Biology subjects

Falson, P.

Publications and source records attributed to Falson, P..

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↗

bioRxiv2024

R6G narrows BmrA conformational spectrum for a more efficient use of ATP

Multidrug ABC transporters harness the energy of ATP binding and hydrolysis to change conformation and thereby translocate substrates out of the cell to detoxify them. While this general access mechanism scheme is well accepted, molecular details of this interplay is still elusive. Rhodamine6G binding on a catalytic mutant of the homodimeric multidrug ABC transporter BmrA triggers a cooperative binding of ATP on the two identical nucleotide-binding-sites, otherwise Michaelian. We investigated this asymmetric behavior via a structural-enzymology approach, solving cryoEM structure of BmrA at defined ATP ratio along the enzymatic transition, highlighting the plasticity of BmrA as it undergoes the transition from inward to outward facing conformations. Analysis of continuous heterogeneity within cryoEM data and structural dynamics, revealed that Rhodamine6G narrows the conformational spectrum explored by the nucleotide-binding-domains, describing the allosteric effect of drug binding that optimizes the ATP-dependent conversion of the transporter to the outward-facing state. Following on these findings, the effect of drug-binding showed an ATPase stimulation and a maximal transport activity of the wild-type protein at the concentration-range where the allosteric transition occurs. Drug diffusion rate is the likely rate-limiting step of the reaction, while drug transport and ATPase activities are in effect uncoupled.

biochemistry↗

bioRxiv2021

Collinearity of alpha-helices or beta strands in membrane proteins causes a characteristic peak centred on 4.9 A resolution in diffraction intensity profiles, inducing higher diffraction anisotropy

Diffraction anisotropy is a phenomenon that impacts more specifically membrane proteins, compared to soluble ones, but the reasons for this discrepancy remained unclear. Often, it is referred to a difference in resolution limits between highest and lowest diffraction limits as a signature for anisotropy. We show in this article that there is no simple correlation between anisotropy and difference in resolution limits, with notably a substantial number of structures displaying various anisotropy with no difference in resolution limits. We further investigated diffraction intensity profiles, and observed a peak centred on 4.9[A] resolution more predominant in membrane proteins. Since this peak is in the region corresponding to secondary structures, we investigated the influence of secondary structure ratio. We showed that secondary structure content has little influence on this profile, while secondary structure collinearity in membrane proteins correlate with a stronger peak. Finally, we could further show that the presence of this peak is linked to higher diffraction anisotropy. SynopsisMembrane protein diffraction anisotropy originates from a peak at 4.9 [A] resolution in intensity profiles, due to secondary structure collinearity.

biophysics↗

bioRxiv2020

Nanodisc, amphipol or detergent belts in cryoEM reconstructions of membrane proteins are similar and correspond to a common ordered solvent layer

To maintain membrane proteins soluble in aqueous solution, amphipathic compounds are used to shield the hydrophobic patch of their membrane insertion, which forms a belt around the protein. This hydrophobic belt is seldom looked at due to the difficulty to visualize it. Cryo-EM is now offering this possibility, where belts are visible in 3D reconstructions. We investigated membrane proteins solved in nanodiscs, amphipols or detergents to analyze whether the nature of the amphipathic compound influences the belt size in 3D reconstructions. We identified belt boundaries in map-density distributions and measured distances for every reconstruction. We showed that all the belts create on average similar reconstructions, whether they originate from the same protein, or from protein from different shapes and structures. There is no difference among detergents or types of nanodisc used. These observations illustrate that the belt observed in 3D reconstructions corresponds to the minimum ordered layer around membrane proteins.

biophysics↗