Biology subjects

Otun, O.

Publications and source records attributed to Otun, O..

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↗

bioRxiv2024

Constitutive activity of an atypical chemokine receptor revealed by inverse agonistic nanobodies

Chemokine stimulation of atypical chemokine receptor 3 (ACKR3) does not activate G proteins but recruits arrestins. It is a chemokine scavenger that indirectly influences responses by restricting the availability of CXCL12, an agonist shared with the canonical receptor CXCR4. ACKR3 is upregulated in numerous disorders. Due to limited insights in chemokine-activated ACKR3 signaling, it is unclear how ACKR3 contributes to pathological phenotypes. One explanation may be that high constitutive activity of ACKR3 drives non-canonical signaling through a basal receptor state. Here we characterize the constitutive action of ACKR3 using novel inverse agonistic nanobodies to suppress basal activity. These new tools promote an inactive receptor conformation which decreased arrestin engagement and inhibited constitutive internalization. Basal, non-chemotactic, breast cancer cell motility was also suppressed, suggesting a role for ACKR3 in this process. The basal receptor activity in pathophysiology may provide a new therapeutic approach for targeting ACKR3.

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↗

bioRxiv2023

Conformational dynamics underlying Atypical Chemokine Receptor 3 activation

Atypical Chemokine Receptor 3 (ACKR3) is a G protein-coupled receptor that does not signal through G proteins. It is known as a chemokine scavenger involved in various pathologies, making it an appealing yet intriguing therapeutic target. Indeed, the structural properties that govern ACKR3 functional selectivity and the overall conformational dynamics of ACKR3 activation are poorly understood. Here we combined Hydrogen/Deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics simulations to examine the binding mode and mechanism of action of various small-molecule ACKR3 ligands of different efficacy for {beta}-arrestin recruitment. Our results show that activation or inhibition of ACKR3 is largely governed by intracellular conformational changes of helix 6, intracellular loop 2 and helix 7, while the DRY motif becomes protected during both processes. Moreover, HDX-MS identifies the binding sites and the allosteric modulation of ACKR3 upon {beta}-arrestin 1 binding. In summary, this study highlights the structure-function relationship of small-molecule ligands, the overall activation dynamics of ACKR3, the binding mode of {beta}-arrestin 1 and the atypical dynamic features in ACKR3 that may contribute to its inability to activate G proteins.

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↗

bioRxiv2020

Ligand-directed covalent labelling of a GPCR with a fluorescent tag

Here, we describe rational design of a compound that covalently and selectively labels a G protein-coupled receptor (GPCR) in living cells with a fluorescent moiety. Using wild-type adenosine A2A receptor as a model system, we show fluorescent labelling without impeding access to the orthosteric binding site and demonstrate its use in endogenously expressing systems. This offers a non-invasive and selective approach to study function and localisation of native GPCRs.

pharmacology and toxicology↗