X-ray structure and enzymatic study of a bacterial NADPH oxidase highlight the activation mechanism of eukaryotic NOX.

NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from Streptococcus pneumoniae, can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2's requirement for activation.

The transport activity of the multidrug ABC transporter BmrA does not require a wide separation of the nucleotide-binding domains.

ATP-binding cassette (ABC) transporters are ubiquitous membrane proteins responsible for the translocation of a wide diversity of substrates across biological membranes. Some of them confer multidrug or antimicrobial resistance to cancer cells and pathogenic microorganisms, respectively. Despite a wealth of structural data gained in the last two decades, the molecular mechanism of these multidrug efflux pumps remains elusive, including the extent of separation between the two nucleotide-binding domains (NBDs) during the transport cycle. Based on recent outward-facing structures of BmrA, a homodimeric multidrug ABC transporter from Bacillus subtilis, we introduced a cysteine mutation near the C-terminal end of the NBDs to analyze the impact of disulfide-bond formation on BmrA function. Interestingly, the presence of the disulfide bond between the NBDs did not prevent the ATPase, nor did it affect the transport of Hoechst 33342 and doxorubicin. Yet, the 7-amino-actinomycin D was less efficiently transported, suggesting that a further opening of the transporter might improve its ability to translocate this larger compound. We solved by cryo-EM the apo structures of the cross-linked mutant and the WT protein. Both structures are highly similar, showing an intermediate opening between their NBDs while their C-terminal extremities remain in close proximity. Distance measurements obtained by electron paramagnetic resonance spectroscopy support the intermediate opening found in these 3D structures. Overall, our data suggest that the NBDs of BmrA function with a tweezers-like mechanism distinct from the related lipid A exporter MsbA.

Purification and characterization of Cdr1, the drug-efflux pump conferring azole resistance in Candida species.

Candida albicans and C. glabrata express exporters of the ATP-binding cassette (ABC) superfamily and address them to their plasma membrane to expel azole antifungals, which cancels out their action and allows the yeast to become multidrug resistant (MDR). In a way to understand this mechanism of defense, we describe the purification and characterization of Cdr1, the membrane ABC exporter mainly responsible for such phenotype in both species. Cdr1 proteins were functionally expressed in the baker yeast, tagged at their C-terminal end with either a His-tag for the glabrata version, cgCdr1-His, or a green fluorescent protein (GFP) preceded by a proteolytic cleavage site for the albicans version, caCdr1-P-GFP. A membrane Cdr1-enriched fraction was then prepared to assay several detergents and stabilizers, probing their level of extraction and the ATPase activity of the proteins as a functional marker. Immobilized metal-affinity and size-exclusion chromatographies (IMAC, SEC) were then carried out to isolate homogenous samples. Overall, our data show that although topologically and phylogenetically close, both proteins display quite distinct behaviors during the extraction and purification steps, and qualify cgCdr1 as a good candidate to characterize this type of proteins for developing future inhibitors of their azole antifungal efflux activity.

Structure, function, and inhibition of catalytically asymmetric ABC transporters: Lessons from the PDR subfamily.

ATP-binding cassette (ABC) superfamily comprises a large group of ubiquitous transmembrane proteins that play a crucial role in transporting a diverse spectrum of substrates across cellular membranes. They participate in a wide array of physiological and pathological processes including nutrient uptake, antigen presentation, toxin elimination, and drug resistance in cancer and microbial cells. ABC transporters couple ATP binding and hydrolysis to undergo conformational changes allowing substrate translocation. Within this superfamily, a set of ABC transporters has lost the capacity to hydrolyze ATP at one of their nucleotide-binding sites (NBS), called the non-catalytic NBS, whose importance became evident with extensive biochemistry carried out on yeast pleiotropic drug resistance (PDR) transporters. Recent single-particle cryogenic electron microscopy (cryo-EM) advances have further catapulted our understanding of the architecture of these pumps. We provide here a comprehensive overview of the structural and functional aspects of catalytically asymmetric ABC pumps with an emphasis on the PDR subfamily. Furthermore, given the increasing evidence of efflux-mediated antifungal resistance in clinical settings, we also discuss potential grounds to explore PDR transporters as therapeutic targets.

Cryo-EM and Molecular Dynamics Simulations Reveal Hidden Conformational Dynamics Controlling Ammonia Transport in Human Asparagine Synthetase.

How dynamical motions in enzymes might be linked to catalytic function is of significant general interest, although almost all relevant experimental data, to date, has been obtained for enzymes with a single active site. Recent advances in X-ray crystallography and cryogenic electron microscopy offer the promise of elucidating dynamical motions for proteins that are not amenable to study using solution-phase NMR methods. Here we use 3D variability analysis (3DVA) of an EM structure for human asparagine synthetase (ASNS) in combination with atomistic molecular dynamics (MD) simulations to detail how dynamic motions of a single side chain mediates interconversion of the open and closed forms of a catalytically relevant intramolecular tunnel, thereby regulating catalytic function. Our 3DVA results are consistent with those obtained independently from MD simulations, which further suggest that formation of a key reaction intermediate acts to stabilize the open form of the tunnel in ASNS to permit ammonia translocation and asparagine formation. This conformational selection mechanism for regulating ammonia transfer in human ASNS contrasts sharply with those employed in other glutamine-dependent amidotransferases that possess a homologous glutaminase domain. Our work illustrates the power of cryo-EM to identify localized conformational changes and hence dissect the conformational landscape of large proteins. When combined with MD simulations, 3DVA is a powerful approach to understanding how conformational dynamics regulate function in metabolic enzymes with multiple active sites.

Conformational space exploration of cryo-EM structures by variability refinement.

Cryo-EM observation of biological samples enables visualization of sample heterogeneity, in the form of discrete states that are separable, or continuous heterogeneity as a result of local protein motion before flash freezing. Variability analysis of this continuous heterogeneity describes the variance between a particle stack and a volume, and results in a map series describing the various steps undertaken by the sample in the particle stack. While this observation is absolutely stunning, it is very hard to pinpoint structural details to elements of the maps. In order to bridge the gap between observation and explanation, we designed a tool that refines an ensemble of structures into all the maps from variability analysis. Using this bundle of structures, it is easy to spot variable parts of the structure, as well as the parts that are not moving. Comparison with molecular dynamics simulations highlights the fact that the movements follow the same directions, albeit with different amplitudes. Ligand can also be investigated using this method. Variability refinement is available in the Phenix software suite, accessible under the program name phenix.varref.

Inhibitors of ABCG2-mediated multidrug resistance: Lead generation through computer-aided drug design.

Human breast cancer resistance protein (BCRP), known also as ABCG2, plays a major role in multiple drug resistance (MDR) in tumor cells. Through this ABC transporter, cancer cells acquire the ability of resistance to structurally and functionally unrelated anticancer drugs. Nowadays, the design of ABCG2 inhibitors as potential agents to enhance the chemotherapy efficacy is an interesting strategy. In this context, we have used computer-aided drug design (CADD) based on available data of a large series of potent inhibitors from our groups as an approach in guiding the design of effective ABCG2 inhibitors. We report therein the results on the use of the FLAPpharm method to elucidate the pharmacophoric features of one of the ABCG2 binding sites involved in the regulation of the basal ATPase activity of the transporter. The predictivity of the model was evaluated by testing three predicted compounds which were found to induce high inhibitory activity of BCRP, in the nanomolar range for the best of them.

The non-Newtonian behavior of detergents during concentration is increased by macromolecules, in trans, and results in their over-concentration.

Concentration of pure membrane proteins in detergent solution results in detergent concentration, albeit in unknown amounts. This phenomenon is observed in every lab working on membrane proteins, but has seldom been investigated. In this study, we explored the behavior of detergents mixed with membrane proteins during the step of sample concentration using centrifugal devices. We show that detergent over-concentrate with the presence of polymers, typically membrane or soluble proteins but also polysaccharides. The over-concentration of detergents depends on centrifugal force applied to the device. With the use of a specific dye, we observed the formation of a mesh on the concentrator device. Importantly, reducing the centrifugal speed allows to reduce the concentration of detergents when mixed to macromolecules, as tested with 3 different membrane proteins. All together, these results highlight the non-Newtonian behavior of detergents and provides a solid framework to investigators to improve drastically biochemical and structural studies of membrane proteins.

Specific Xray diffraction patterns of membrane proteins caused by secondary structure collinearity.

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 single 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 Å 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. These results bring to light a specific diffraction of membrane protein crystals, which calls for a specific handling by crystallographic software. It also brings an explanation for investigators struggling with their anisotropic data.

Overexpression of the ABC Transporter BmrA Within Intracellular Caveolae in Escherichia coli.

We describe here the overproduction and oriented membrane insertion of membrane protein inside intracellular vesicles named heterologous caveolae within E. coli. The method is described with BmrA, a multidrug efflux pump from Bacillus subtilis. BmrA is produced in these vesicles, thanks to the coexpression with the canine caveolin-1ß, one of the two isoforms of caveolin-1. Enriched by sucrose gradient, the caveolae-containing fraction allows to probe the ATPase and Hoechst 33342 transport activities, the latter displaying a higher specific activity than the same without caveolin-1ß.

Substrate-bound and substrate-free outward-facing structures of a multidrug ABC exporter.

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 three ATP-Mg2+-bound outward-facing conformations of the Bacillus subtilis (homodimeric) BmrA by x-ray crystallography and single-particle cryo-electron microscopy (EM) in detergent solution, one of them with rhodamine 6G (R6G), a substrate exported by BmrA when overexpressed in B. subtilis. 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. They induce a rearrangement of TM1-2, highlighting a local flexibility that we confirmed by hydrogen/deuterium exchange and molecular dynamics simulations. In the absence of R6G, simulations show a fast postrelease occlusion of the cavity driven by hydrophobicity, while when present, R6G can move within the cavity, maintaining it open.

The Det.Belt Server: A Tool to Visualize and Estimate Amphipathic Solvent Belts around Membrane Proteins.

Detergents wrap around membrane proteins to form a belt covering the hydrophobic part of the protein serving for membrane insertion and interaction with lipids. The number of detergent monomers forming this belt is usually unknown to investigators, unless dedicated detergent quantification is undertaken, which for many projects is difficult to setup. Yet, having an approximate knowledge of the amount of detergent forming the belt is extremely useful, to better grasp the protein of interest in interaction with its direct environment rather than picturing the membrane protein "naked". We created the Det.Belt server to dress up membrane proteins and represent in 3D the bulk made by detergent molecules wrapping in a belt. Many detergents are included in a database, allowing investigators to screen in silico the effect of different detergents around their membrane protein. The input number of detergents is changeable with fast recomputation of the belt for interactive usage. Metrics representing the belt are readily available together with scripts to render quality 3D images for publication. The Det.Belt server is a tool for biochemists to better grasp their sample.

CryoEM reconstructions of membrane proteins solved in several amphipathic solvents, nanodisc, amphipol and detergents, yield amphipathic belts of similar sizes corresponding 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 amphipathic 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.

Molecular analysis of the massive GSH transport mechanism mediated by the human Multidrug Resistant Protein 1/ABCC1.

The transporter Multidrug Resistance Protein 1 (MRP1, ABCC1) is implicated in multidrug resistant (MDR) phenotype of cancer cells. Glutathione (GSH) plays a key role in MRP1 transport activities. In addition, a ligand-stimulated GSH transport which triggers the death of cells overexpressing MRP1, by collateral sensitivity (CS), has been described. This CS could be a way to overcome the poor prognosis for patients suffering from a chemoresistant cancer. The molecular mechanism of such massive GSH transport and its connection to the other transport activities of MRP1 are unknown. In this context, we generated MRP1/MRP2 chimeras covering different regions, MRP2 being a close homolog that does not trigger CS. The one encompassing helices 16 and 17 led to the loss of CS and MDR phenotype without altering basal GSH transport. Within this region, the sole restoration of the original G1228 (D1236 in MRP2) close to the extracellular loop between the two helices fully rescued the CS (massive GSH efflux and cell death) but not the MDR phenotype. The flexibility of that loop and the binding of a CS agent like verapamil could favor a particular conformation for the massive transport of GSH, not related to other transport activities of MRP1.

Au courant computation of the PDB to audit diffraction anisotropy of soluble and membrane proteins.

This data article makes available the informed computation of the whole Protein Data Bank (PDB) to investigate diffraction anisotropy on a large scale and to perform statistics. This data has been investigated in detail in "X-ray diffraction reveals the intrinsic difference in the physical properties of membrane and soluble proteins" [1]. Diffraction anisotropy is traditionally associated with absence of contacts in-between macromolecules within the crystals in a given direction of space. There are however many case that do not follow this empirical rule. To investigate and sort out this discrepancy, we computed diffraction anisotropy for every entry of the PDB, and put them in context of relevant metrics to compare X-ray diffraction in reciprocal space to the crystal packing in real space. These metrics were either extracted from PDB files when available (resolution, space groups, cell parameters, solvent content), or calculated using standard procedures (anisotropy, crystal contacts, presence of ligands). More specifically, we separated entries to compare soluble vs membrane proteins, and further separated the later in subcategories according to their insertion in the membrane, function, or type of crystallization (Type I vs Type II crystal packing). This informed database is being made available to investigators in the raw and curated formats that can be re-used for further downstream studies. This dataset is useful to test ideas and to ascertain hypothesis based on statistical analysis.

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.

X-ray diffraction reveals the intrinsic difference in the physical properties of membrane and soluble proteins.

Membrane proteins are distinguished from soluble proteins by their insertion into biological membranes. This insertion is achieved via a noticeable arrangement of hydrophobic amino acids that are exposed at the surface of the protein, and renders the interaction with the aliphatic tails of lipids more energetically favorable. This important difference between these two categories of proteins is the source of the need for a specific handling of membrane proteins, which transpired in the creation of new tools for their recombinant expression, purification and even crystallization. Following this line, we show here that crystals of membrane proteins display systematically higher diffraction anisotropy than those of soluble proteins. This phenomenon dramatically hampers structure solution and refinement, and has a strong impact on the quality of electron-density maps. A farther search for origins of this phenomenon showed that the type of crystallization, and thus the crystal packing, has no impact on anisotropy, nor does the nature or function of the membrane protein. Membrane proteins fully embedded within the membrane display equal anisotropy compared to the ones with extra membranous domains or fusions with soluble proteins. Overall, these results overturn common beliefs and call for a specific handling of their diffraction data.

Gradient reconstitution of membrane proteins for solid-state NMR studies.

We here adapted the GRecon method used in electron microscopy studies for membrane protein reconstitution to the needs of solid-state NMR sample preparation. We followed in detail the reconstitution of the ABC transporter BmrA by dialysis as a reference, and established optimal reconstitution conditions using the combined sucrose/cyclodextrin/lipid gradient characterizing GRecon. We established conditions under which quantitative reconstitution of active protein at low lipid-to-protein ratios can be obtained, and also how to upscale these conditions in order to produce adequate amounts for NMR. NMR spectra recorded on a sample produced by GRecon showed a highly similar fingerprint as those recorded previously on samples reconstituted by dialysis. GRecon sample preparation presents a gain in time of nearly an order of magnitude for reconstitution, and shall represent a valuable alternative in solid-state NMR membrane protein sample preparation.

Multidrug ABC transporter Cdr1 of Candida albicans harbors specific and overlapping binding sites for human steroid hormones transport.

The present study examines the kinetics of steroids efflux mediated by the Candida drug resistance protein 1 (Cdr1p) and evaluates their interaction with the protein. We exploited our in-house mutant library for targeting the 252 residues forming the twelve transmembrane helices (TMHs) of Cdr1p. The screening revealed 65 and 58 residues critical for ß-estradiol and corticosterone transport, respectively. Notably, up to 83% critical residues for corticosterone face the lipid interface compared to 54% for ß-estradiol. Molecular docking identified a possible peripheral corticosterone-binding site made of 8/14 critical/non-critical residues between TMHs 3, 4 and 6. ß-estradiol transport was severely hampered by alanine replacements of Cdr1p core residues involving TMHs 2, 5 and 8, in a binding site made of 10/14 critical residues mainly shared with rhodamine 6G with which it competes. By contrast, TMH11 was poorly impacted, although being part of the core domain. Finally, we observed the presence of several contiguous stretches of 3-5 critical residues in TMHs 2, 5 and 10 that points to a rotation motion of these helices during the substrate transport cycle. The selective structural arrangement of the steroid-binding pockets in the core region and at the lipid-TMD interface, which was never reported before, together with the possible rotation of some TMHs may be the structural basis of the drug-transport mechanism achieved by these type II ABC transporters.

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.