Biology subjects

Boumendjel, A.

Publications and source records attributed to Boumendjel, A..

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↗

bioRxiv2026

Isoprenoid quinone profiling in complex biological samples using a novel semi-quantitative HPLC-MS/MS method

Isoprenoid quinones are ubiquitous redox lipids that mediate electron transfer in various cellular processes across all domains of life. These molecules also serve as taxonomic and metabolic markers, facilitating the characterisation of microbial communities. However, their structural diversity and extreme hydrophobicity are challenging for comprehensive detection and quantification in complex biological matrices. In this study, we present a semi-quantitative HPLC-MS/MS method that enables the sensitive analysis of the widest range of quinones reported to date. Using a 16-quinone standard mixture, we optimised separation within a 14-minute HPLC gradient and achieved femtomole-level sensitivity in targeted analyses. When applied to sewage sludges sampled weekly over three weeks, our method detected 57 distinct quinones, revealing stage-specific quinone profiles that reflect shifts in bacterial communities during wastewater treatment. This rapid and sensitive workflow provides a robust tool for accurate quinone profiling in complex samples, opening avenues for the discovery of novel quinones through untargeted approaches. By pushing the boundaries of quinone profiling, our method holds significant promise for advancing microbial ecology, environmental monitoring, and biotechnological applications. HighlightsO_LIuHPLC-Orbitrap method for the semi-quantitative profiling of isoprenoid quinones C_LIO_LIAnalysis of the widest range of isoprenoid quinones to date C_LIO_LIFemtomole-level sensitivity in just 14 minutes of chromatographic separation C_LIO_LIDetection of 57 quinones in complex wastewater sludge matrices C_LIO_LIMost comprehensive set of quinone standards including purified microbial quinones C_LI

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↗

bioRxiv2024

A novel quinone biosynthetic pathway illuminates the evolution of aerobic metabolism

The dominant organisms in modern oxic ecosystems rely on respiratory quinones with high redox potential (HPQs) for electron transport in aerobic respiration and photosynthesis. The diversification of quinones, from low redox potential in anaerobes to HPQs in aerobes, is assumed to have followed Earths surface oxygenation [~]2.3 billion years ago. However, the evolutionary origins of HPQs remain unresolved. Here, we characterize the structure and biosynthetic pathway of a novel ancestral HPQ, methyl-plastoquinone, that is unique to bacteria of the phylum Nitrospirota. Methyl-plastoquinone is structurally related to the two previously known HPQs, plastoquinone from Cyanobacteriota/chloroplasts and ubiquinone from Pseudomonadota/mitochondria, respectively. We demonstrate a common origin of the three HPQ biosynthetic pathways that predates the emergence of Nitrospirota, Cyanobacteriota, and Pseudomonadota. An ancestral HPQ biosynthetic pathway evolved [≥] 3.4 billion years ago in an extinct lineage and was laterally transferred to these three phyla [~]2.5-3.2 billion years ago. We show that Cyanobacteriota and Pseudomonadota were ancestrally aerobic and thus propose that aerobic metabolism using HPQs significantly predates Earths surface oxygenation. Two of the three HPQ pathways were later obtained by eukaryotes through endosymbiosis forming chloroplasts and mitochondria, enabling their rise to dominance in modern oxic ecosystems. Significance statementOxygenic photosynthesis and aerobic respiration by bacteria and eukaryotes rely on respiratory quinones with high redox potential that facilitate membrane-bound electron transport. These quinones are integral to aerobic metabolism and therefore the evolution of aerobic metabolism and quinone biosynthesis must be intertwined. Only two types of high redox potential quinones have been described in bacteria and eukaryotes. Here, we describe the structure and biosynthetic pathway of a third type, methyl-plastoquinone, that is exclusive to bacteria of the phylum Nitrospirota. We then use phylogenetic analysis to show that the three high redox potential quinones have a single evolutionary origin and are much older than previously considered, predating the Great Oxygenation Event, when significant amounts of O2 first accumulated in the atmosphere.

microbiology↗

bioRxiv2021

Drug-bound and -free outward-facing structures of a multidrug ABC exporter point to a swing mechanism

Multidrug ABC transporters translocate drugs across membranes by a mechanism for which the molecular features of drug release are so far unknown. Here, we resolved two ATP-Mg2+-bound outward-facing (OF) conformations of the Bacillus subtilis (homodimeric) BmrA, one by X-ray crystallography without drug, and another by single-particle cryo-EM with rhodamine 6G (R6G). Two R6G molecules bind to the drug-binding cavity at the level of the outer leaflet, between transmembrane (TM) helices 1-2 of one monomer and TM5-6 of the other. R6G induces a rearrangement of TM1-2, highlighting a flexibility that was confirmed by H/D exchange and molecular dynamics simulations. The latter also shows a fast post-release occlusion of the cavity driven by hydrophobicity. Altogether, these data support a new swing mechanism for drug transport.

biochemistry↗