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.

Zwitterionic fluorinated detergents: From design to membrane protein applications.

We report herein the synthesis of zwitterionic sulfobetaine (SB) and dimethylamine oxide (AO) detergents whose alkyl chain is made of either a perfluorohexyl (F6H3) or a perfluoropentyl (F5H5) group linked to a hydrogenated spacer arm. In aqueous solution, the critical micellar concentrations (CMCs) measured by surface tensiometry (SFT) and isothermal titration calorimetry (ITC) were found in the millimolar range (1.3-2.4 mM). The morphologies of the aggregates were evaluated by dynamic light scattering (DLS), analytical ultracentrifugation (AUC), small-angle X-ray scattering (SAXS), and transmission electron microscopy (TEM), demonstrating that the two perfluoropentyl derivatives formed small micelles less than 10 nm in diameter, whereas the perfluorohexyl derivatives formed larger and more heterogeneous micelles. The two SB detergents were able to solubilize synthetic lipid vesicles in a few hours; by contrast, the perfluoropentyl AO induced much faster solubilization, whereas the perfluorohexyl AO did not show any solubilization. All detergents were tested for their abilities to stabilize three membrane proteins, namely, bacteriorhodopsin (bR), the Bacillus subtilis ABC transporter BmrA, and the Streptococcus pneumoniae enzyme SpNOX. The SB detergents outperformed the AO derivatives as well as their hydrogenated analogs in stabilizing these proteins. Among the four new compounds, F5H5SB combines many desirable properties for membrane-protein study, as it is a powerful yet gentle detergent.

Membrane-Bound Flavocytochrome MsrQ Is a Substrate of the Flavin Reductase Fre in Escherichia coli.

MsrPQ is a new type of methionine sulfoxide reductase (Msr) system found in bacteria. It is specifically involved in the repair of periplasmic methionine residues that are oxidized by hypochlorous acid. MsrP is a periplasmic molybdoenzyme that carries out the Msr activity, whereas MsrQ, an integral membrane-bound hemoprotein, acts as the physiological partner of MsrP to provide electrons for catalysis. Although MsrQ (YedZ) was associated since long with a protein superfamily named FRD (ferric reductase domain), including the eukaryotic NADPH oxidases and STEAP proteins, its biochemical properties are still sparsely documented. Here, we have investigated the cofactor content of the E. coli MsrQ and its mechanism of reduction by the flavin reductase Fre. We showed by electron paramagnetic resonance (EPR) spectroscopy that MsrQ contains a single highly anisotropic low-spin (HALS) b-type heme located on the periplasmic side of the membrane. We further demonstrated that MsrQ holds a flavin mononucleotide (FMN) cofactor that occupies the site where a second heme binds in other members of the FDR superfamily on the cytosolic side of the membrane. EPR spectroscopy indicates that the FMN cofactor can accommodate a radical semiquinone species. The cytosolic flavin reductase Fre was previously shown to reduce the MsrQ heme. Here, we demonstrated that Fre uses the FMN MsrQ cofactor as a substrate to catalyze the electron transfer from cytosolic NADH to the heme. Formation of a specific complex between MsrQ and Fre could favor this unprecedented mechanism, which most likely involves transfer of the reduced FMN cofactor from the Fre active site to MsrQ.

NADPH Oxidases (NOX): An Overview from Discovery, Molecular Mechanisms to Physiology and Pathology.

The reactive oxygen species (ROS)-producing enzyme NADPH oxidase (NOX) was first identified in the membrane of phagocytic cells. For many years, its only known role was in immune defense, where its ROS production leads to the destruction of pathogens by the immune cells. NOX from phagocytes catalyzes, via one-electron trans-membrane transfer to molecular oxygen, the production of the superoxide anion. Over the years, six human homologs of the catalytic subunit of the phagocyte NADPH oxidase were found: NOX1, NOX3, NOX4, NOX5, DUOX1, and DUOX2. Together with the NOX2/gp91phox component present in the phagocyte NADPH oxidase assembly itself, the homologs are now referred to as the NOX family of NADPH oxidases. NOX are complex multidomain proteins with varying requirements for assembly with combinations of other proteins for activity. The recent structural insights acquired on both prokaryotic and eukaryotic NOX open new perspectives for the understanding of the molecular mechanisms inherent to NOX regulation and ROS production (superoxide or hydrogen peroxide). This new structural information will certainly inform new investigations of human disease. As specialized ROS producers, NOX enzymes participate in numerous crucial physiological processes, including host defense, the post-translational processing of proteins, cellular signaling, regulation of gene expression, and cell differentiation. These diversities of physiological context will be discussed in this review. We also discuss NOX misregulation, which can contribute to a wide range of severe pathologies, such as atherosclerosis, hypertension, diabetic nephropathy, lung fibrosis, cancer, or neurodegenerative diseases, giving this family of membrane proteins a strong therapeutic interest.

Interdomain Flexibility within NADPH Oxidase Suggested by SANS Using LMNG Stealth Carrier.

Small angle neutron scattering (SANS) provides a method to obtain important low-resolution information for integral membrane proteins (IMPs), challenging targets for structural determination. Specific deuteration furnishes a "stealth" carrier for the solubilized IMP. We used SANS to determine a structural envelope of SpNOX, the Streptococcus pneumoniae NADPH oxidase (NOX), a prokaryotic model system for exploring structure and function of eukaryotic NOXes. SpNOX was solubilized in the detergent lauryl maltose neopentyl glycol, which provides optimal SpNOX stability and activity. Using deuterated solvent and protein, the lauryl maltose neopentyl glycol was experimentally undetected in SANS. This affords a cost-effective SANS approach for obtaining novel structural information on IMPs. Combining SANS data with molecular modeling provided a first, to our knowledge, structural characterization of an entire NOX enzyme. It revealed a distinctly less compact structure than that predicted from the docking of homologous crystal structures of the separate transmembrane and dehydrogenase domains, consistent with a flexible linker connecting the two domains.

Assemblies of lauryl maltose neopentyl glycol (LMNG) and LMNG-solubilized membrane proteins.

Laurylmaltose neopentylglycol (LMNG) bears two linked hydrophobic chains of equal length and two hydrophilic maltoside groups. It arouses a strong interest in the field of membrane protein biochemistry, since it was shown to efficiently solubilize and stabilize membrane proteins often better than the commonly used dodecylmaltopyranoside (DDM), and to allow structure determination of some challenging membrane proteins. However, LMNG was described to form large micelles, which could be unfavorable for structural purposes. We thus investigated its auto-assemblies and the association state of different membrane proteins solubilized in LMNG by analytical ultracentrifugation, size exclusion chromatography coupled to light scattering, centrifugation on sucrose gradient and/or small angle scattering. At high concentrations (in the mM range), LMNG forms long rods, and it stabilized the membrane proteins investigated herein, i.e. a bacterial multidrug transporter, BmrA; a prokaryotic analogous of the eukaryotic NADPH oxidases, SpNOX; an E. coli outer membrane transporter, FhuA; and the halobacterial bacteriorhodopsin, bR. BmrA, in the Apo and the vanadate-inhibited forms showed reduced kinetics of limited proteolysis in LMNG compared to DDM. Both SpNOX and BmrA display an increased specific activity in LMNG compared to DDM. The four proteins form LMNG complexes with their usual quaternary structure and with usual amount of bound detergent. No heterogeneous complexes related to the large micelle size of LMNG alone were observed. In conditions where LMNG forms assemblies of large size, FhuA crystals diffracting to 4.0 Šwere obtained by vapor diffusion. LMNG large micelle size thus does not preclude membrane protein homogeneity and crystallization.

The NOX Family of Proteins Is Also Present in Bacteria.

Transmembrane NADPH oxidase (NOX) enzymes have been so far only characterized in eukaryotes. In most of these organisms, they reduce molecular oxygen to superoxide and, depending on the presence of additional domains, are called NOX or dual oxidases (DUOX). Reactive oxygen species (ROS), including superoxide, have been traditionally considered accidental toxic by-products of aerobic metabolism. However, during the last decade it has become evident that both O2•- and H2O2 are key players in complex signaling networks and defense. A well-studied example is the production of O2•- during the bactericidal respiratory burst of phagocytes; this production is catalyzed by NOX2. Here, we devised and applied a novel algorithm to search for additional NOX genes in genomic databases. This procedure allowed us to discover approximately 23% new sequences from bacteria (in relation to the number of NOX-related sequences identified by the authors) that we have added to the existing eukaryotic NOX family and have used to build an expanded phylogenetic tree. We cloned and overexpressed the identified nox gene from Streptococcus pneumoniae and confirmed that it codes for an NADPH oxidase. The membrane of the S. pneumoniae NOX protein (SpNOX) shares many properties with its eukaryotic counterparts, such as affinity for NADPH and flavin adenine dinucleotide, superoxide dismutase and diphenylene iodonium inhibition, cyanide resistance, oxygen consumption, and superoxide production. Traditionally, NOX enzymes in eukaryotes are related to functions linked to multicellularity. Thus, the discovery of a large family of NOX-related enzymes in the bacterial world brings up fascinating questions regarding their role in this new biological context.IMPORTANCE NADPH oxidase (NOX) enzymes have not yet been reported in bacteria. Here, we carried out computational and experimental studies to provide the first characterization of a prokaryotic NOX. Out of 996 prokaryotic proteins showing NOX signatures, we initially selected, cloned, and overexpressed four of them. Subsequently, and based on preliminary testing, we concentrated our efforts on Streptococcus SpNOX, which shares many biochemical characteristics with NOX2, the referent model of NOX enzymes. Our work makes possible, for the first time, the study of pure forms of this important family of enzymes, allowing for biophysical and molecular characterization in an unprecedented way. Similar advances regarding other membrane protein families have led to new structures, further mechanistic studies, and the improvement of inhibitors. In addition, biological functions of these newly described bacterial enzymes will be certainly discovered in the near future.

A Two-component NADPH Oxidase (NOX)-like System in Bacteria Is Involved in the Electron Transfer Chain to the Methionine Sulfoxide Reductase MsrP.

MsrPQ is a newly identified methionine sulfoxide reductase system found in bacteria, which appears to be specifically involved in the repair of periplasmic proteins oxidized by hypochlorous acid. It involves two proteins: a periplasmic one, MsrP, previously named YedY, carrying out the Msr activity, and MsrQ, an integral b-type heme membrane-spanning protein, which acts as the specific electron donor to MsrP. MsrQ, previously named YedZ, was mainly characterized by bioinformatics as a member of the FRD superfamily of heme-containing membrane proteins, which include the NADPH oxidase proteins (NOX/DUOX). Here we report a detailed biochemical characterization of the MsrQ protein from Escherichia coli We optimized conditions for the overexpression and membrane solubilization of an MsrQ-GFP fusion and set up a purification scheme allowing the production of pure MsrQ. Combining UV-visible spectroscopy, heme quantification, and site-directed mutagenesis of histidine residues, we demonstrated that MsrQ is able to bind two b-type hemes through the histidine residues conserved between the MsrQ and NOX protein families. In addition, we identify the E. coli flavin reductase Fre, which is related to the dehydrogenase domain of eukaryotic NOX enzymes, as an efficient cytosolic electron donor to the MsrQ heme moieties. Cross-linking experiments as well as surface Plasmon resonance showed that Fre interacts with MsrQ to form a specific complex. Taken together, these data support the identification of the first prokaryotic two-component protein system related to the eukaryotic NOX family and involved in the reduction of periplasmic oxidized proteins.

Biophysical and physiological characterization of ZraP from Escherichia coli, the periplasmic accessory protein of the atypical ZraSR two-component system.

The ZraSR system belongs to the family of TCSs (two-component signal transduction systems). In Escherichia coli, it was proposed to participate in zinc balance and to protect cytoplasmic zinc overload by sequestering this metal ion into the periplasm. This system controls the expression of the accessory protein ZraP that would be a periplasmic zinc scavenger. ZraPSR is functionally homologous with CpxPAR that integrates signals of envelope perturbation, including misfolded periplasmic proteins. The auxiliary periplasmic regulator CpxP inhibits the Cpx pathway by interacting with CpxA. Upon envelope stress sensing, the inhibitory function of CpxP is relieved, resulting in CpxR activation. Similarly to CpxPAR, ZraPSR probably plays a role in envelope stress response as a zinc-dependent chaperone activity was demonstrated for ZraP in Salmonella. We have purified ZraP from E. coli and shown that it is an octamer containing four interfacial metal-binding sites contributing to dimer stability. These sites are located close to the N-terminus, whereas the C-terminus is involved in polymerization of the protein to form a tetramer of dimers. In vitro, ZraP binds copper with a higher affinity than zinc and displays chaperone properties partially dependent on zinc binding. In vivo, zinc-bound ZraP is a repressor of the expression of the zraPSR operon. However, we have demonstrated that none of the Zra proteins are involved in zinc or copper resistance. We propose an integrated mechanism in which zinc is a marker of envelope stress perturbation and ZraPSR TCS is a sentinel sensing and responding to zinc entry into the periplasm.

Response of CnrX from Cupriavidus metallidurans CH34 to nickel binding.

Resistance to high concentration of nickel ions is mediated in Cupriavidus metallidurans by the CnrCBA transenvelope efflux complex. Expression of the cnrCBA genes is regulated by the transmembrane signal transduction complex CnrYXH. Together, the metal sensor CnrX and the transmembrane antisigma factor CnrY control the availability of the extracytoplasmic function sigma factor CnrH. Release of CnrH from sequestration by CnrY at the cytoplasmic side of the membrane depends essentially on the binding of the agonist metal ion Ni(ii) to the periplasmic metal sensor domain of CnrX. CnrH availability leads to transcription initiation at the promoters cnrYp and cnrCp and to the expression of the genes in the cnrYXHCBA nickel resistance determinant. The first steps of signal propagation by CnrX rely on subtle metal-dependent allosteric modifications. To study the nickel-mediated triggering process by CnrX, we have altered selected residues, F66, M123, and Y135, and explored the physiological consequences of these changes with respect to metal resistance, expression of a cnrCBA-lacZ reporter fusion and protein production. M123C- and Y135F-CnrXs have been further characterized in vitro by metal affinity measurements and crystallographic structure analysis. Atomic-resolution structures of metal-bound M123C- and Y135F-CnrXs showed that Ni(ii) binds two of the three canonical conformations identified and that Ni(ii) sensing likely proceeds by conformation selection.

The X-ray structure of NccX from Cupriavidus metallidurans 31A illustrates potential dangers of detergent solubilization when generating and interpreting crystal structures of membrane proteins.

The x-ray structure of NccX, a type II transmembrane metal sensor, from Cupriavidus metallidurans 31A has been determined at a resolution of 3.12 Å. This was achieved after solubilization by dodecylphosphocholine and purification in the presence of the detergent. NccX crystal structure did not match the model based on the extensively characterized periplasmic domain of its closest homologue CnrX. Instead, the periplasmic domains of NccX appeared collapsed against the hydrophobic transmembrane segments, leading to an aberrant topology incompatible with membrane insertion. This was explained by a detergent-induced redistribution of the hydrophobic interactions among the transmembrane helices and a pair of hydrophobic patches keeping the periplasmic domains together in the native dimer. Molecular dynamics simulations performed with the full-length protein or with the transmembrane segments were used along with in vivo homodimerization assays (TOXCAT) to evaluate the determinants of the interactions between NccX protomers. Taken as a whole, computational and experimental results are in agreement with the structural model of CnrX where a cradle-shaped periplasmic metal sensor domain is anchored into the inner membrane by two N-terminal helices. In addition, they show that the main determinant of NccX dimerization is the periplasmic soluble domain and that the interaction between transmembrane segments is highly dynamic. The present work introduces a new crystal structure for a transmembrane protein and, in line with previous studies, substantiates the use of complementary theoretical and in vivo investigations to rationalize a three-dimensional structure obtained in non-native conditions.

The crystal structure of the anti-σ factor CnrY in complex with the σ factor CnrH shows a new structural class of anti-σ factors targeting extracytoplasmic function σ factors.

Gene expression in bacteria is regulated at the level of transcription initiation, a process driven by σ factors. The regulation of σ factor activity proceeds from the regulation of their cytoplasmic availability, which relies on specific inhibitory proteins called anti-σ factors. With anti-σ factors regulating their availability according to diverse cues, extracytoplasmic function σ factors (σ(ECF)) form a major signal transduction system in bacteria. Here, structure:function relationships have been characterized in an emerging class of minimal-size transmembrane anti-σ factors, using CnrY from Cupriavidus metallidurans CH34 as a model. This study reports the 1.75-Å-resolution structure of CnrY cytosolic domain in complex with CnrH, its cognate σ(ECF), and identifies a small hydrophobic knob in CnrY as the major determinant of this interaction in vivo. Unsuspected structural similarity with the molecular switch regulating the general stress response in α-proteobacteria unravels a new class of anti-σ factors targeting σ(ECF). Members of this class carry out their function via a 30-residue stretch that displays helical propensity but no canonical structure on its own.

C-terminal engineering of CXCL12 and CCL5 chemokines: functional characterization by electrophysiological recordings.

Chemokines are chemotactic cytokines comprised of 70-100 amino acids. The chemokines CXCL12 and CCL5 are the endogenous ligands of the CXCR4 and CCR5 G protein-coupled receptors that are also HIV co-receptors. Biochemical, structural and functional studies of receptors are ligand-consuming and the cost of commercial chemokines hinders their use in such studies. Here, we describe methods for the expression, refolding, purification, and functional characterization of CXCL12 and CCL5 constructs incorporating C-terminal epitope tags. The model tags used were hexahistidines and Strep-Tag for affinity purification, and the double lanthanoid binding tag for fluorescence imaging and crystal structure resolution. The ability of modified and purified chemokines to bind and activate CXCR4 and CCR5 receptors was tested in Xenopus oocytes expressing the receptors, together with a Kir3 G-protein activated K(+) channel that served as a reporter of receptor activation. Results demonstrate that tags greatly influence the biochemical properties of the recombinant chemokines. Besides, despite the absence of any evidence for CXCL12 or CCL5 C-terminus involvement in receptor binding and activation, we demonstrated unpredictable effects of tag insertion on the ligand apparent affinity and efficacy or on the ligand dissociation. These tagged chemokines should constitute useful tools for the selective purification of properly-folded chemokines receptors and the study of their native quaternary structures.

Spectroscopic characterization of the metal-binding sites in the periplasmic metal-sensor domain of CnrX from Cupriavidus metallidurans CH34.

CnrX, the dimeric metal sensor of the three-protein transmembrane signal transduction complex CnrYXH of Cupriavidus metallidurans CH34, contains one metal-binding site per monomer. Both Ni and Co elicit a biological response and bind the protein in a 3N2O1S coordination sphere with a nearly identical octahedral geometry as shown by the X-ray structure of CnrXs, the soluble domain of CnrX. However, in solution CnrXs is titrated by 4 Co-equiv and exhibits an unexpected intense band at 384 nm that was detected neither by single-crystal spectroscopy nor under anaerobiosis. The data from a combination of spectroscopic techniques (spectrophotometry, electron paramagnetic resonance, X-ray absorption spectroscopy) showed that two sites correspond to those identified by crystallography. The two extra binding sites accommodate Co(II) in an octahedral geometry in the absence of oxygen and are occupied in air by a mixture of low-spin Co(II) as well as EPR-silent Co(III). These extra sites, located at the N-terminus of the protein, are believed to participate to the formation of peroxo-bridged dimers. Accordingly, we hypothesize that the intense band at 384 nm relies on the formation of a binuclear µ-peroxo Co(III) complex. These metal binding sites are not physiologically relevant since they are not detected in full-length NccX, the closest homologue of CnrX. X-ray absorption spectroscopy demonstrates that NccX stabilizes Co(II) in two-binding sites similar to those characterized by crystallography in its soluble counterpart. Nevertheless, the original spectroscopic properties of the extra Co-binding sites are of interest because they are susceptible to be detected in other Co-bound proteins.