Carmen Galián, Damien Ficheux, Antoine Leydier, Pierre Falson, Jean-Michel Jault, David Flot, Moez Rhimi, Frédéric Huché, Nushin Aghajari, Attilio Di Pietro, Rima Matar-Merheb, Richard Kahn, Anthony W. Coleman, Elodie Desuzinges-Mandon, Centre National de la Recherche Scientifique (CNRS), Université de Lyon (COMUE), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), European Synchrotron Radiation Facility (ESRF), Laboratoire des Multimatériaux et Interfaces (LMI), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), CNRS (Centre National de la Recherche Scientifique, www.cnrs.fr), ANR (Agence Nationale de la Recherche, www.agence- nationale-recherche.fr) ANR-06-PCVI-0019-01, ANR-09-PIRI-0002-01, ANR-06-BLANC-0420, CIBLE (www.rhonealpes.fr) Cible n u 06 022966 01/02, PRIME (Projets de Recherche Interne Multi-Equipes, www.ibcp.fr) IBCP-Prime 2006, IBCP Prime 2007, Ligue Nationale contre le Cancer (www.ligue-cancer.net), label led team:'Laboratoire des Protéines de Résistance aux Agents Chimiothérapeutiques'., MICrobiologie de l'ALImentation au Service de la Santé (MICALIS), AgroParisTech-Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), ANR-06-BLANC-0420CIBLE (www.rhonealpes.fr) Cible nu 06 022966 01/02PRIME (Projets de Recherche Interne Multi-Equipes, www.ibcp.fr) IBCP-Prime 2006IBCP Prime 2007Ligue Nationale contre le Cancer (www.ligue-cancer.net), labelledteam: 'Laboratoire des Protéines de Résistance aux Agents Chimio-thérapeutiques'. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript, ANR (Agence Nationale de la Recherche, www.agencenationale-recherche.fr) ANR-06-PCVI-0019-01, ANR-09-PIRI-0002-01, ANR-06-PCVI-0019,Stabicalix,Design of calix[n]arenic detergents for structural and functional studies of ABC transporters(2006), ANR-06-BLAN-0420,MDR ATPases,Functional and structural mechanism of multidrug ATPase pumps(2006), Rhimi, Moez, Leydier, Antoine, Huché, Frédéric, Coleman, Anthony W, Falson, Pierre, Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
International audience; Background: Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation.Methodology/principal findings: Anionic calix[4]arene based detergents (C4Cn, n=1-12) were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5-24 nm, with the critical micellar concentration (CMC) being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein), a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM). They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux) much more efficiently than SDS (sodium dodecyl sulphate), FC12 (Foscholine 12) or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein.Conclusion/significance: These compounds seem promising to extract in a functional state membrane proteins obeying the positive inside rule. In that context, they may contribute to the membrane protein crystallization field.