Solubilization and Stabilization of Native Membrane Proteins for Drug Discovery.

Membrane proteins (MPs) are stable in their native lipid environment. To enable structural and functional investigations, MPs need to be extracted from the membrane. This is a critical step that represents the main obstacle for MP biochemistry and structural biology. Here we describe detergent solubilization screening of MPs using dot-blot and Western-blot analyses. Good solubilization conditions are ranked for their best capacity to stabilize MPs using thermal shift assay. The protein functionality is evaluated by radioligand binding (for G-protein-coupled receptor) and ATPase activity (ABC Transporter) and finally the aggregation status as well as protein homogeneity are assessed by Native-polyacrylamide gel, chemical cross-linking, and size exclusion chromatography.

NACHO Engages N-Glycosylation ER Chaperone Pathways for α7 Nicotinic Receptor Assembly.

The α7 nicotinic acetylcholine receptor participates in diverse aspects of brain physiology and disease. Neurons tightly control α7 assembly, which relies upon NACHO, an endoplasmic reticulum (ER)-localized integral membrane protein. By constructing α7 chimeras and mutants, we find that NACHO requires the α7 ectodomain to promote receptor assembly and surface trafficking. Also critical are two amino acids in the α7 second transmembrane domain. NACHO-mediated assembly is independent and separable from that induced by cholinergic ligands or RIC-3 protein, the latter of which acts on the large α7 intracellular loop. Proteomics indicates that NACHO associates with the ER oligosaccharyltransferase machinery and with calnexin. Accordingly, NACHO-mediated effects on α7 assembly and channel function require N-glycosylation and calnexin chaperone activity. These studies identify ER pathways that mediate α7 assembly by NACHO and provide insights into novel pharmacological strategies for these crucial nicotinic receptors.

CX3CL1 homo-oligomerization drives cell-to-cell adherence.

During inflammatory response, blood leukocytes adhere to the endothelium. This process involves numerous adhesion molecules, including a transmembrane chemokine, CX3CL1, which behaves as a molecular cluster. How this cluster assembles and whether this association has a functional role remain unknown. The analysis of CX3CL1 clusters using native electrophoresis and single molecule fluorescence kinetics shows that CX3CL1 is a homo-oligomer of 3 to 7 monomers. Fluorescence recovery after photobleaching assays reveal that the CX3CL1-transmembrane domain peptide self-associates in both cellular and acellular lipid environments, while its random counterpart (i.e. peptide with the same residues in a different order) does not. This strongly indicates that CX3CL1 oligomerization is driven by its intrinsic properties. According to the molecular modeling, CX3CL1 does not associate in compact bundles but rather with monomers linearly assembled side by side. Finally, the CX3CL1 transmembrane peptide inhibits both the CX3CL1 oligomerization and the adhesive function, while its random counterpart does not. This demonstrates that CX3CL1 oligomerization is mandatory for its adhesive potency. Our results provide a new direction to control CX3CL1-dependent cellular adherence in key immune processes.

Lactobionamide-based fluorinated detergent for functional and structural stabilization of membrane proteins.

Membrane proteins (MPs) are important drug discovery targets for a wide range of diseases. Conventional detergents such as n-Dodecyl ß-D-maltoside have been used largely and efficiently to solubilize MPs with varying degrees of success concerning MPs functionality and stability. Fluorinated surfactants (FSs) have shown a great potential for the stabilization of various MPs. However, so far only a limited number of reports have demonstrated the ability of FSs to solubilize MPs from biological membranes. We report herein the use of a fluorinated lactobionamide-based detergent named FLAC6 for functional and structural stabilization of membrane proteins. We first demonstrated that FLAC6 efficiently solubilized three membrane proteins i.e. the native adenosine receptor A2AR, a G protein-coupled receptor, and two native transporters AcrB and BmrA. The resulting affinity purified MPs were highly pure, homogenous and aggregates free. Furthermore, the functionality of each MP was well maintained. Finally, striking overstabilization features were observed. Indeed, the Tm of native A2AR, AcrB and BmrA could be improved by 7, ~9 and ~ 23 °C, respectively when FLAC6 was used instead of the reference detergent. This work illustrates that FLAC6 is an efficient tool to maintain structural and functional integrities of different MPs belonging to different classes, providing a new avenue for functional stabilization of highly druggable and challenging membrane proteins involved in unmet medical needs.

Novel calixarene-based surfactant enables low dose split inactivated vaccine protection against influenza infection.

Influenza A viruses cause major morbidity and represent a severe global health problem. Current influenza vaccines are mainly egg-based products requiring the split of whole viruses using classical detergents such as Triton X-100, which implies certain limitations. Here, we report the use of the novel calixarene-based surfactant CALX133ACE as an alternative to classical detergents for influenza inactivated split vaccine preparation. We confirmed that CALX133ACE-based split HA antigens are fully functional and quantifiable by the "gold standard" method SRID. Additionally, as in the case of the Triton X-100-based split, the CALX133ACE-based split antigens are stable for at least 6 months at 4 °C. Moreover, immunization of mice with CALX133ACE-based split NYMC X-179A (H1N1) antigens harboring 10 to 30-fold less antigen than the commercialized trivalent inactivated vaccines Vaxigrip® or Fluviral® induced comparable efficient protection and neutralizing antibody responses against A(H1N1)pdm09 infection. Taken together, our results demonstrate for the first time the use of a calixarene-based detergent as an efficient splitting agent for the production of optimized influenza split antigens, paving the way for significant improvement in the vaccine manufacturing process, notably with regard to the current regulation on the prohibition of endocrine disruptors, such as Triton X-100.

Biochemical and biophysical characterization of purified native CD20 alone and in complex with rituximab and obinutuzumab.

CD20 is a B-lymphocyte specific integral membrane protein, an activated-glycosylated phosphoprotein expressed on the surface of B-cells and a clinically validated target of monoclonal antibodies such as rituximab, ocrelizumab, ofatumumab and obinutuzumab in the treatment of all B cell lymphomas and leukemias as well as autoimmune diseases. Here, we report the extraction and purification of native CD20 from SUDHL4 and RAMOS cell lines. To improve the protein yield, we applied a calixarene-based detergent approach to solubilize, stabilize and purify native CD20 from HEK293 cells. Size Exclusion Chromatography (SEC) and Analytical Ultracentrifugation show that purified CD20 was non-aggregated and that CD20 oligomerization is concentration dependent. Negative stain electron microscopy and atomic force microscopy revealed homogenous populations of CD20. However, no defined structure could be observed. Interestingly, micellar solubilized and purified CD20 particles adopt uniformly confined nanodroplets which do not fuse and aggregate. Finally, purified CD20 could bind to rituximab and obinutuzumab as demonstrated by SEC, and Surface Plasmon Resonance (SPR). Specificity of binding was confirmed using CD20 antibody mutants to human B-cell lymphoma cells. The strategy described in this work will help investigate CD20 binding with newly developed antibodies and eventually help to optimize them. This approach may also be applicable to other challenging membrane proteins.

Stabilization of Human Multidrug Resistance Protein 4 (MRP4/ABCC4) Using Novel Solubilization Agents.

Membrane proteins (MPs) are important drug discovery targets for a wide range of diseases. However, elucidating the structure and function of native MP is notoriously challenging as their original structure has to be maintained once removed from the lipid bilayer. Conventionally, detergents have been used to solubilize MP with varying degrees of success concerning MP stability. To try to address this, new, more stabilizing agents have been developed, such as calixarene-based detergents and styrene-maleic acid (SMA) copolymer. Calixarene-based detergents exhibit enhanced solubilizing and stabilizing properties compared with conventional detergents, whereas SMA is able to extract MPs with their surrounding lipids, forming a nanodisc structure. Here we report a comparative study using classical detergents, calixarene-based detergents, and SMA to assess the solubilization and stabilization of the human ABC transporter MRP4 (multidrug resistance protein 4/ABCC4). We show that both SMA and calixarene-based detergents have a higher solubility efficiency (at least 80%) than conventional detergents, and show striking overstabilization features of MRP4 (up to 70 °C) with at least 30 °C stability improvement in comparison with the best conventional detergents. These solubilizing agents were successfully used to purify aggregate-free, homogenous and stable MRP4, with sevenfold higher yield for C4C7 calixarene detergent in comparison with SMA. This work paves the way to MRP4 structural and functional investigations and illustrates once more the high value of using calixarene-based detergent or SMA as versatile and efficient tools to study MP, and eventually enable drug discovery of challenging and highly druggable targets.

Functional Expression of Multidrug Resistance Protein 4 MRP4/ABCC4.

To study the function and structure of membrane proteins, high quantities of pure and stable protein are needed. One of the first hurdles in accomplishing this is expression of the membrane protein at high levels and in a functional state. Membrane proteins are naturally expressed at low levels, so finding a suitable host for overexpression is imperative. Multidrug resistance protein 4 (MRP4) or ATP-binding cassette subfamily C member 4 (ABCC4) is a multi-transmembrane protein that is able to transport a range of organic anionic compounds (both endogenous and xenobiotic) out of the cell. This versatile transporter has been linked with extracellular signaling pathways and cellular protection, along with conferring drug resistance in cancers. Here we report the use of MRP4 as a case study to be expressed in three different expression systems: mammalian, insect, and yeast cells, to gain the highest yield possible. Interestingly, using the baculovirus expression system with Sf9 insect cells produced the highest protein yields. Vesicular transport assays were used to confirm that MRP4 expressed in Sf9 was functional using a fluorescent cAMP analogue (fluo-cAMP) instead of the traditional radiolabeled substrates. MRP4 transported fluo-cAMP in an ATP-dependent manner. The specificity of functional expression of MRP4 was validated by the use of nonhydrolyzable ATP analogues and MRP4 inhibitor MK571. Functionally expressed MRP4 in Sf9 cells can now be used in downstream processes such as solubilization and purification in order to better understand its function and structure.

Hybrid Double-Chain Maltose-Based Detergents: Synthesis and Colloidal and Biochemical Evaluation.

Four hybrid double-chain surfactants with a maltose polar head were synthesized. The apolar domain consists of a hydrogenated chain, and a partially fluorinated chain made of a propyl hydrogenated spacer terminated by a perfluorinated core of various lengths. Their water solubility was found to be lower than 1 g/L irrespective of the length of both chains. The self-assembling properties of pure hybrids in water were studied by dynamic light scattering and transmission electron microscopy, which revealed the formation of two populations of aggregates with diameters of 8-50 nm and 80-300 nm. When mixed with the classical detergent n-dodecylmaltoside (DDM), the four hybrids were well soluble and formed small mixed micelles. DDM/hybrid mixtures were further evaluated for the extraction of the full-length, wild-type human GPCR adenosine receptor (A2AR), and the bacterial transporter AcrB. The solubilization of A2AR showed extraction efficiencies ranging from 40 to 70%, while that of AcrB reached 60-90%. Finally, three of the hybrids exhibited significant thermostabilization when present as additives. The derivative with a C12-hydrogenated chain and a C4F9-fluorinated chain emerged as the most potent additive exhibiting both good extraction yields of A2AR and AcrB and thermostabilization of A2AR by ∼7 °C.

Hydrogenated Diglucose Detergents for Membrane-Protein Extraction and Stabilization.

We report herein the design and synthesis of a novel series of alkyl glycoside detergents consisting of a nonionic polar headgroup that comprises two glucose moieties in a branched arrangement (DG), onto which octane-, decane-, and dodecanethiols were grafted leading to ODG, DDG, and DDDG detergents, respectively. Micellization in aqueous solution was studied by isothermal titration calorimetry, 1H NMR spectroscopy, and surface tensiometry. Critical micellar concentration values were found to decrease by a factor of ∼10 for each pair of methylene groups added to the alkyl chain, ranging from ∼0.05 to 9 mM for DDDG and ODG, respectively. Dynamic light scattering and analytical ultracentrifugation sedimentation velocity experiments were used to investigate the size and composition of the micellar aggregates, showing that the aggregation number significantly increased from ∼40 for ODG to ∼80 for DDDG. All new compounds were able to solubilize membrane proteins (MPs) from bacterial membranes, insect cells, as well as the Madin-Darby canine kidney cells. In particular, native human adenosine receptor (A2AR) and bacterial transporter (BmrA) were solubilized efficiently. Striking thermostability improvements of +13 and +8 °C were observed when ODG and DDG were, respectively, applied to wild-type and full-length A2AR. Taken together, this novel detergent series shows promising detergent potency for solubilization and stabilization of membrane proteins (MPs) and thus makes a valuable addition to the chemical toolbox available for extracting and handling these important but challenging MP targets.

Externalized Keratin 8: A Target at the Interface of Microenvironment and Intracellular Signaling in Colorectal Cancer Cells.

Accumulating evidence supports the remarkable presence at the membrane surface of cancer cells of proteins, which are normally expressed in the intracellular compartment. Although these proteins, referred to as externalized proteins, represent a highly promising source of accessible and druggable targets for cancer therapy, the mechanisms via which they impact cancer biology remain largely unexplored. The aim of this study was to expose an externalized form of cytokeratin 8 (eK8) as a key player of colorectal tumorigenesis and characterize its mode of action. To achieve this, we generated a unique antagonist monoclonal antibody (D-A10 MAb) targeting an eight-amino-acid-long domain of eK8, which enabled us to ascertain the pro-tumoral activity of eK8 in both KRAS-mutant and wild-type colorectal cancers (CRC). We showed that this pro-tumoral activity involves a bidirectional eK8-dependent control of caspase-mediated apoptosis in vivo and of the plasminogen-induced invasion process in cellulo. Furthermore, we demonstrated that eK8 is anchored at the plasma membrane supporting this dual function. We, therefore, identified eK8 as an innovative therapeutic target in CRC and provided a unique MAb targeting eK8 that displays anti-neoplastic activities that could be useful to treat CRC, including those harboring KRAS mutations.

Fluorinated diglucose detergents for membrane-protein extraction.

Fluorinated surfactants have scarcely been explored for the direct extraction of proteins from membranes because fluorination is believed to abrogate detergency. However, we have recently shown that a commercially available fluorinated surfactant readily solubilizes lipid membranes, thereby suggesting that fluorination per se does not interfere with detergent activity. In this work, we developed new fluorinated surfactants that exhibit detergency in terms of both lipid-vesicle solubilization and membrane-protein extraction. The compounds made and tested contain two glucose moieties as polar headgroup, a hydrogenated thioether linker, and a perfluorinated alkyl tail with either 4, 6, or 8 carbon atoms. The physicochemical properties of the micelles formed by the three fluorinated surfactants were evaluated by NMR spectroscopy, surface tensiometry, isothermal titration calorimetry, dynamic light scattering, small-angle X-ray scattering, and analytical ultracentrifugation. At 25 °C, micellization was mainly entropy-driven, and the CMC values were found to decrease with chain length of the fluorinated tail, whereas the aggregation number increased with chain length. Remarkably, all three surfactants were found to solubilize lipid vesicles and extract a broad range of proteins from Escherichia coli membranes. These findings demonstrate, for the first time, that nonionic fluorinated surfactants could be further exploited for the direct extraction and solubilization of membrane proteins.

Enabling STD-NMR fragment screening using stabilized native GPCR: A case study of adenosine receptor.

Structural studies of integral membrane proteins have been limited by the intrinsic conformational flexibility and the need to stabilize the proteins in solution. Stabilization by mutagenesis was very successful for structural biology of G protein-coupled receptors (GPCRs). However, it requires heavy protein engineering and may introduce structural deviations. Here we describe the use of specific calixarenes-based detergents for native GPCR stabilization. Wild type, full length human adenosine A2A receptor was used to exemplify the approach. We could stabilize native, glycosylated, non-aggregated and homogenous A2AR that maintained its ligand binding capacity. The benefit of the preparation for fragment screening, using the Saturation-Transfer Difference nuclear magnetic resonance (STD-NMR) experiment is reported. The binding of the agonist adenosine and the antagonist caffeine were observed and competition experiments with CGS-21680 and ZM241385 were performed, demonstrating the feasibility of the STD-based fragment screening on the native A2A receptor. Interestingly, adenosine was shown to bind a second binding site in the presence of the agonist CGS-21680 which corroborates published results obtained with molecular dynamics simulation. Fragment-like compounds identified using STD-NMR showed antagonistic effects on A2AR in the cAMP cellular assay. Taken together, our study shows that stabilization of native GPCRs represents an attractive approach for STD-based fragment screening and drug design.

The yin and yang of solubilization and stabilization for wild-type and full-length membrane protein.

Membrane proteins (MP) are stable in their native lipid environment. To enable structural and functional investigations, MP need to be extracted from the membrane. This is a critical step that represents the main obstacle for MP biochemistry and structural biology. General guidelines and rules for membrane protein solubilization remain difficult to establish. This review aims to provide the reader with a comprehensive overview of the general concepts of MP solubilization and stabilization as well as recent advances in detergents innovation. Understanding how solubilization and stabilization are intimately linked is key to facilitate MP isolation toward fundamental structural and functional research as well as drug discovery applications. How to manage the tour de force of destabilizing the lipid bilayer and stabilizing MP at the same time is the holy grail of successful isolation and investigation of such a delicate and fascinating class of proteins.

Glycosyl-Substituted Dicarboxylates as Detergents for the Extraction, Overstabilization, and Crystallization of Membrane Proteins.

To tackle the problems associated with membrane protein (MP) instability in detergent solutions, we designed a series of glycosyl-substituted dicarboxylate detergents (DCODs) in which we optimized the polar head to clamp the membrane domain by including, on one side, two carboxyl groups that form salt bridges with basic residues abundant at the membrane-cytoplasm interface of MPs and, on the other side, a sugar to form hydrogen bonds. Upon extraction, the DCODs 8 b, 8 c, and 9 b preserved the ATPase function of BmrA, an ATP-binding cassette pump, much more efficiently than reference or recently designed detergents. The DCODs 8 a, 8 b, 8 f, 9 a, and 9 b induced thermal shifts of 20 to 29 °C for BmrA and of 13 to 21 °C for the native version of the G-protein-coupled adenosine receptor A2A R. Compounds 8 f and 8 g improved the diffraction resolution of BmrA crystals from 6 to 4 Å. DCODs are therefore considered to be promising and powerful tools for the structural biology of MPs.

Molecular architecture of potassium chloride co-transporter KCC2.

KCC2 is a neuron specific K+-Cl- co-transporter that controls neuronal chloride homeostasis, and is critically involved in many neurological diseases including brain trauma, epilepsies, autism and schizophrenia. Despite significant accumulating data on the biology and electrophysiological properties of KCC2, structure-function relationships remain poorly understood. Here we used calixarene detergent to solubilize and purify wild-type non-aggregated and homogenous KCC2. Specific binding of inhibitor compound VU0463271 was demonstrated using surface plasmon resonance (SPR). Mass spectrometry revealed glycosylations and phosphorylations as expected from functional KCC2. We show by electron microscopy (EM) that KCC2 exists as monomers and dimers in solution. Monomers are organized into "head" and "core" domains connected by a flexible "linker". Dimers are asymmetrical and display a bent "S-shape" architecture made of four distinct domains and a flexible dimerization interface. Chemical crosslinking in reducing conditions shows that disulfide bridges are involved in KCC2 dimerization. Moreover, we show that adding a tag to the C-terminus is detrimental to KCC2 function. We postulate that the conserved KCC2 C-ter may be at the interface of dimerization. Taken together, our findings highlight the flexible multi-domain structure of KCC2 with variable anchoring points at the dimerization interface and an important C-ter extremity providing the first in-depth functional architecture of KCC2.

A Novel View on the Role of Intracellular Tails in Surface Delivery of the Potassium-Chloride Cotransporter KCC2.

A plethora of neurological disorders are associated with alterations in the expression and localization of potassium-chloride cotransporter type 2 (KCC2), making KCC2 a critical player in neuronal function and an attractive target for therapeutic treatment. The activity of KCC2 is determined primarily by the rates of its surface insertion and internalization. Currently the domains of KCC2 dictating its trafficking and endocytosis are unknown. Here, using live-cell immunolabeling and biotinylation of KCC2 proteins expressed in murine neuroblastoma N2a cells, human embryonic kidney 293 cells, or primary cultures of rat hippocampal neurons, we identified a novel role for the intracellular N and C termini in differentially regulating KCC2 surface expression. We report that the N terminus is required for KCC2 insertion into the plasma membrane, whereas the C terminus is critical for the membrane stability of KCC2. Our results provide novel insights into the structure-function role of specific KCC2 domains and open perspectives in exploring structural organization of this protein.

Quantification of Detergents Complexed with Membrane Proteins.

Most membrane proteins studies require the use of detergents, but because of the lack of a general, accurate and rapid method to quantify them, many uncertainties remain that hamper proper functional and structural data analyses. To solve this problem, we propose a method based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) that allows quantification of pure or mixed detergents in complex with membrane proteins. We validated the method with a wide variety of detergents and membrane proteins. We automated the process, thereby allowing routine quantification for a broad spectrum of usage. As a first illustration, we show how to obtain information of the amount of detergent in complex with a membrane protein, essential for liposome or nanodiscs reconstitutions. Thanks to the method, we also show how to reliably and easily estimate the detergent corona diameter and select the smallest size, critical for favoring protein-protein contacts and triggering/promoting membrane protein crystallization, and to visualize the detergent belt for Cryo-EM studies.

Novel systematic detergent screening method for membrane proteins solubilization.

Membrane proteins play crucial role in many cellular processes including cell adhesion, cell-cell communication, signal transduction and transport. To better understand the molecular basis of such central biological machines and in order to specifically study their biological and medical role, it is necessary to extract them from their membrane environment. To do so, it is challenging to find the best solubilization condition. Here we describe, a systematic screening method called BMSS (Biotinylated Membranes Solubilization & Separation) that allow screening 96 conditions at once. Streptavidine magnetic beads are used to separate solubilized proteins from remaining biotinylated membranes after solubilization. Relative quantification of dot blots help to select the best conditions to be confirmed by classical ultra-centrifugation and western blot. Classical detergents with different physical-chemical characteristics, novel calixarene based detergents and combination of both, were used for solubilization trials to obtain broad spectrum of conditions. Here, we show the application of BMSS to discover solubilization conditions of a GPCR target (MP-A) and a transporter (MP-B). The selected conditions allowed the solubilization and purification of non-aggregated and homogenous native membrane proteins A and B. Taken together, BMSS represent a rapid, reproducible and high throughput assessment of solubilization toward biochemical/functional characterization, biophysical screening and structural investigations of membrane proteins of high biological and medical relevance.