Biology subjects

Baccouch, R.

Publications and source records attributed to Baccouch, R..

5 recordsLinked to original sources

bioRxiv2026

The immunosuppressant tacrolimus (FK506) inhibits C. glabrata Cdr1 efflux pump function by stabilizing the inward-facing conformation.

The pathogenic yeast Candida glabrata is intrinsically resistant to azole antifungals through the overexpression of the multidrug transporter Cdr1. CgCdr1 detoxifies the yeast by expelling azoles out of the cell, thereby decreasing their intracellular concentration. Tacrolimus (FK506), one of the most widely used immunosuppressant medications used world-wide, has been identified as a broad-spectrum inhibitor of Cdr1 homologs in several Candida species. However, its mechanism of action remains unknown. We solved the cryoEM structure of CgCdr1 in complex with FK506, with or without ATP. The structure revealed that FK506 binds within the drug-binding site of CgCdr1, occupying the space occupied by Itraconazole. The hydrophobic face of FK506 stacks against the TMD1 and forms hydrogen bonds with TMD2, stabilizing a different conformation from the one adopted in FK-binding-proteins. FK506 binding triggered structural rearrangements bringing the nucleotide-binding-domains closer to the trans-membrane-domains, while stabilizing the inward-facing conformation. While ATP can still bind to the catalytic nucleotide-binding site, FK506 prevents the conformational transition required for ATP hydrolysis, thereby effectively blocking azole transport. Inter-particle variability analysis (3DVA) revealed significant conformational flexibility of FK506 within the binding pocket, with minimal transporter mobility. It allowed to visualize the conformational space occupied by the inhibitor within its binding-pocket, serving as a useful tool for inhibitor rational design. Overall, these findings demonstrate that FK506s inhibition extends beyond competitive binding, involving allosteric modulation of the ATPase cycle. Significance statementThe pathogenic yeast Candida glabrata exhibits intrinsic resistance to azole antifungals via the multidrug transporter Cdr1, which expels azoles from the cell. Tacrolimus (FK506), a widely used immunosuppressant, inhibits Cdr1 homologs across Candida species, yet its mechanism remained unknown. Here, we resolved the cryoEM structure of CgCdr1 in complex with FK506, revealing that FK506 binds to the drug-binding site, like itraconazole. Its hydrophobic face interacts with TMD1, while hydrogen bonds form with TMD2. FK506 stabilizes the inward-facing conformation, preventing ATP hydrolysis despite ATP binding, thereby blocking azole transport. Variability analysis highlighted FK506s conformational flexibility within the pocket, offering insights for rational inhibitor design. These findings demonstrate that FK506s inhibition involves both competitive binding and allosteric modulation of the ATPase cycle.

biochemistry↗

bioRxiv2025

CryoEM of ATP-driven dynamics and itraconazole binding in a fungal ABC pump

Azole resistance in Candida species is often caused by the overexpression of Cdr1. Despite its clinical relevance, the structural basis for its ATP-driven efflux pump function remains elusive. We present four high-resolution cryo-EM structures for Candida glabrata Cdr1 under active turnover conditions in the absence and presence of ATP-Mg{superscript 2}, itraconazole, and vanadate. Additional transient cryo-EM structures were unveiled by 3D variability analysis offering a detailed view of the step-by-step transitions triggered by ATP-hydrolysis. The motion cascade starts with a 4 [A] piston-like retraction of the C-helix from the {gamma}-phosphate/vanadate of the hydrolyzed ATP. This causes the nearby transmembrane helix-1 (TMH-1) to open the drug-binding site via lateral displacement and unwinding of the inner-leaflet region of TMH-2. A reverse squeeze-and-push motion of TMH-2 possibly drives substrate extrusion. High resolution structures also reveal how itraconazole adapts its shape to fit into the drug-binding site. Our findings provide a dynamic structural framework for Cdr1-mediated azole resistance and the conserved chemo-mechanical cycle of ABC proteins, including non-membranous members.

biochemistry↗

bioRxiv2025

Membrane partition and structural reorganization induced by anti-psychotics with distinct clinical profiles

Antipsychotics (APs) are used in the treatment of severe mental disorders. Their mechanism of action involves interaction with multiple brain targets, notably the dopamine D2 receptors (D2R), where they compete with dopamine. Due to their lipophilic nature, APs also partition and accumulate in lipid membranes, particularly around the D2R and in synaptic vesicles. When intercalated into brain membranes, APs slowly accumulate and act as a reservoir, allowing their rapid release on demand to modulate neurotransmitter signaling. They also modify the physicochemical and mechanical properties of the lipid bilayer. These modifications can subsequently affect the conformational changes of embedded membrane proteins like the D2R. The present study investigated two major APs with different pharmacological and clinical profiles: chlorpromazine, which exerts its clinical activity mainly through a strong antagonistic action at the D2R, and clozapine, the weakest D2R antagonist of all APs. Surprisingly, although D2R antagonism is usually associated with AP potency, clozapine has repeatedly demonstrated clinical superior efficacy to all APs and is therefore recommended for treatment-resistant schizophrenia. The current work aims to extend the classical AP receptor mediated paradigmatic mode of action to their potential and unique membrane remodeling properties by thoroughly comparing their partitioning and impact on the physicochemical properties of the lipid membrane. Lipid model membranes mimicking synaptic vesicles have been investigated using a combination of several biophysical methods. The study aims to determine how the partitioning of the two APs modifies membrane order, phase transition, thickness, elasticity, phase separation, membrane integrity and charge. Differences have been demonstrated between these two compounds, which may further differ both over time as they accumulate as well as depending on their pre- or post-synaptic location.

biophysics↗

bioRxiv2023

Membrane lipid poly-unsaturation selectively affects ligand induced dopamine D2 receptor internalization

The brain is highly enriched in poly-unsaturated fatty acids (PUFAs) and their deficiency has been associated with several neuropsychiatric disorders. Here, we demonstrate that the Dopamine receptor D2 (D2R), a class A G protein coupled receptor (GPCR) which is a main target of antipsychotics, displays unique sensitivity to membrane PUFA composition. We found that membrane enrichment with either of two distinct PUFAs significantly impairs agonist-induced D2R endocytosis in HEK-293 cells and cortical neurons. This treatment does not affect clathrin-mediated endocytosis or the internalization of several other GPCRs. Moreover, we show that D2R clustering at endocytic pits is not affected, but that recruitment of {beta}-arrestin2 is strongly impaired and endocytic vesicle formation is slowed down. Finally, mutation of key residues in intracellular loop 2 abolishes the sensitivity of D2R endocytosis to PUFA enrichment. We conclude that D2R trafficking is uniquely dependent on membrane PUFAs, which could influence its role in the control of brain function and behavior.

cell biology↗

bioRxiv2022

Impact of membrane lipid polyunsaturation on dopamine D2 receptor ligand binding and signaling

The heterogenous and dynamic constitution of the membrane fine-tunes signal transduction. In particular, the polyunsaturated fatty acid (PUFA) tails of phospholipids influence the biophysical properties of the membrane, production of second messengers, or membrane partitioning. Few evidence mostly originating from studies of rhodopsin suggest that PUFAs directly modulate the conformational dynamic of transmembrane proteins. However, whether such properties translate to other G protein-coupled receptors remains unclear. We focused on the dopamine D2 receptor (D2R), a main target of antipsychotics. Membrane enrichment in n-3, but not n-6, PUFAs potentiates ligand binding. Molecular dynamics simulations show that the D2R preferentially interacts with n-3 over n-6 PUFAs. Furthermore, even though this mildly affects signalling in heterologous systems, in vivo n-3 PUFA deficiency blunts the effects of D2R ligands. These results suggest that n-3 PUFAs act as allosteric modulators of the D2R and provide a putative mechanism for their potentiating effect on antipsychotic efficacy.

cell biology↗