Lacapere, J.-J. | Search bioRxiv

bioRxiv2024

Fluor NMR study of amino acid derived ligand to study TSPO

Translocator protein (TSPO, 18 kDa), previously known as peripheral-type benzodiazepine receptor, is an evolutionarily conserved membrane protein involved in various physiological processes and patho-physiological conditions. The endogeneous TSPO ligand is a polypeptide of 9 kDa, but dipeptides with biological activity have been previously synthesized and characterized. Herein, we synthesized a phenyl alanine derived ligand with a 19F labelling which opens prospective for 19F-MRI and potential 18F-PET applications. We characterized the coexistence of two conformers that are not equally sensitive to the media used for membrane protein studies. Interaction studies with the recombinant mouse TSPO (mTSPO) in different membrane-mimicking environments are presented using 19F NMR enabling structure/function characterizations. A change in the mTSPO environment from pure detergent to lipid/detergent mixture reveals different exchange rates between bound and free ligand forms. Competition experiments with the high-affinity drug ligand (R)-PK 11195 suggests that phenyl alanine derived ligand binds in the same protein cavity. HighlightsO_LIFluor labelling of ligands easily reveals the presence of conformers C_LIO_LIFluorinated phenyl alanine derived ligand interacts with TSPO C_LIO_LIFluor NMR enables characterization of TSPO ligand interactions C_LIO_LIFluor NMR facilitates exchange rate studies between free and bound ligand states C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/618300v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@1913e31org.highwire.dtl.DTLVardef@8a3c30org.highwire.dtl.DTLVardef@175881borg.highwire.dtl.DTLVardef@13aaa7d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2019

CX3CL1 oligomerization is required for efficient CX3CR1-specific cell adherence

During inflammatory response, blood leukocytes adhere to the endothelium. This process involves numerous adhesion molecules, including a transmembrane chemokine, CX3CL1. We previously found that CX3CL1 clusters in oligomers. How this cluster assembles and whether it has a functional role remain unknown. Using various biochemical and biophysical approaches, we show that CX3CL1 clusters are homo-oligomers with 3 to 7 CX3CL1 molecules. We demonstrate that the transmembrane domain peptide self-associates at a similar level in both cellular and acellular lipid environments while its random counterpart (a scrambled peptide) does not. Hence, oligomerization is mainly driven by the transmembrane domain intrinsic properties. Molecular modeling suggests that transmembrane peptide oligomers are mostly made of monomers linearly assembled side by side. Using a new adherence assay, we demonstrate that, functionally, oligomerization is mandatory for the adhesive potency of CX3CL1. Our results indicate that CX3CL1-dependent cellular adherence in key immune processes can be controlled by disrupting clusters using heterotopic peptides, which, in turn, alter the adhesive function of the membrane CX3CL1 without affecting the function of the CX3CL1 soluble form.

cell biology↗