Tripartite efflux pumps built around ATP-binding cassette (ABC) transporters are membrane protein machineries that perform vectorial export of a large variety of drugs and virulence factors from Gram ...negative bacteria, using ATP-hydrolysis as energy source. Determining the number of ATP molecules consumed per transport cycle is essential to understanding the efficiency of substrate transport. Using a reconstituted pump in a membrane mimic environment, we show that MacAB-TolC from Escherichia coli couples substrate transport to ATP-hydrolysis with high efficiency. Contrary to the predictions of the currently prevailing "molecular bellows" model of MacB-operation, which assigns the power stroke to the ATP-binding by the nucleotide binding domains of the transporter, by utilizing a novel assay, we report clear synchronization of the substrate transfer with ATP-hydrolysis, suggesting that at least some of the power stroke for the substrate efflux is provided by ATP-hydrolysis. Our findings narrow down the window for energy consumption step that results in substrate transition into the TolC-channel, expanding the current understanding of the efflux cycle of the MacB-based tripartite assemblies. Based on that we propose a modified model of the MacB cycle within the context of tripartite complex assembly.
The NADPH oxidase is the sole enzymatic complex that produces, in a controlled way, superoxide anions. In phagocytes, it is constituted by the assembly of four cytosolic (p67phox, p47phox, p40phox ...and Rac) and two membrane (p22phox and Nox2) proteins. In response to pro‐inflammatory mediators, the NADPH oxidase is activated. In cells, arachidonic acid (cis‐AA), released by activated phospholipase A2, also plays a role in activation of the NADPH oxidase complex, but the mechanism of action of cis‐AA is still a matter for debate. In cell‐free systems, cis‐AA is commonly used for activation. We have shown previously that trans‐AA isomers were unable to activate the NADPH oxidase complex. Here, we aim to evaluate the structural changes in p47phox and p67phox induced by AA. The structural impact of both AA isomers on both cytosolic proteins was investigated by the accessibility of the thiol group and by circular dichroism in the far‐UV for global folds. cis‐AA induces secondary structure changes of p47phox and p67phox, while the trans isomer does not, suggesting that the changes observed are of importance for the activation process of these proteins. While five of the nine thiol groups in p67phox and all of them in p47phox have low access to the solvent when proteins are alone in solution, all of them become fully accessible when proteins are together. In conclusion, the secondary structures of p47phox and p67phox are both dependent on the presence of the partner protein in solution and on the presence of the activator molecule cis‐AA.
Structure of resting/activated p47phox and p67phox: when not activated, the interactions between both proteins increase their flexibility but not their secondary structures. Upon arachidonic acid activation, their secondary structures individually are modified but not the global architecture of heterodimer. Protein activation depends not only on arachidonic acid but also on the presence of the cytosolic partner protein.
ABC transporters have long been known to mediate resistance phenotypes in all kingdoms of life, and ATP-driven tripartite efflux pump from Gram-negative bacteria have attracted increasing interest. ...We give a special focus on MacAB TolC, a prototypical member of the recently described Type VII ABC transporter superfamily, from Escherichia coli. We provide original experimental evidence for the in vitro, substrate-induced ATPase activity and show a maximal activity when the tripartite pump is fully assembled in lipid nanodiscs. These results are evaluated and interpreted in the context of the structural and functional data that have accumulated over the years.
•MacAB TolC shows maximal activity when fully assembled and reconstituted in lipids.•MacB is under strict dependance of the hydrophobic environment.•There is an intricate mutual interdependency among MacB, MacA and TolC.•The Periplasmic Core Domain plays a pivotal role in the functioning of the pump.
The NADPH-oxidase complex, which plays beneficial or detrimental role in the inflammatory and degenerative diseases, is a membrane multi-subunit complex tightly regulated in order to produce ...superoxide anions, precursor of oxygen reactive species (ROS), in cells. The flavocytochrome b558 (Cytb558) is the catalytic core of the NADPH oxidase which consists of two membrane proteins gp91phox (highly glycosylated) and p22phox. In this work we took advantage of heterologous yeast cells engineered to express wild-type bovine Cytb558 to analyze the properties of the NADPH oxidase activity during the biosynthesis processing steps of gp91phox and p22phox within endoplasmic reticulum (ER) and plasma membrane (Pmb). Our data showed that, in yeast, the heterodimerization at the endoplasmic reticulum membranes was concomitant with high level glycosylation of gp91phox and the heme acquisition. This study also demonstrated that the phagocyte NADPH oxidase was active at ER membranes and that this activity was surprisingly higher at the ER compared to the Pmb membranes. We have correlated these findings with the presence of sterols in the plasma membranes and their absence in ER membranes. This correlation was confirmed by decreased superoxide anion production rates in proteoliposomes supplemented with ergosterol or cholesterol. Our data support the idea that membrane environment might be determinant for ROS regulation and that sterols could directly interact with the membrane proteins of the NADPH oxidase constraining its capacity to produce superoxide anions.
•p22phox dependence of high-level glycosylation and heme acquisition of Cytb558.•Phagocyte NADPH oxidase is active at endoplasmic reticulum membranes.•Sterol membrane content modulates the NADPH oxidase activity.•Sterols constraint the superoxide anion production rate by Cytb558.•Potential cholesterol-binding sites in p22phox and gp91phox sequences.
Proteoliposome reconstitution is a method of choice for the investigation of membrane proteins as it allows their manipulation in the desired hydrophobic environment and allows one to tackle their ...study from both functional and structural points of view. Methods for their rapid and efficient reconstitution have been known for a long time but the quality and dispersity of the resulting suspensions is often overlooked. Here we describe our routine for the obtention of monodisperse populations of proteoliposomes as well as for the quantitation of protein per liposome.
Free C-terminus of p22phox allows a proper folding but a toxic effect on the yeast growth(right) and an increase oxidase activity capacity (left).
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•Free- or tagged-p22phox termini ...determine production of mature and active heterodimer.•Overexpression of mature and well-folded His-p22phox decreased yeast cell biomass.•Instability of N-terminus free p22phox-His is counterbalanced by co-expression of gp91phox.•Free C-terminus of p22phox contributes to efficient NADPH oxidase.
The flavocytochrome b558 (Cytb558), heterodimer of two membrane proteins gp91phox and p22phox, corresponds to the catalytic part of the phagocyte NADPH-oxidase. This complex plays a crucial role of the innate immune system by generating superoxide anion, the precursor of toxic reactive oxygen species. Cytb558 lacks of precise structural knowledge due to an inefficient production of pure protein. Aiming to optimize its production level in Pichia Pastoris, we explored the impact of the His-tag location on the p22phox-termini. Expressed alone with N-terminus fusion-tag, a production of stable and mature p22phox proteins was observed at the plasma membrane which was associated to a decrease of the yeast biomass (membrane limitation). In contrast, the C-terminus tagged p22phox was not stably expressed and was likely shuttled elsewhere to avoid any troubles of the biomass. The stability of the C-terminus tagged p22phox was restored when the protein was co-expressed with gp91phox. Our data support the idea that Pichia pastoris presents the criteria of tangible cell factory for the production of the recombinant membrane Cytb558 but an investigation in details on the optimal introduction of the tag was a prerequisite to obtain efficient production of mature proteins and active NADPH-oxidase complexes.
The NADPH oxidase NOX2 complex consists of assembled cytosolic and redox membrane proteins. In mammalian cells, natural arachidonic acid (cis-AA), released by activated phospholipase-A2, plays an ...important role in the activation of the NADPH oxidase, but the mechanism of action of cis-AA is still a matter of debate. In cell-free systems, cis-AA is commonly used for activation although its structural effects are still unclear. Undoubtedly cis-AA participates in the synergistic multi-partner assembly that can be hardly studied at the molecular level in vivo due to cellular complexity. The capacity of this anionic amphiphilic fatty acid to activate the oxidase is mainly explained by its ability to disrupt intramolecular bonds, mimicking phosphorylation events in cell signaling and therefore allowing protein-protein interactions. Interestingly the geometric isomerism of the fatty acid and its purity are crucial for optimal superoxide production in cell-free assays. Indeed, optimal NADPH oxidase assembly was hampered by the substitution of the cis form by the trans forms of AA isomers (Souabni et al., BBA-Biomembranes 1818:2314-2324, 2012). Structural analysis of the changes induced by these two compounds, by circular dichroism and by biochemical methods, revealed differences in the interaction between subunits. We describe how the specific geometry of AA plays an important role in the activation of the NOX2 complex.
Membrane protein (MP) complexes play key roles in all living cells. Their structural characterisation is hampered by difficulties in purifying and crystallising them. Recent progress in electron ...microscopy (EM) have revolutionised the field, not only by providing higher-resolution structures for previously characterised MPs but also by yielding first glimpses into the structure of larger and more challenging complexes, such as bacterial secretion systems. However, the resolution of pioneering EM structures may be difficult and their interpretation requires clues regarding the overall organisation of the complexes. In this context, we present BAmSA, a new method for localising transmembrane (TM) regions in MP complexes, using a general procedure that allows tagging them without resorting to neither genetic nor chemical modification. Labels bound to TM regions can be visualised directly on raw negative-stain EM images, on class averages, or on three-dimensional reconstructions, providing a novel strategy to explore the organisation of MP complexes.
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•Transmembrane regions in low and medium-resolution models of membrane proteins are currently identified by indirect means.•Monovalent Streptavidin bound to biotin molecules on biotinylated amphipols/protein complexes was visualised by EM.•Two protocols were used to label successfully the membrane region of both cytochrome bc1 and CsgG with different yields.•mSA particles were visualised by negative stain-EM on raw images, class averages and/or three-dimensional reconstructions.
The NADPH oxidase is the sole enzymatic complex that produces, in a controlled way, superoxide anions. In phagocytes, it is constituted by the assembly of four cytosolic (p67(phox) , p47(phox) , ...p40(phox) and Rac) and two membrane (p22(phox) and Nox2) proteins. In response to pro-inflammatory mediators, the NADPH oxidase is activated. In cells, arachidonic acid (cis-AA), released by activated phospholipase A2, also plays a role in activation of the NADPH oxidase complex, but the mechanism of action of cis-AA is still a matter for debate. In cell-free systems, cis-AA is commonly used for activation. We have shown previously that trans-AA isomers were unable to activate the NADPH oxidase complex. Here, we aim to evaluate the structural changes in p47(phox) and p67(phox) induced by AA. The structural impact of both AA isomers on both cytosolic proteins was investigated by the accessibility of the thiol group and by circular dichroism in the far-UV for global folds. cis-AA induces secondary structure changes of p47(phox) and p67(phox) , while the trans isomer does not, suggesting that the changes observed are of importance for the activation process of these proteins. While five of the nine thiol groups in p67(phox) and all of them in p47(phox) have low access to the solvent when proteins are alone in solution, all of them become fully accessible when proteins are together. In conclusion, the secondary structures of p47(phox) and p67(phox) are both dependent on the presence of the partner protein in solution and on the presence of the activator molecule cis-AA.
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