Structure of cytosine transport protein CodB provides insight into nucleobase-cation symporter 1 mechanism.

CodB is a cytosine transporter from the Nucleobase-Cation-Symport-1 (NCS1) transporter family, a member of the widespread LeuT superfamily. Previous experiments with the nosocomial pathogen Pseudomonas aeruginosa have shown CodB as also important for the uptake of 5-fluorocytosine, which has been suggested as a novel drug to combat antimicrobial resistance by suppressing virulence. Here we solve the crystal structure of CodB from Proteus vulgaris, at 2.4 Å resolution in complex with cytosine. We show that CodB carries out the sodium-dependent uptake of cytosine and can bind 5-fluorocytosine. Comparison of the substrate-bound structures of CodB and the hydantoin transporter Mhp1, the only other NCS1 family member for which the structure is known, highlight the importance of the hydrogen bonds that the substrates make with the main chain at the breakpoint in the discontinuous helix, TM6. In contrast to other LeuT superfamily members, neither CodB nor Mhp1 makes specific interactions with residues on TM1. Comparison of the structures provides insight into the intricate mechanisms of how these proteins transport substrates across the plasma membrane.

Controlled propulsion of micro/nanomotors: operational mechanisms, motion manipulation and potential biomedical applications.

Inspired by natural mobile microorganisms, researchers have developed micro/nanomotors (MNMs) that can autonomously move by transducing different kinds of energies into kinetic energy. The rapid development of MNMs has created tremendous opportunities for biomedical fields including diagnostics, therapeutics, and theranostics. Although the great progress has been made in MNM research, at a fundamental level, the accepted propulsion mechanisms are still a controversial matter. In practical applications such as precision nanomedicine, the precise control of the motion, including the speed and directionality, of MNMs is also important, which makes advanced motion manipulation desirable. Very recently, diverse MNMs with different propulsion strategies, morphologies, sizes, porosities and chemical structures have been fabricated and applied for various uses. Herein, we thoroughly summarize the physical principles behind propulsion strategies, as well as the recent advances in motion manipulation methods and relevant biomedical applications of these MNMs. The current challenges in MNM research are also discussed. We hope this review can provide a bird's eye overview of the MNM research and inspire researchers to create novel and more powerful MNMs.

Structural basis of carnitine monooxygenase CntA substrate specificity, inhibition, and intersubunit electron transfer.

Microbial metabolism of carnitine to trimethylamine (TMA) in the gut can accelerate atherosclerosis and heart disease, and these TMA-producing enzymes are therefore important drug targets. Here, we report the first structures of the carnitine oxygenase CntA, an enzyme of the Rieske oxygenase family. CntA exists in a head-to-tail α3 trimeric structure. The two functional domains (the Rieske and the catalytic mononuclear iron domains) are located >40 Å apart in the same monomer but adjacent in two neighboring monomers. Structural determination of CntA and subsequent electron paramagnetic resonance measurements uncover the molecular basis of the so-called bridging glutamate (E205) residue in intersubunit electron transfer. The structures of the substrate-bound CntA help to define the substrate pocket. Importantly, a tyrosine residue (Y203) is essential for ligand recognition through a π-cation interaction with the quaternary ammonium group. This interaction between an aromatic residue and quaternary amine substrates allows us to delineate a subgroup of Rieske oxygenases (group V) from the prototype ring-hydroxylating Rieske oxygenases involved in bioremediation of aromatic pollutants in the environment. Furthermore, we report the discovery of the first known CntA inhibitors and solve the structure of CntA in complex with the inhibitor, demonstrating the pivotal role of Y203 through a π-π stacking interaction with the inhibitor. Our study provides the structural and molecular basis for future discovery of drugs targeting this TMA-producing enzyme in human gut.

Structural basis of trehalose recognition by the mycobacterial LpqY-SugABC transporter.

The Mycobacterium tuberculosis (Mtb) LpqY-SugABC ATP-binding cassette transporter is a recycling system that imports trehalose released during remodeling of the Mtb cell-envelope. As this process is essential for the virulence of the Mtb pathogen, it may represent an important target for tuberculosis drug and diagnostic development, but the transporter specificity and molecular determinants of substrate recognition are unknown. To address this, we have determined the structural and biochemical basis of how mycobacteria transport trehalose using a combination of crystallography, saturation transfer difference NMR, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays, and the synthesis of trehalose analogs. This analysis pinpoints key residues of the LpqY substrate binding lipoprotein that dictate substrate-specific recognition and has revealed which disaccharide modifications are tolerated. These findings provide critical insights into how the essential Mtb LpqY-SugABC transporter reuses trehalose and modified analogs and specifies a framework that can be exploited for the design of new antitubercular agents and/or diagnostic tools.

Colour scales with climate in North American ratsnakes: a test of the thermal melanism hypothesis using community science images.

Animal colour is a complex trait shaped by multiple selection pressures that can vary across geography. The thermal melanism hypothesis predicts that darker coloration is beneficial to animals in colder regions because it allows for more rapid solar absorption. Here, we use community science images of three closely related species of North American ratsnakes (genus Pantherophis) to examine if climate predicts colour variation across range-wide scales. We predicted that darker individuals are found in colder regions and higher elevations, in accordance with the thermal melanism hypothesis. Using an unprecedented dataset of over 8000 images, we found strong support for temperature as a key predictor of darker colour, supporting thermal melanism. We also found that elevation and precipitation are predictive of colour, but the direction and magnitude of these effects were more variable across species. Our study is the first to quantify colour variation in Pantherophis ratsnakes, highlighting the value of community science images for studying range-wide colour variation.

Engineering of Yolk/Core-Shell Structured Nanoreactors for Thermal Hydrogenations.

Heterogeneous hydrogenation reactions are of great importance for chemical upgrading and synthesis, but still face the challenges of controlling selectivity and long-term stability. To improve the catalytic performance, many hydrogenation reactions utilize special yolk/core-shell nanoreactors (YCSNs) with unique architectures and advantageous properties. This work presents the developmental and technological challenges in the preparation of YCSNs that are potentially useful for hydrogenation reactions, and provides a summary of the properties of these materials. The work also addresses the scientific challenges in applications of these YCSNs in various gas and liquid-phase hydrogenation reactions. The catalyst structures, catalytic performance, structure-performance relationships, reaction mechanisms, and unsolved problems are discussed too. Also, a brief outlook and opportunities for future research in this field are presented. This work on the advancements in YCSNs might inspire the creation of new materials with desired structures for achieving maximal hydrogenation performances.

Atomic Pyridinic Nitrogen Sites Promoting Levulinic Acid Hydrogenations over Double-Shelled Hollow Ru/C Nanoreactors.

Nitrogen-doped nanocarbons are widely used as supports for metal-heterogeneous catalytic conversions. When nitrogen-doped nanocarbon supports are used to disperse metallic nanoparticles (MNPs), the nitrogen dopant can enhance MNPs electron density to reach higher catalytic activity and promote MNPs stability through anchoring effects. However, the precise identification of active nitrogen species between N-dopants and reactants is rarely reported. Herein, a proof-of-concept study on the active N species for levulinic acid hydrogenation is reported. A double-shell structured carbon catalyst (DSC) is designed with selectively locating ultrafine Ru NPs only on inner carbon shell, specifically, different N species on the external carbon shell. Through the design of such a nanostructure, it is demonstrated that the alkaline pyridinic N species on the outer shell serves as an anchor point for the spontaneous binding of the acidic reactant. The pyridinic N content can be modulated from 7.4 to 29.2 mg gcat-1 by selecting different precursors. Finally, the Ru-DSC-CTS (using chitosan as the precursor) catalyst achieves a 99% conversion of levulinic acid under 70 °C and 4 MPa hydrogen pressure for 1 h. This work sheds light on the design of nanoreactors at the atomic scale and investigates heterogeneous catalysis at the molecular level.

Nanospatial Charge Modulation of Monodispersed Polymeric Microsphere Photocatalysts for Exceptional Hydrogen Peroxide Production.

Photocatalysis offers a sustainable strategy for hydrogen peroxide (H2 O2 ) production, which is an essential oxidant and emerging energy carrier in modern chemical industry. The development of polymer-based photocatalysts to produce H2 O2 has great potential but is limited by lower efficiency due to the limitation of light utilization and the low charge separation efficiency. Herein, a series of monodispersed mesoporous resorcinol-formaldehyde resin spheres (MRFS) are reported with a rational designed spatial charge distribution, exhibiting wide light absorption with a solar-to-chemical conversion (SCC) efficiency of 1.1%. Surface photovoltage microscopy (SPVM) measurements unraveled the charge separation in nanospace with uneven distribution of donor (D) and acceptor (A) sites. A density functional theory (DFT) calculation elucidated the origin of photogenerated electrons and holes. Moreover, MRFS demonstrates photocatalytic water oxidation ability. The findings in this work open a new avenue for the development of porous polymeric photocatalysts toward highly efficient solar energy conversion.

The Leishmania PABP1-eIF4E4 interface: a novel 5'-3' interaction architecture for trans-spliced mRNAs.

Trans-splicing of trypanosomatid polycistronic transcripts produces polyadenylated monocistronic mRNAs modified to form the 5' cap4 structure (m7Gpppm36,6,2'Apm2'Apm2'Cpm23,2'U). NMR and X-ray crystallography reveal that Leishmania has a unique type of N-terminally-extended cap-binding protein (eIF4E4) that binds via a PAM2 motif to PABP1. This relies on the interactions of a combination of polar and charged amino acid side-chains together with multiple hydrophobic interactions, and underpins a novel architecture in the Leishmania cap4-binding translation factor complex. Measurements using microscale thermophoresis, fluorescence anisotropy and surface plasmon resonance characterize the key interactions driving assembly of the Leishmania translation initiation complex. We demonstrate that this complex can accommodate Leishmania eIF4G3 which, unlike the standard eukaryotic initiation complex paradigm, binds tightly to eIF4E4, but not to PABP1. Thus, in Leishmania, the chain of interactions 5'cap4-eIF4E4-PABP1-poly(A) bridges the mRNA 5' and 3' ends. Exceptionally, therefore, by binding tightly to two protein ligands and to the mRNA 5' cap4 structure, the trypanosomatid N-terminally extended form of eIF4E acts as the core molecular scaffold for the mRNA-cap-binding complex. Finally, the eIF4E4 N-terminal extension is an intrinsically disordered region that transitions to a partly folded form upon binding to PABP1, whereby this interaction is not modulated by poly(A) binding to PABP1.

Light-Activated Electron Transfer and Catalytic Mechanism of Carnitine Oxidation by Rieske-Type Oxygenase from Human Microbiota.

Oxidation of quaternary ammonium substrate, carnitine by non-heme iron containing Acinetobacter baumannii (Ab) oxygenase CntA/reductase CntB is implicated in the onset of human cardiovascular disease. Herein, we develop a blue-light (365 nm) activation of NADH coupled to electron paramagnetic resonance (EPR) measurements to study electron transfer from the excited state of NADH to the oxidized, Rieske-type, [2Fe-2S]2+ cluster in the AbCntA oxygenase domain with and without the substrate, carnitine. Further electron transfer from one-electron reduced, Rieske-type [2Fe-2S]1+ center in AbCntA-WT to the mono-nuclear, non-heme iron center through the bridging glutamate E205 and subsequent catalysis occurs only in the presence of carnitine. The electron transfer process in the AbCntA-E205A mutant is severely affected, which likely accounts for the significant loss of catalytic activity in the AbCntA-E205A mutant. The NADH photo-activation coupled with EPR is broadly applicable to trap reactive intermediates at low temperature and creates a new method to characterize elusive intermediates in multiple redox-centre containing proteins.

Strategies for successful isolation of a eukaryotic transporter.

The isolation of integral membrane proteins for structural analysis remains challenging and this is particularly the case for eukaryotic membrane proteins. Here we describe our efforts to isolate OsBOR3, a boron transporter from Oryza sativa. OsBOR3 was expressed as both full length and a C-terminally truncated form lacking residues 643-672 (OsBOR3Δ1-642). While both express well as C-terminal GFP fusion proteins in Saccharomyces cerevisiae, the full length protein isolates poorly in the detergent dodecyl-ß-d-maltoside (DDM). The OsBOR3Δ1-642 isolated in DDM in large quantities but was contaminated with GFP tagged protein, indicated incomplete protease removal of the tag. Addition of the reducing agent dithiothreitol (DTT) had no effect on isolation. Detergent screening indicated that the neopentyl glycol detergents, LMNG, UDMNG and DMNG conferred greater stability on the OsBOR3Δ1-642 than DDM. Isolation of OsBOR3Δ1-642 in LMNG both in the presence and absence of DTT produced large quantities of protein but contaminated with GFP tagged protein. Isolation of OsBOR3Δ1-642 in DMNG + DTT resulted in protein sample that does not contain any detectable GFP but elutes at a higher retention volume than that seen for protein isolated in either DDM or LMNG. Mass spectrometry confirmed that the LMNG and DMNG purified protein is OsBOR3Δ1-642 indicating that the DMNG isolated protein is monomer compared to the dimer isolated using LMNG. This was further supported by single particle electron microscopic analysis revealing that the DMNG protein particles are roughly half the size of the LMNG protein particles.

Structural and functional determination of homologs of the Mycobacterium tuberculosis N-acetylglucosamine-6-phosphate deacetylase (NagA).

The Mycobacterium tuberculosis (Mtb) pathogen encodes a GlcNAc-6-phosphate deacetylase enzyme, NagA (Rv3332), that belongs to the amidohydrolase superfamily. NagA enzymes catalyze the deacetylation of GlcNAc-6-phosphate (GlcNAc6P) to glucosamine-6-phosphate (GlcN6P). NagA is a potential antitubercular drug target because it represents the key enzymatic step in the generation of essential amino-sugar precursors required for Mtb cell wall biosynthesis and also influences recycling of cell wall peptidoglycan fragments. Here, we report the structural and functional characterization of NagA from Mycobacterium smegmatis (MSNagA) and Mycobacterium marinum (MMNagA), close relatives of Mtb Using a combination of X-ray crystallography, site-directed mutagenesis, and biochemical and biophysical assays, we show that these mycobacterial NagA enzymes are selective for GlcNAc6P. Site-directed mutagenesis studies revealed crucial roles of conserved residues in the active site that underpin stereoselective recognition, binding, and catalysis of substrates. Moreover, we report the crystal structure of MSNagA in both ligand-free form and in complex with the GlcNAc6P substrate at 2.6 and 2.0 Å resolutions, respectively. The GlcNAc6P complex structure disclosed the precise mode of GlcNAc6P binding and the structural framework of the active site, including two divalent metals located in the α/ß binuclear site. Furthermore, we observed a cysteine residue located on a flexible loop region that occludes the active site. This cysteine is unique to mycobacteria and may represent a unique subsite for targeting mycobacterial NagA enzymes. Our results provide critical insights into the structural and mechanistic properties of mycobacterial NagA enzymes having an essential role in amino-sugar and nucleotide metabolism in mycobacteria.

A two-domain elevator mechanism for sodium/proton antiport.

Sodium/proton (Na(+)/H(+)) antiporters, located at the plasma membrane in every cell, are vital for cell homeostasis. In humans, their dysfunction has been linked to diseases, such as hypertension, heart failure and epilepsy, and they are well-established drug targets. The best understood model system for Na(+)/H(+) antiport is NhaA from Escherichia coli, for which both electron microscopy and crystal structures are available. NhaA is made up of two distinct domains: a core domain and a dimerization domain. In the NhaA crystal structure a cavity is located between the two domains, providing access to the ion-binding site from the inward-facing surface of the protein. Like many Na(+)/H(+) antiporters, the activity of NhaA is regulated by pH, only becoming active above pH 6.5, at which point a conformational change is thought to occur. The only reported NhaA crystal structure so far is of the low pH inactivated form. Here we describe the active-state structure of a Na(+)/H(+) antiporter, NapA from Thermus thermophilus, at 3 Å resolution, solved from crystals grown at pH 7.8. In the NapA structure, the core and dimerization domains are in different positions to those seen in NhaA, and a negatively charged cavity has now opened to the outside. The extracellular cavity allows access to a strictly conserved aspartate residue thought to coordinate ion binding directly, a role supported here by molecular dynamics simulations. To alternate access to this ion-binding site, however, requires a surprisingly large rotation of the core domain, some 20° against the dimerization interface. We conclude that despite their fast transport rates of up to 1,500 ions per second, Na(+)/H(+) antiporters operate by a two-domain rocking bundle model, revealing themes relevant to secondary-active transporters in general.

Molecular mechanism of ligand recognition by membrane transport protein, Mhp1.

The hydantoin transporter Mhp1 is a sodium-coupled secondary active transport protein of the nucleobase-cation-symport family and a member of the widespread 5-helix inverted repeat superfamily of transporters. The structure of Mhp1 was previously solved in three different conformations providing insight into the molecular basis of the alternating access mechanism. Here, we elucidate detailed events of substrate binding, through a combination of crystallography, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays, and the design and synthesis of novel ligands. We show precisely where 5-substituted hydantoin substrates bind in an extended configuration at the interface of the bundle and hash domains. They are recognised through hydrogen bonds to the hydantoin moiety and the complementarity of the 5-substituent for a hydrophobic pocket in the protein. Furthermore, we describe a novel structure of an intermediate state of the protein with the external thin gate locked open by an inhibitor, 5-(2-naphthylmethyl)-L-hydantoin, which becomes a substrate when leucine 363 is changed to an alanine. We deduce the molecular events that underlie acquisition and transport of a ligand by Mhp1.

G-protein-coupled receptor inactivation by an allosteric inverse-agonist antibody.

G-protein-coupled receptors are the largest class of cell-surface receptors, and these membrane proteins exist in equilibrium between inactive and active states. Conformational changes induced by extracellular ligands binding to G-protein-coupled receptors result in a cellular response through the activation of G proteins. The A(2A) adenosine receptor (A(2A)AR) is responsible for regulating blood flow to the cardiac muscle and is important in the regulation of glutamate and dopamine release in the brain. Here we report the raising of a mouse monoclonal antibody against human A(2A)AR that prevents agonist but not antagonist binding to the extracellular ligand-binding pocket, and describe the structure of A(2A)AR in complex with the antibody Fab fragment (Fab2838). This structure reveals that Fab2838 recognizes the intracellular surface of A(2A)AR and that its complementarity-determining region, CDR-H3, penetrates into the receptor. CDR-H3 is located in a similar position to the G-protein carboxy-terminal fragment in the active opsin structure and to CDR-3 of the nanobody in the active ß(2)-adrenergic receptor structure, but locks A(2A)AR in an inactive conformation. These results suggest a new strategy to modulate the activity of G-protein-coupled receptors.

The prevalence of Coxiella burnetii shedding in dairy goats at the time of parturition in an endemically infected enterprise and associated milk yield losses.

BACKGROUND: This was a panel study of the prevalence of C. burnetii infection in does in an endemic dairy goat enterprise in Victoria, Australia. Our first objective was to determine the prevalence of does shedding C. burnetii at the time of parturition and to quantify the concentration of genome equivalents (GE) present in each C. burnetii positive sample. Our second objective was to determine the proportion of positive does that were persistent shedders. Our final objective was to quantify the association between C. burnetii qPCR status at the time of kidding and daily milk volumes produced during the subsequent lactation. RESULTS: Vaginal swabs (n= 490) were collected from does at the time of kidding and analysed using a quantitative polymerase chain reaction (qPCR) assay. Shedding of C. burnetii was detected in 15% (95% CI: 12% to 18%) of the sampled does. Does were classified as qPCR-negative, qPCR-positive low and qPCR-positive high based on the estimated concentration of GE from the qPCR. Persistent shedding at relatively low concentrations was detected in 20% (95% CI: 10% to35%) of shedding does sampled again at their subsequent parturition. After controlling for possible confounders and adjusting for variation in daily milk yields at the individual doe level, daily milk yields for qPCR-positive high does were reduced by 17% (95% CI: 3% to 32%) compared to qPCR-negative does (p= 0.02). CONCLUSIONS: Shedding concentrations of C. burnetii were highly skewed, with a relatively small group of does shedding relatively high quantities of C. burnetii. Further, high shedding does had reduced milk yields compared to qPCR-negative does. Early detection and culling of high shedding does would result in increased farm profitability and reduce the risk of Q fever transmission.

A longitudinal study of serological responses to Coxiella burnetii and shedding at kidding among intensively-managed goats supports early use of vaccines.

Vaccination against Coxiella burnetii, the cause of Q fever, is reportedly the only feasible strategy of eradicating infection in ruminant herds. Preventive vaccination of seronegative goats is more effective in reducing shedding of C. burnetii than vaccinating seropositive goats. The age at which goats born on heavily-contaminated farms first seroconvert to C. burnetii has not yet been documented. In a 16-month birth cohort study, the age at which goats seroconverted against C. burnetii was investigated; 95 goats were bled every 2 weeks and tested for antibodies against C. burnetii. Risk factors for seroconversion were explored and goats shedding C. burnetii were identified by testing vaginal swabs taken at the goats' first kidding using a com1 polymerase chain reaction assay. The first surge in the number of goats with IgM to C. burnetii was observed at week 9. Thus, a first vaccination not later than 8 weeks of age to control C. burnetii in highly contaminated environments is indicated. The odds of seroconversion were 2.0 times higher [95% confidence interval (CI) 1.2, 3.5] in kids born by does with serological evidence of recent infection (IgM seropositive) compared to kids born by IgM seronegative does, suggesting either in utero transmission or peri-parturient infection. The rate of seroconversion was 4.5 times higher (95% CI 2.1, 9.8) during than outside the kidding season, highlighting the risk posed by C. burnetii shed during kidding, even to goats outside the kidding herd. Shedding of C. burnetii at kidding was detected in 15 out of 41 goats infected before breeding.

Crystal structure of a bacterial homologue of the bile acid sodium symporter ASBT.

High cholesterol levels greatly increase the risk of cardiovascular disease. About 50 per cent of cholesterol is eliminated from the body by its conversion into bile acids. However, bile acids released from the bile duct are constantly recycled, being reabsorbed in the intestine by the apical sodium-dependent bile acid transporter (ASBT, also known as SLC10A2). It has been shown in animal models that plasma cholesterol levels are considerably lowered by specific inhibitors of ASBT, and ASBT is thus a target for hypercholesterolaemia drugs. Here we report the crystal structure of a bacterial homologue of ASBT from Neisseria meningitidis (ASBT(NM)) at 2.2 Å. ASBT(NM) contains two inverted structural repeats of five transmembrane helices. A core domain of six helices harbours two sodium ions, and the remaining four helices pack in a row to form a flat, 'panel'-like domain. Overall, the architecture of the protein is remarkably similar to the sodium/proton antiporter NhaA, despite having no detectable sequence homology. The ASBT(NM) structure was captured with the substrate taurocholate present, bound between the core and panel domains in a large, inward-facing, hydrophobic cavity. Residues near this cavity have been shown to affect the binding of specific inhibitors of human ASBT. The position of the taurocholate molecule, together with the molecular architecture, suggests the rudiments of a possible transport mechanism.

Alternating access mechanism in the POT family of oligopeptide transporters.

Short chain peptides are actively transported across membranes as an efficient route for dietary protein absorption and for maintaining cellular homeostasis. In mammals, peptide transport occurs via PepT1 and PepT2, which belong to the proton-dependent oligopeptide transporter, or POT family. The recent crystal structure of a bacterial POT transporter confirmed that they belong to the major facilitator superfamily of secondary active transporters. Despite the functional characterization of POT family members in bacteria, fungi and mammals, a detailed model for peptide recognition and transport remains unavailable. In this study, we report the 3.3-Å resolution crystal structure and functional characterization of a POT family transporter from the bacterium Streptococcus thermophilus. Crystallized in an inward open conformation the structure identifies a hinge-like movement within the C-terminal half of the transporter that facilitates opening of an intracellular gate controlling access to a central peptide-binding site. Our associated functional data support a model for peptide transport that highlights the importance of salt bridge interactions in orchestrating alternating access within the POT family.

Transporter oligomerization: form and function.

Transporters are integral membrane proteins with central roles in the efficient movement of molecules across biological membranes. Many transporters exist as oligomers in the membrane. Depending on the individual transport protein, oligomerization can have roles in membrane trafficking, function, regulation and turnover. For example, our recent studies on UapA, a nucleobase ascorbate transporter, from Aspergillus nidulans, have revealed both that dimerization of this protein is essential for correct trafficking to the membrane and the structural basis of how one UapA protomer can affect the function of the closely associated adjacent protomer. Here, we review the roles of oligomerization in many particularly well-studied transporters and transporter families.