Biology subjects

Bechara, C.

Publications and source records attributed to Bechara, C..

6 recordsLinked to original sources

bioRxiv2026

Distinct atypical chemokine receptor 1 determinants underlie bacterial toxins recognition and pore formation

Atypical chemokine receptor 1 (ACKR1) is one of the most promiscuous receptors in the human chemokine system, engaging structurally diverse chemokines through a conformationally flexible N-terminal tail. This same interface is exploited by pathogens, including Plasmodium vivax and Staphylococcus aureus (SA), via a compact sulfotyrosine code. Among pathogenic proteins recognizing ACKR1, the SA leukocidin pair HlgAB is a notable exception. HlgAB-mediated pore formation is only weakly competed by chemokines, the Duffy binding protein, or antibodies targeting the receptor's N-terminus, leaving open how HlgAB engage ACKR1. Combining structural biology approaches with cell-based assays, we show that HlgA and HlgB engage ACKR1 sulfated N-terminus with distinct affinities and site hierarchies, with a single higher-affinity site for sulfated tyrosine 41 present in HlgA but absent in HlgB. Unexpectedly, this N-terminal engagement is dispensable for pore formation; productive lysis instead requires a separate interaction between the toxins and ACKR1 extracellular vestibule. These results define a two-step recognition mechanism, toxin capture by the sulfotyrosine N-terminus followed by vestibule-dependent pore formation, and extend the view that ACKR1 promiscuity arises from distributed, ligand-specific use of multiple receptor surfaces rather than a single adaptable interface.

biochemistry↗

bioRxiv2026

Structural and evolutionary insights into DAF-12 interactions with transcriptional coactivators in parasitic nematodes

Parasitic nematodes infect billions of humans and livestock worldwide, causing major health and economic burdens, while the spread of anthelmintic resistance threatens current control strategies. A critical step in parasite infection is the resumption of development of infective third-stage larvae (iL3) upon host entry, a process controlled by the nuclear receptor DAF-12. Activation of DAF-12 by dafachronic acids promotes developmental progression and reproductive maturation, making this receptor an attractive therapeutic target. However, the molecular mechanisms governing DAF-12 activation, particularly transcriptional coactivator recruitment, remain poorly understood. Here, we combined biophysical, cellular, structural, and bioinformatic approaches to investigate coactivator recognition by DAF-12 from the parasitic nematodes Brugia malayi and Haemonchus contortus. Crystal structures of ligand-bound DAF-12 ligand-binding domains in complex with coactivator-derived peptides reveal conserved features of ligand-dependent coactivator recruitment shared with mammalian nuclear receptors. In addition, we uncover previously unrecognized interaction features, including motif-specific contacts that extend beyond the canonical LXXLL binding mode of coactivators and distinct patterns of DAF-12 conservation across nematode clades. Structure-guided analyses redefine the interaction motif of the only described parasite-specific coactivator DIP-1 and suggest novel candidate motifs for DAF-12-interacting proteins. Together, these findings establish the structural basis of coactivator binding to nematode DAF-12 and provide mechanistic insight into the transcriptional regulation underlying parasite development. These results expand current understanding of nuclear receptor signaling in parasitic nematodes and provide a framework for the future design of strategies aimed at disrupting DAF-12 activation as a potential antiparasitic approach. Author SummaryParasitic nematodes infect billions of people and livestock worldwide, causing major health and economic burdens, while increasing resistance threatens current treatments. These parasites rely on a developmental switch that allows infectious larvae to resume growth inside their host, a process controlled by the nuclear receptor DAF-12. Blocking this pathway could prevent parasites from establishing infection. However, the molecular mechanisms regulating DAF-12 activation remain poorly understood. Here, we investigate how DAF-12 from two parasitic nematodes, Brugia malayi and Haemonchus contortus, interacts with transcriptional coactivators that enable gene activation, using a combination of biophysical, cellular, structural, and bioinformatic approaches. We identified conserved features of ligand-dependent coactivator recruitment shared with mammalian nuclear receptors as well as nematode-specific interaction mechanisms that vary across evolutionary clades. These findings provide new insights into the structural basis of coactivator binding to DAF-12 and advance our understanding of a key pathway controlling parasitic nematode development.

biochemistry↗

bioRxiv2024

Physical interaction with Ephrin B1 promotes CXCR4 intracellular localization and oncogenic potential

Chemokine receptor 4 (CXCR4) is a member of the chemokine receptor exclusively activated by the chemokine CXCL12. While CXCR4 regulates numerous physiological processes associated with cell migration and embryogenesis, its overexpression has been involved in various cancer types. Studies suggest that intracellular CXCR4 expression rather than CXCR4-operated signaling underlies its pro-tumorigenic functions. Given the role of GPCR interacting proteins in their trafficking and subcellular localization, we characterized the CXCR4 interactome using an affinity purification coupled to mass spectrometry (AP-MS) strategy. The most abundant protein identified in the CXCR4 interactome is Ephrin B1, a member of the Ephrin protein family that shares several functions with CXCR4, such as the regulation of cell migration and proliferation. Further studies showed that interaction between CXCR4 and Ephrin B1 is direct and enhanced by treating cell with CXCL12. They also indicated that Ephrin B1 prevents CXCR4 N-glycosylation, decreases CXCR4 cell surface expression and consistently inhibits CXCL12-induced CXCR4 coupling to Gi1-3 and the recruitment of {beta}-arrestins 1 and 2. Conversely, Ephrin B1 signals to Erk1,2 through CXCR4 activation and mediates the decrease in Death Receptor 5 expression elicited by intracellular CXCR4. Collectively, these findings identify Ephrin B1 as a key mediator of CXCR4 tumorigenic signaling.

biochemistry↗

bioRxiv2022

Cryo-EM structure of the agonist-bound Hsp90-XAP2-AHR complex

SummaryLiving organisms have developed protein sensors helping them to adapt to their environment1. The aryl hydrocarbon receptor (AHR) is an emblematic member of this class of proteins, and a ligand-dependent transcription factor that mediates a broad spectrum of (patho)physiological processes in response to numerous substances including pollutants, natural products and metabolites2. However, in the absence of high-resolution structural data, a molecular understanding of how AHR is activated by such diverse compounds is lacking. Here we present a 2.85 [A] cryo-electron microscopy structure of the cytosolic complex comprising AHR bound to the ligand indirubin, the chaperone Hsp90 and the co-chaperone XAP2. The structure reveals a closed Hsp90 dimer with AHR threaded through its lumen. XAP2 directly interacts with Hsp90 and the AHR ligand-binding domain, thereby acting as a brace stabilizing the entire complex. Importantly, we provide the first experimental visualization of the AHR PAS-B domain bound to a ligand, revealing a unique organization of the ligand-binding pocket and the structural determinants of ligand-binding specificity and promiscuity of the receptor. By providing unprecedented structural details of the molecular initiating event leading to AHR activation, our study rationalizes prior biochemical data and provides a framework for future mechanistic studies and structure-guided drug design.

biochemistry↗

bioRxiv2021

Structural insights into recognition of chemokine receptors by Staphylococcus aureus leukotoxins

Staphylococcus aureus (SA) leukocidin LukED belongs to a family of bicomponent pore forming toxins that play important roles in SA immune evasion and nutrient acquisition. LukED targets specific G protein-coupled chemokine receptors to lyse human erythrocytes and leukocytes. The first recognition step of receptors is critical for specific cell targeting and lysis. The structural and molecular bases for this mechanism are not well understood but could constitute essential information to guide antibiotic development. Here, we characterized the interaction of LukE with chemokine receptors ACKR1, CCR2 and CCR5 using a combination of structural, pharmacological and computational approaches. First, crystal structures of LukE in complex with a small molecule mimicking sulfotyrosine side chain (p-cresyl sulfate) and with peptides containing sulfotyrosines issued from receptor sequences revealed the location of receptor sulfotyrosine binding sites in the toxins. Then, by combining the available experimental information with protein docking, classical and accelerated weight histogram (AWH) molecular dynamics we propose models of the ACKR1-LukE and CCR5-LukE complexes. This work provides novel insights into chemokine receptor recognition by leukotoxins and suggests that the conserved sulfotyrosine binding pocket could be a target of choice for future drug development.

biochemistry↗

bioRxiv2021

Mechanisms of GPCR hijacking by Staphylococcus aureus

Atypical chemokine receptor 1 (ACKR1) is a G protein-coupled receptor (GPCR) targeted by Staphylococcus aureus (SA) bi-component pore-forming leukotoxins to promote bacterial growth and immune evasion. Here we have developed an integrative molecular pharmacology and structural biology approach in order to characterize the effect of leukotoxins HlgA and HlgB on ACKR1 structure and function. Interestingly, we found that both components HlgA and HlgB compete with endogenous chemokines through a direct binding to ACKR1 captured by native mass spectrometry (MS). Unexpectedly, HDX-MS analysis revealed that toxin binding allosterically modulates the intracellular G protein-binding domain of the receptor, resulting in dissociation of ACKR1-G protein complexes in living cells. Altogether, our study brings important molecular insights into the initial steps of leukotoxins targeting a host GPCR. Our findings may open the way to develop antibiotics inhibiting host receptors binding, a mechanism of action less prone to resistance.

biochemistry↗