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Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are multidomain transmembrane proteins, which facilitate the transport of various substances across cell membranes using energy derived from ATP hydrolysis. They are important drug targets since they mediate decreased drug susceptibility during pharmacological treatments. For the methylotrophic yeast

Dieter Oesterhelt, the biochemist who identified the first known microbial rhodopsin, died on 28 November 2022 at age 82. Dieter’s discovery of the color and function of the so-called purple membrane of the halophilic bacterium Halobacterium salinarum—and the availability, simplicity, and stability of its single protein, bacteriorhodopsin—laid the foundations for the membrane protein research field that he launched in 1971. Microbial rhodopsins act as light-driven pumps, channels, and enzymes, making the field of optogenetics, in which light controls the activity of targets such as neurons, possible.

The treatment of infectious diseases caused by multidrug-resistant pathogens is a major clinical challenge of the 21st century. The membrane-embedded respiratory cytochrome bd-type oxygen reductase is a critical survival factor utilized by pathogenic bacteria during infection, proliferation and the transition from acute to chronic states. Escherichia coli encodes for two cytochrome bd isoforms that are both involved in respiration under oxygen limited conditions. Mechanistic and structural differences between cydABX (Ecbd-I) and appCBX (Ecbd-II) operon encoded cytochrome bd variants have remained elusive in the past. Here, we demonstrate that cytochrome bd-II catalyzes oxidation of benzoquinols while possessing additional specificity for naphthoquinones. Our data show that although menaquinol-1 (MK1) is not able to directly transfer electrons onto cytochrome bd-II from E. coli, it has a stimulatory effect on its oxygen reduction rate in the presence of ubiquinol-1. We further determined cryo-EM structures of cytochrome bd-II to high resolution of 2.1 Å. Our structural insights confirm that the general architecture and substrate accessible pathways are conserved between the two bd oxidase isoforms, but two notable differences are apparent upon inspection: (i) Ecbd-II does not contain a CydH-like subunit, thereby exposing heme b595 to the membrane environment and (ii) the AppB subunit harbors a structural demethylmenaquinone-8 molecule instead of ubiquinone-8 as found in CydB of Ecbd-I. Our work completes the structural landscape of terminal respiratory oxygen reductases of E. coli and suggests that structural and functional properties of the respective oxidases are linked to quinol-pool dependent metabolic adaptations in E. coli.

Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are multidomain transmembrane proteins, which facilitate the transport of various substances across cell membranes using energy derived from ATP hydrolysis. They are important drug targets since they mediate decreased drug susceptibility during pharmacological treatments. For the methylotrophic yeastPichia pastoris, a model organism that is a widely used host for protein expression, the role and function of its ABC transporters is unexplored. In this work, we investigated thePichiaABC-B transporter STE6-2p. Functional investigations revealed that STE6-2p is capable of transporting rhodamines in vivo and is active in the presence of verapamil and triazoles in vitro. A phylogenetic analysis displays homology among multidrug resistance (MDR) transporters from pathogenic fungi to human ABC-B transporters. Further, we present high-resolution single-particle electron cryomicroscopy structures of an ABC transporter fromP. pastorisin the apo conformation (3.1 Å) and in complex with verapamil and adenylyl imidodiphosphate (AMP-PNP) (3.2 Å). An unknown density between transmembrane helices 4, 5, and 6 in both structures suggests the presence of a sterol-binding site of unknown function.

Identification and sequence determination by mass spectrometry have become routine analyses for soluble proteins. Membrane proteins, however, remain challenging targets due to their hydrophobicity and poor annotation. In particular small membrane proteins often remain unnoticed as they are largely inaccessible to Bottom-Up proteomics. Recent advances in structural biology, though, have led to multiple membrane protein complex structures being determined at sufficiently high resolution to detect uncharacterized, small subunits. In this work we offer a guide for the mass spectrometric characterization of solvent extraction-based purifications of small membrane proteins isolated from protein complexes and cellular membranes. We first demonstrate our Top-Down MALDI-MS/MS approach on a Photosystem II preparation, analyzing target protein masses between 2.5 and 9 kDa with high accuracy and sensitivity. Then we apply our technique to purify and sequence the mycobacterial ATP synthase

Bacteria utilize small extracellular molecules to communicate in order to collectively coordinate their behaviors in response to the population density. Autoinducer-2 (AI-2), a universal molecule for both intra- and inter-species communication, is involved in the regulation of biofilm formation, virulence, motility, chemotaxis, and antibiotic resistance. While many studies have been devoted to understanding the biosynthesis and sensing of AI-2, very little information is available on its export. The protein TqsA from Escherichia coli, which belongs to the AI-2 exporter superfamily, has been shown to export AI-2. Here, we report the cryogenic electron microscopic structures of two AI-2 exporters (TqsA and YdiK) from E. coli at 3.35 Å and 2.80 Å resolutions, respectively. Our structures suggest that the AI-2 exporter exists as a homo-pentameric complex. In silico molecular docking and native mass spectrometry experiments were employed to demonstrate the interaction between AI-2 and TqsA, and the results highlight the functional importance of two helical hairpins in substrate binding. We propose that each monomer works as an independent functional unit utilizing an elevator-type transport mechanism.

Respiratory chain complexes convert energy by coupling electron flow to transmembrane proton translocation. Owing to a lack of atomic structures of cytochrome bc 1 complex (Complex III) from thermophilic bacteria, little is known about the adaptations of this macromolecular machine to hyperthermophilic environments. In this study, we purified the cytochrome bc1 complex of Aquifex aeolicus, one of the most extreme thermophilic bacteria known, and determined its structure with and without an inhibitor at 3.3 Å resolution. Several residues unique for thermophilic bacteria were detected that provide additional stabilization for the structure. An extra transmembrane helix at the N‐terminus of cyt. c 1 was found to greatly enhance the interaction between cyt. b and cyt. c 1, and to bind a phospholipid molecule to stabilize the complex in the membrane. These results provide the structural basis for the hyperstability of the cytochrome bc1 complex in an extreme thermal environment., A hot take on thermophiles: The hyperstability of respiratory complex III from Aquifex aeolicus provides a suitable environment for the internal electron transfer reaction at high temperature.

Multidrug and toxic compound extrusion (MATE) transporters are widespread in all domains of life. Bacterial MATE transporters confer multidrug resistance by utilizing an electrochemical gradient of H+ or Na+ to export xenobiotics across the membrane. Despite the availability of X-ray structures of several MATE transporters, a detailed understanding of the transport mechanism has remained elusive. Here we report the crystal structure of a MATE transporter from Aquifex aeolicus at 2.0-A resolution. In light of its phylogenetic placement outside of the diversity of hitherto-described MATE transporters and the lack of conserved acidic residues, this protein may represent a subfamily of prokaryotic MATE transporters, which was proven by phylogenetic analysis. Furthermore, the crystal structure and substrate docking results indicate that the substrate binding site is located in the N bundle. The importance of residues surrounding this binding site was demonstrated by structure-based site-directed mutagenesis. We suggest that Aq_128 is functionally similar but structurally diverse from DinF subfamily transporters. Our results provide structural insights into the MATE transporter, which further advances our global understanding of this important transporter family.

Cytochrome c oxidases are among the most important and fundamental enzymes of life. Integrated into membranes they use four electrons from cytochrome c molecules to reduce molecular oxygen (dioxygen) to water. Their catalytic cycle has been considered to start with the oxidized form. Subsequent electron transfers lead to the E-state, the R-state (which binds oxygen), the P-state (with an already split dioxygen bond), the F-state and the O-state again. Here, we determined structures of up to 1.9 Å resolution of these intermediates by single particle cryo-EM. Our results suggest that in the O-state the active site contains a peroxide dianion and in the P-state possibly an intact dioxygen molecule, the F-state may contain a superoxide anion. Thus, the enzyme’s catalytic cycle may have to be turned by 180 degrees., Cytochrome c oxidase is a fundamental enzyme of life and its mechanism is not fully understood yet. Here, the authors present four cryo-EM structures of different intermediate states, which suggest an alternative cytochrome c oxidase reaction cycle.

Bacteria utilize small extracellular molecules to communicate in order to collectively coordinate their behaviors in response to the population density. Autoinducer-2 (AI-2), a universal molecule for both intra- and inter-species communication, is involved in the regulation of biofilm formation, virulence, motility, chemotaxis and antibiotic resistance. While many studies have been devoted to understanding the biosynthesis and sensing of AI-2, very little information is available on its export. The protein TqsA from E. coli, which belongs to a large underexplored membrane transporter family, the AI-2 exporter superfamily, has been shown to export AI-2. Here, we report the cryogenic electron microscopic structures of two AI-2 exporters (TqsA and YdiK) from E. coli at 3.35 Å and 2.80 Å resolutions, respectively. Our structures suggest that the AI-2 exporter exists as a homo-pentameric complex. In silico molecular docking and native mass spectrometry experiments were employed to demonstrate the interaction between AI-2 and TqsA, and the results highlight the functional importance of two helical hairpins in substrate binding. We propose that each monomer works as an independent functional unit utilizing an elevator-type transport mechanism. This study emphasizes the structural distinctiveness of this family of pentameric transporters and provides fundamental insights for the ensuing studies.

Multidrug and toxic compound extrusion (MATE) transporters are widespread in all domains of life. Bacterial MATE transporters confer multidrug resistance by utilizing an electrochemical gradient of H(+) or Na(+) to export xenobiotics across the membrane. Despite the availability of X-ray structures of several MATE transporters, a detailed understanding of the transport mechanism has remained elusive. Here we report the crystal structure of a MATE transporter from Aquifex aeolicus at 2.0-Å resolution. In light of its phylogenetic placement outside of the diversity of hitherto-described MATE transporters and the lack of conserved acidic residues, this protein may represent a subfamily of prokaryotic MATE transporters, which was proven by phylogenetic analysis. Furthermore, the crystal structure and substrate docking results indicate that the substrate binding site is located in the N bundle. The importance of residues surrounding this binding site was demonstrated by structure-based site-directed mutagenesis. We suggest that Aq_128 is functionally similar but structurally diverse from DinF subfamily transporters. Our results provide structural insights into the MATE transporter, which further advances our global understanding of this important transporter family.

Background The osmolyte and antioxidant taurine plays an important role in regulation of cellular volume, oxidative status and Ca2+-homeostasis. Taurine uptake in human cells is regulated by the Na+- and Cl−-dependent taurine transporter TauT. In order to gain deeper structural insights about the substrate binding pocket of TauT, a HEK293 cell line producing a GFP-TauT fusion protein was generated. Methods Transport activity was validated using cell-based [3H]-taurine transport assays. We determined the Km and IC50 values of taurine, β-alanine and γ-aminobutyrate. Additionally we were able to identify structurally similar compounds as potential new substrates or inhibitors of the TauT transporter. Substrate induced cytotoxicity was analyzed using a cell viability assay. Results In this study we show competitive effects of the 3-pyridinesulfonate, 2-aminoethylhydrogen sulfate, 5-aminovalerate, β-aminobutyrate, piperidine-4-sulfonate, 2-aminoethylphosphate and homotaurine. We demonstrate that taurine uptake can be inhibited by a phosphate. Furthermore our studies revealed that piperidine-4-sulfonate interacts with TauT with a higher affinity than γ-aminobutyrate and imidazole-4-acetate. Conclusion We propose that piperidine-4-sulfonate may serve as a potential lead structure for the design of novel drug candidates required for specific modulation of the TauT transporter in therapy of neurodegenerative diseases.

Peroxiredoxins (Prxs) are thiol peroxidases that scavenge various peroxide substrates such as hydrogen peroxide (H2O2), alkyl hydroperoxides and peroxinitrite. They also function as chaperones and are involved in signal transduction by H2O2 in eukaryotic cells. The genome of Aquifex aeolicus, a microaerophilic, hyperthermophilic eubacterium, encodes four Prxs, among them an alkyl hydroperoxide reductase AhpC2 which was found to be closely related to archaeal 1-Cys peroxiredoxins. We determined the crystal structure of AhpC2 at 1.8 A resolution and investigated its oligomeric state in solution by electron microscopy. AhpC2 is arranged as a toroid-shaped dodecamer instead of the typically observed decamer. The basic folding topology and the active site structure are conserved and possess a high structural similarity to other 1-Cys Prxs. However, the C-terminal region adopts an opposite orientation. AhpC2 contains three cysteines, Cys49, Cys212, and Cys218. The peroxidatic cysteine CP49 was found to be hyperoxidized to the sulfonic acid ( SO3H) form, while Cys212 forms an intra-monomer disulfide bond with Cys218. Mutagenesis experiments indicate that Cys212 and Cys218 play important roles in the oligomerization of AhpC2. Based on these structural characteristics, we proposed the catalytic mechanism of AhpC2. This study provides novel insights into the structure and reaction mechanism of 1-Cys peroxiredoxins.

Cytochrome bd oxidase is a bacterial terminal oxygen reductase that was suggested to enable adaptation to different environments and to confer resistance to stress conditions. An electrocatalytic study of the cyt bd oxidases from Escherichia coli, Corynebacterium glutamicum and Geobacillus thermodenitrificans gives evidence for a different reactivity towards oxygen. An inversion of the redox potential values of the three hemes is found when comparing the enzymes from different bacteria. This inversion can be correlated with different protonated glutamic acids as evidenced by reaction induced FTIR spectroscopy. The influence of the microenvironment of the hemes on the reactivity towards oxygen is discussed.

The heme‐copper oxidase superfamily comprises cytochrome c and ubiquinol oxidases. These enzymes catalyze the transfer of electrons from different electron donors onto molecular oxygen. A B‐family cytochrome c oxidase from the hyperthermophilic bacterium Aquifex aeolicus was discovered previously to be able to use both cytochrome c and naphthoquinol as electron donors. Its molecular mechanism as well as the evolutionary significance are yet unknown. Here we solved its 3.4 Å resolution electron cryo‐microscopic structure and discovered a novel dimeric structure mediated by subunit I (CoxA2) that would be essential for naphthoquinol binding and oxidation. The unique structural features in both proton and oxygen pathways suggest an evolutionary adaptation of this oxidase to its hyperthermophilic environment. Our results add a new conceptual understanding of structural variation of cytochrome c oxidases in different species., The 3.4 Å structure of cytochrome c oxidase from the hyperthermophilic bacterium Aquifex aeolicus (AaCcO) has been solved. The molecular mechanism that AaCcO uses involves both cytochrome c and quinol as electron donors through the native quinol molecules (NQs) bound at the dimeric interface.

Heteromeric amino acid transporters (HATs) comprise a group of membrane proteins that belong to the solute carrier (SLC) superfamily. They are formed by two different protein components: a light chain subunit from an SLC7 family member and a heavy chain subunit from the SLC3 family. The light chain constitutes the transport subunit whereas the heavy chain mediates trafficking to the plasma membrane and maturation of the functional complex. Mutation, malfunction, and dysregulation of HATs are associated with a wide range of pathologies or represent the direct cause of inherited and acquired disorders. Here we report the cryogenic electron microscopy structure of the neutral and basic amino acid transport complex (b([0,+])AT1-rBAT) which reveals a heterotetrameric protein assembly composed of two heavy and light chain subunits, respectively. The previously uncharacterized interaction between two HAT units is mediated via dimerization of the heavy chain subunits and does not include participation of the light chain subunits. The b((0,+))AT1 transporter adopts a LeuT fold and is captured in an inward-facing conformation. We identify an amino-acid–binding pocket that is formed by transmembrane helices 1, 6, and 10 and conserved among SLC7 transporters.

Heme A is a vital redox cofactor unique for the terminal cytochrome c oxidase in mitochondria and many microorganisms. It plays a key role in oxygen reduction by serving as an electron carrier and as the oxygen-binding site. Heme A is synthesized from heme O by an integral membrane protein, heme A synthase (HAS). Defects in HAS impair cellular respiration and have been linked to various human diseases, e.g., fatal infantile hypertrophic cardiomyopathy and Leigh syndrome. HAS exists as a stable oligomeric complex, and studies have shown that oligomerization of eukaryotic HAS is necessary for its proper function. However, the molecular architecture of the HAS oligomeric complex has remained uncharacterized. The present study shows that HAS forms trimers and reveals how the oligomeric arrangement contributes to the complex stability and flexibility, enabling HAS to perform its catalytic function effectively. This work provides the basic understanding for future studies on heme A biosynthesis., The integral membrane protein heme A synthase (HAS) catalyzes the biosynthesis of heme A, which is a prerequisite for cellular respiration in a wide range of aerobic organisms. Previous studies have revealed that HAS can form homo-oligomeric complexes, and this oligomerization appears to be evolutionarily conserved among prokaryotes and eukaryotes and is shown to be essential for the biological function of eukaryotic HAS. Despite its importance, little is known about the detailed structural properties of HAS oligomers. Here, we aimed to address this critical issue by analyzing the oligomeric state of HAS from Aquifex aeolicus (AaHAS) using a combination of techniques, including size exclusion chromatography coupled with multiangle light scattering (SEC-MALS), cross-linking, laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS), and single-particle electron cryomicroscopy (cryo-EM). Our results show that HAS forms a thermostable trimeric complex. A cryo-EM density map provides information on the oligomerization interface of the AaHAS trimer. These results provide structural insights into HAS multimerization and expand our knowledge of this important enzyme.

The respiratory chain of Escherichia coli contains two different types of terminal oxidase that are differentially regulated as a response to changing environmental conditions. These oxidoreductases catalyze the reduction of molecular oxygen to water and contribute to the proton motive force. The cytochrome bo 3 oxidase (cyt bo 3) acts as the primary terminal oxidase under atmospheric oxygen levels, whereas the bd‐type oxidase is most abundant under microaerobic conditions. In E. coli, both types of respiratory terminal oxidase (HCO and bd‐type) use ubiquinol‐8 as electron donor. Here, we assess the inhibitory potential of newly designed and synthesized 3‐alkylated Lawson derivatives through L‐proline‐catalyzed three‐component reductive alkylation (TCRA). The inhibitory effects of these Lawson derivatives on the terminal oxidases of E. coli (cyt bo 3 and cyt bd‐I) were tested potentiometrically. Four compounds were able to reduce the oxidoreductase activity of cyt bo 3 by more than 50 % without affecting the cyt bd‐I activity. Moreover, two inhibitors for both cyt bo 3 and cyt bd‐I oxidase could be identified. Based on molecular‐docking simulations, we propose binding modes of the new Lawson inhibitors. The molecular fragment benzyl enhances the inhibitory potential and selectivity for cyt bo 3, whereas heterocycles reduce this effect. This work extends the library of 3‐alkylated Lawson derivatives as selective inhibitors for respiratory oxidases and provides molecular probes for detailed investigations of the mechanisms of respiratory‐chain enzymes of E. coli., To b or not to b: Alkylated hydroxynaphthoquinone derivatives, synthesized by reductive alkylation and tested for their inhibitory effect on bacterial respiratory oxidases, showed selectivity for one particular cytochrome b oxidase. Enzyme activity assays and docking analysis provided insights into their binding site. Alkylation with cyclic residues like benzyl groups only increased the inhibitory effect of one oxidase.

The NADH:ubiquinone oxidoreductase (complex I) is the first enzyme of the respiratory chain and the entry point for most electrons. Generally, the bacterial complex I consists of 14 core subunits, homologues of which are also found in complex I of mitochondria. In complex I preparations from the hyperthermophilic bacterium Aquifex aeolicus we have identified 20 partially homologous subunits by combining MALDI-TOF and LILBID mass spectrometry methods. The subunits could be assigned to two different complex I isoforms, named NQOR1 and NQOR2. NQOR1 consists of subunits NuoA(2), NuoB, NuoD(2), NuoE, NuoF, NuoG, Nuol(1), NuoH(1), NuoJ(1), NuoL(1), NuoM(1) and NuoN(1), with an entire mass of 504.17 kDa. NQOR2 comprises subunits NuoA(1), NuoB, NuoD(1), NuoE, NuoF, NuoG, NuoH(2), NuoI(2), NuoJ(1), NuoK(1), NuoL(2), NuoM(2) and NuoN(2), with a total mass of 523.99 kDa. Three Fe-S clusters could be identified by EPR spectroscopy in a preparation containing predominantly NQOR1. These were tentatively assigned to a binuclear center N1, and two tetranuclear centers, N2 and N4. The redox midpoint potentials of N1 and N2 are 273 mV and 184 mV, respectively. Specific activity assays indicated that NQOR1 from cells grown under low concentrations of oxygen was the more active form. Increasing the concentration of oxygen in the bacterial cultures induced formation of NQOR2 showing the lower specific activity.

Na+/H+ antiporters comprise a family of membrane proteins evolutionarily conserved in all kingdoms of life and play an essential role in cellular ion homeostasis. The NhaA crystal structure of Escherichia coli has become the paradigm for this class of secondary active transporters. However, structural data are only available at low pH, where NhaA is inactive. Here, we adapted hydrogen/deuterium-exchange mass spectrometry (HDX-MS) to analyze conformational changes in NhaA upon Li+ binding at physiological pH. Our analysis revealed a global conformational change in NhaA with two sets of movements around an immobile binding site. Based on these results, we propose a model for the ion translocation mechanism that explains previously controversial data for this antiporter. Furthermore, these findings contribute to our understanding of related human transporters that have been linked to various diseases.

The Cbb3-type cytochrome c oxidases (Cbb3-CcOs), the second most abundant CcOs, catalyze the reduction of molecular oxygen to water, even at micromolar oxygen concentrations. In Pseudomonas stutzeri ZoBell, two tandemly organized cbb3-operons encode the isoforms Cbb3-1 and Cbb3-2 both possessing subunits CcoN, CcoO and CcoP. However, only the cbb3-2 operon contains an additional ccoQ gene. CcoQ consists of 62 amino acids and is predicted to possess one transmembrane spanning helix. The physiological role of CcoQ was investigated based on a CcoQ-deletion mutant and wild-type Cbb3-2 crystals not containing subunit CcoQ. Cbb3-2 isolated from the deletion mutant is inactive and appears as a dispersed band on blue native-PAGE gels. Surprisingly, in the absence of ccoQ, Cbb3-1 also shows a strongly reduced activity. Our data suggest that CcoQ primarily functions as an assembly factor for Cbb3-2 but is also required for correct assembly of Cbb3-1. In contrast, once correctly assembled, Cbb3-1 and Cbb3-2 possess a full enzymatic activity even in the absence of CcoQ.

PMID: 27664317; Cytochrome cbb3 (also known as C-type) oxidases belong to the family of heme-copper terminal oxidases which couple at the end of the respiratory chain the reduction of molecular oxygen into water and the pumping of protons across the membrane. They are expressed most often at low pressure of O2 and they exhibit a low homology of sequence with the cytochrome aa3 (A-type) oxidases found in mitochondria. Their binuclear active site comprises a high-spin heme b3 associated with a CuB center. The protein also contains one low-spin heme b and 3 hemes c. We address here the redox properties of cbb3 oxidases from three organisms, Rhodobacter sphaeroides, Vibrio cholerae and Pseudomonas stutzeri by means of electrochemical and spectroscopic techniques. We show that the redox potential of the heme b3 exhibits a relatively low midpoint potential, as in related cytochrome c-dependent nitric oxide reductases. Potential implications for the coupled electron transfer and proton uptake mechanism of C-type oxidases are discussed.

Hemes switch spots in a terminal oxidase Reduction of molecular oxygen to water is the driving force for respiration in aerobic organisms and is catalyzed by several distinct integral membrane complexes. These include an exclusively prokaryotic enzyme, cytochrome bd–type quinol oxidase, which is a potential antimicrobial target. Safarian et al. determined a high-resolution cryo–electron microscopy structure of this enzyme from the enteric bacterium Escherichia coli . Comparison to a homolog reveals a complete relocation of the site of oxygen binding and reduction caused by a change in the arrangement of heme cofactors and channels in the protein scaffold. This switch illustrates the diversity of structure and function in this family of enzymes and might reflect different biochemical roles of these homologs. Science , this issue p. 100

Multidrug and toxic compound extrusion (MATE) transporters mediate excretion of xenobiotics and toxic metabolites, thereby conferring multidrug resistance in bacterial pathogens and cancer cells. Structural information on the alternate conformational states and knowledge of the detailed mechanism of MATE transport are of great importance for drug development. However, the structures of MATE transporters are only known in V-shaped outward-facing conformations. Here, we present the crystal structure of a MATE transporter from Pyrococcus furiosus (PfMATE) in the long-sought-after inward-facing state, which was obtained after crystallization in the presence of native lipids. Transition from the outward-facing state to the inward-facing state involves rigid body movements of transmembrane helices (TMs) 2–6 and 8–12 to form an inverted V, facilitated by a loose binding of TM1 and TM7 to their respective bundles and their conformational flexibility. The inward-facing structure of PfMATE in combination with the outward-facing one supports an alternating access mechanism for the MATE family transporters.

Multidrug and toxic compound extrusion (MATE) transporters exist in all three domains of life. They confer multidrug resistance by utilizing H(+) or Na(+) electrochemical gradients to extrude various drugs across the cell membranes. The substrate binding and the transport mechanism of MATE transporters is a fundamental process but so far not fully understood. Here we report a detailed substrate binding study of NorM_PS, a representative MATE transporter from Pseudomonas stutzeri Our results indicate that NorM_PS is a proton-dependent multidrug efflux transporter. Detailed binding studies between NorM_PS and 4',6-diamidino-2-phenylindole (DAPI) were performed by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and spectrofluorometry. Two exothermic binding events were observed from ITC data, and the high-affinity event was directly correlated with the extrusion of DAPI. The affinities are about 1 μm and 0.1 mm for the high and low affinity binding, respectively. Based on our homology model of NorM_PS, variants with mutations of amino acids that are potentially involved in substrate binding, were constructed. By carrying out the functional characterization of these variants, the critical amino acid residues (Glu-257 and Asp-373) for high-affinity DAPI binding were determined. Taken together, our results suggest a new substrate-binding site for MATE transporters.

Peering into a membrance oxidase Microorganisms have evolved a number of enzymes to reduce oxygen and prevent oxidative stress. Cytochrome bd oxidases serve this role and also protect pathogenic bacteria from nitric acid; however, this class of enzymes so far has eluded high-resolution crystallography. Safarian et al. were able to resolve the three-dimensional structure of cytochrome bd oxidase from a thermophilic bacterium (see the Perspective by Cook and Poole). The overall structure and triangular arrangement of its heme cofactors bear little structural resemblance to those of other membrane-spanning oxidases, despite serving a similar function. Science , this issue p. 583 ; see also p. 518

Solute carrier (SLC) 26 or sulfate permease (SulP) anion transporters, belong to a phylogenetically ancient family of secondary active transporters. Members of the family are involved in several human genetic diseases and cell physiological processes. Despite their importance, the substrates for transport by this family of proteins have been poorly characterized. In this study, recombinant StmYchM/DauA, a SulP from Salmonella typhimurium was purified to homogeneity and functionally characterized. StmYchM/DauA was found to be a dimer in solution as determined by size exclusion chromatography coupled to multiple angle light scattering. We report a functional characterization of the SulP proteins in two membrane mimetic systems and reveal a dual nature of anionic substrates for SulP. StmYchM/DauA functionally incorporated into nanodiscs could bind fumarate with millimolar affinities (KD = 4.6 ± 0.29 mM) as detected by intrinsic tryptophan fluorescence quench studies. In contrast, electrophysiological experiments performed in reconstituted liposomes indicate a strong bicarbonate transport in the presence of chloride but no detectable electrogenic fumarate transport. We hence suggest that while SulP acts as an electrogenic bicarbonate transporter, fumarate may serve as substrate under different conditions indicating multiple functions of SulP.

Cell-free systems exploit the transcription and translation machinery of cells from different origins to produce proteins in a defined chemical environment. Due to its open nature, cell-free protein production is a versatile tool to introduce specific labels such as heavy isotopes, non-natural amino acids and tags into the protein while avoiding cell toxicity. In particular, radiolabelled peptides and proteins are valuable tools for the functional characterization of protein-protein interactions and for studying binding kinetics. In this study we evaluated cell-free protein production for the generation of radiolabelled ligands for G protein-coupled receptors (GPCRs). These receptors are seven-transmembrane-domain receptors activated by a plethora of extracellular stimuli including peptide ligands. Many GPCR peptide ligands contain disulphide bonds and are thus inherently difficult to produce in bacterial expression hosts or in Escherichia coli-based cell-free systems. Here, we established an adapted E. coli-based cell-free translation system for the production of disulphide bond-containing GPCR peptide ligands and specifically introduce tritium labels for detection. The bacterial oxidoreductase DsbA is used as a chaperone to favour the formation of disulphide bonds and to enhance the yield of correctly folded proteins and peptides. We demonstrate the correct folding and formation of disulphide bonds and show high-affinity ligand binding of the produced radio peptide ligands to the respective receptors. Thus, our system allows the fast, cost-effective and reliable synthesis of custom GPCR peptide ligands for functional and structural studies.

The crystal structure of the enzyme previously characterized as a type-2 NADH:menaquinone oxidoreductase (NDH-2) from Thermus thermophilus has been solved at a resolution of 2.9 Å and revealed that this protein is, in fact, a coenzyme A-disulfide reductase (CoADR). Coenzyme A (CoASH) replaces glutathione as the major low molecular weight thiol in Thermus thermophilus and is maintained in the reduced state by this enzyme (CoADR). Although the enzyme does exhibit NADH:menadione oxidoreductase activity expected for NDH-2 enzymes, the specific activity with CoAD as an electron acceptor is about 5-fold higher than with menadione. Furthermore, the crystal structure contains coenzyme A covalently linked Cys44, a catalytic intermediate (Cys44-S-S-CoA) reduced by NADH via the FAD cofactor. Soaking the crystals with menadione shows that menadione can bind to a site near the redox active FAD, consistent with the observed NADH:menadione oxidoreductase activity. CoADRs from other species were also examined and shown to have measurable NADH:menadione oxidoreductase activity. Although a common feature of this family of enzymes, no biological relevance is proposed. The CoADR from T. thermophilus is a soluble homodimeric enzyme. Expression of the recombinant TtCoADR at high levels in E. coli results in a small fraction that co-purifies with the membrane fraction, which was used previously to isolate the enzyme wrongly identified as a membrane-bound NDH-2. It is concluded that T. thermophilus does not contain an authentic NDH-2 component in its aerobic respiratory chain.

Multidrug resistance (MDR) in bacterial pathogens has become a severe threat to public health. Membrane transporters of the multidrug and toxic compound extrusion (MATE) family contribute critically to MDR, making them promising drug targets. Despite recent advances, structures in different conformations and the mechanistic details of their antiport cycle are still elusive. Here we studied NorM_PS, a representative MATE transporter from Pseudomonas stutzeri, using biochemical assays in combination with hydrogen/deuterium exchange-mass spectrometry. Our results confirm that the antiport is proton dependent and electroneutral with a stoichiometry of two protons per one doubly positively charged substrate. We investigated the conformational dynamics upon substrate binding, and our hydrogen/deuterium exchange-mass spectrometry analysis revealed an occlusion in the proposed binding site as well as a closure of the cytoplasmic cavity and formation of a periplasmic cavity. Together with the results of selected variants (D38N, D373N and Q376A), we propose a six-step rocker-switch model for NorM_PS, which also increases our understanding of related MATE transporters and may help to fight the burden of MDR.