existing interactions rather than conferring strict specificity, without requiring a fixed phosphoserine spacing. Instead, favored phosphopeptides combined a key phosphoserine with upstream acidic residues. Structural modeling suggests that this phosphoserine occupies the canonical inositol phosphate binding pocket, while adjacent acidic residues make delocalized electrostatic contacts. Thus, PH domains can recognize various patterns of protein negative charge, expanding their potential regulatory roles beyond membrane targeting.

biochemistry↗

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↗

bioRxiv2023

Distinct interactions stabilize EGFR dimers and higher-order oligomers in cell membranes

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) with important roles in many cellular processes as well as cancer and other diseases. EGF binding promotes EGFR dimerization and autophosphorylation through interactions that are well understood structurally. However, it is not clear how these dimers relate to higher-order EGFR oligomers detected at the cell surface. We used single-particle tracking (SPT) and Forster resonance energy transfer (FRET) imaging to examine how each domain within EGFR contributes to receptor dimerization and the rate of its diffusion in the cell membrane. We show that the EGFR extracellular region is sufficient to drive receptor dimerization, but that the EGF-induced EGFR slow-down seen by SPT requires formation of higher order oligomers, mediated in part by the intracellular tyrosine kinase domain - but only when in its active conformation. Our data thus provide important insight into higher-order EGFR interactions required for EGF signaling.

biophysics↗

bioRxiv2021

Phosphatidylserine binding regulates TIM-3 effects on T cell receptor signaling

Co-signaling receptors for the T cell receptor are important therapeutic targets, with blocking co-inhibitory receptors such as PD-1 now central in immuno-oncology. Advancing additional therapeutic immune modulation approaches requires understanding ligand regulation of other co-signaling receptors. One poorly understood therapeutic target is TIM-3 (T cell immunoglobulin and mucin domain containing-3). Which ligands are relevant for TIM-3 signaling is unclear, and different studies have reported it as co-inhibitory or co-stimulatory. Here, we show that TIM-3 promotes NF-{kappa}B signaling and IL-2 secretion following T cell receptor stimulation in Jurkat cells, and is regulated by phosphatidylserine (PS) binding. TIM-3 signaling is stimulated by PS exposed constitutively in cultured Jurkat cells, and can be blocked by mutating the PS-binding site or by occluding this site with an antibody. We also find that TIM-3 signaling alters CD28 phosphorylation. Our findings help clarify conflicting literature results with TIM-3, and inform its exploitation as a therapeutic target.

biochemistry↗

bioRxiv2021

Glioblastoma mutations impair ligand discrimination by EGFR

The epidermal growth factor receptor (EGFR) is frequently mutated in human cancer, and is an important therapeutic target. EGFR inhibitors have been successful in lung cancer, where the intracellular tyrosine kinase domain is mutated, but not in glioblastoma multiforme (GBM) - where mutations (or deletions) occur exclusively in the EGFR extracellular region. Wild-type EGFR is known to elicit distinct signals in response to different growth factor ligands, exhibiting biased agonism. We recently showed that individual ligands stabilize distinct receptor dimer structures, which signal with different kinetics to specify outcome. EGF induces strong symmetric dimers that signal transiently to promote proliferation. Epiregulin (EREG) induces weak asymmetric dimers that generate sustained signaling and differentiation. Intriguingly, several GBM mutation hotspots coincide with residues that define the asymmetric and symmetric dimer structures. Here, we show that common extracellular GBM mutations prevent EGFR from distinguishing between EGF and EREG based on dimer structure and stability - allowing strong dimers to form with both ligands. Crystal structures show that the R84K mutation symmetrizes EREG-driven dimers, whereas the A265V mutation remodels key dimerization sites. Our results suggest that modulating EGFRs biased agonism plays an important role in GBM, and suggest new approaches for correcting aberrant EGFR signaling in cancer.

biochemistry↗

bioRxiv2021

ROR and RYK extracellular region structures suggest that receptor tyrosine kinases have distinct WNT-recognition modes

SUMMARYWNTs play key roles in development and disease, by binding both Frizzled (FZD) seven-pass transmembrane receptors and numerous co-receptors that include the ROR and RYK receptor tyrosine kinases (RTKs). We describe crystal structures and WNT-binding characteristics of extracellular regions from the Drosophila ROR and RYK orthologs Nrk (neurospecific receptor tyrosine kinase) and Derailed-2 (Drl-2). RORs bind WNTs though a FZD-related cysteine-rich domain (CRD), and RYKs through a WNT-inhibitory factor (WIF) domain. Our structures suggest that neither the Nrk CRD nor the Drl-2 WIF domain can accommodate the acyl chain typically attached to WNTs. The Nrk CRD contains a deeply buried bound fatty acid, unlikely to be exchangeable with a WNT acyl chain. The Drl-2 WIF domain lacks the lipid-binding site seen in WIF-1. We also show that DWnt-5, which regulates Drosophila ROR and RYK orthologs, lacks an acyl chain. Together with analysis of WNT/receptor interaction sites, these structures provide new insight into how WNTs recruit their RTK co-receptors into signaling complexes.

biochemistry↗