The C-Terminal Domain of the Arabinosyltransferase Mycobacterium tuberculosis EmbC Is a Lectin-Like Carbohydrate Binding Module

The D-arabinan-containing polymers arabinogalactan (AG) and lipoarabinomannan (LAM) are essential components of the unique cell envelope of the pathogen Mycobacterium tuberculosis. Biosynthesis of AG and LAM involves a series of membrane-embedded arabinofuranosyl (Araf) transferases whose structures are largely uncharacterised, despite the fact that several of them are pharmacological targets of ethambutol, a frontline drug in tuberculosis therapy. Herein, we present the crystal structure of the C-terminal hydrophilic domain of the ethambutol-sensitive Araf transferase M. tuberculosis EmbC, which is essential for LAM synthesis. The structure of the C-terminal domain of EmbC (EmbCCT) encompasses two sub-domains of different folds, of which subdomain II shows distinct similarity to lectin-like carbohydrate-binding modules (CBM). Co-crystallisation with a cell wall-derived di-arabinoside acceptor analogue and structural comparison with ligand-bound CBMs suggest that EmbCCT contains two separate carbohydrate binding sites, associated with subdomains I and II, respectively. Single-residue substitution of conserved tryptophan residues (Trp868, Trp985) at these respective sites inhibited EmbC-catalysed extension of LAM. The same substitutions differentially abrogated binding of di- and penta-arabinofuranoside acceptor analogues to EmbCCT, linking the loss of activity to compromised acceptor substrate binding, indicating the presence of two separate carbohydrate binding sites, and demonstrating that subdomain II indeed functions as a carbohydrate-binding module. This work provides the first step towards unravelling the structure and function of a GT-C-type glycosyltransferase that is essential in M. tuberculosis. Author Summary Top Tuberculosis (TB), an infectious disease caused by the bacillus Mycobacterium tuberculosis, burdens large swaths of the world population. Treatment of active TB typically requires administration of an antibiotic cocktail over several months that includes the drug ethambutol. This front line compound inhibits a set of arabinosyltransferase enzymes, called EmbA, EmbB and EmbC, which are critical for the synthesis of arabinan, a vital polysaccharide in the pathogen's unique cell envelope. How precisely ethambutol inhibits arabinosyltransferase activity is not clear, in part because structural information of its pharmacological targets has been elusive. Here, we report the high-resolution structure of the C-terminal domain of the ethambutol-target EmbC, a 390-amino acid fragment responsible for acceptor substrate recognition. Combining the X-ray crystallographic analysis with structural comparisons, site-directed mutagenesis, activity and ligand binding assays, we identified two regions in the C-terminal domain of EmbC that are capable of binding acceptor substrate mimics and are critical for activity of the full-length enzyme. Our results begin to define structure-function relationships in a family of structurally uncharacterised membrane-embedded glycosyltransferases, which are an important target for tuberculosis therapy

Control of serine integrase recombination directionality by fusion with the directionality factor

Bacteriophage serine integrases are extensively used in biotechnology and synthetic biology for assembly and rearrangement of DNA sequences. Serine integrases promote recombination between two different DNA sites, attP and attB, to form recombinant attL and attR sites. The ‘reverse’ reaction requires another phage-encoded protein called the recombination directionality factor (RDF) in addition to integrase; RDF activates attL × attR recombination and inhibits attP × attB recombination. We show here that serine integrases can be fused to their cognate RDFs to create single proteins that catalyse efficient attL × attR recombination in vivo and in vitro, whereas attP × attB recombination efficiency is reduced. We provide evidence that activation of attL × attR recombination involves intra-subunit contacts between the integrase and RDF moieties of the fusion protein. Minor changes in the length and sequence of the integrase–RDF linker peptide did not affect fusion protein recombination activity. The efficiency and single-protein convenience of integrase–RDF fusion proteins make them potentially very advantageous for biotechnology/synthetic biology applications. Here, we demonstrate efficient gene cassette replacement in a synthetic metabolic pathway gene array as a proof of principle

Differentiating criminal networks in the illegal wildlife trade: organized, corporate and disorganized crime

Historically, the poaching of wildlife was portrayed as a small-scale local activity in which only small numbers of wildlife would be smuggled illegally by collectors or opportunists. Nowadays, this image has changed: criminal networks are believed to be highly involved in wildlife trafficking, which has become a significant area of illicit activity. Even though wildlife trafficking has become accepted as a major area of crime and an important topic and criminologists have examined a variety of illegal wildlife markets, research that specifically focusses on the involvement of different criminal networks and their specific nature is lacking. The concept of a ‘criminal network’ or ‘serious organized crime’ is amorphous – getting used interchangeably and describes all crime that is structured rather than solely reflecting crime that fits within normative definitions of ‘organized’ crime. In reality, criminal networks are diverse. As such, we propose categories of criminal networks that are evidenced in the literature and within our own fieldwork: (1) organized crime groups (2) corporate crime groups and (3) disorganized criminal networks. Whereas there are instances when these groups act alone, this article will (also) discuss the overlap and interaction that occurs between our proposed categories and discuss the complicated nature of the involved criminal networks as well as predictions as to the future of these networks

A digital waveguide-based approach for Clavinet modeling and synthesis

The Clavinet is an electromechanical musical instrument produced in the mid-twentieth century. As is the case for other vintage instruments, it is subject to aging and requires great effort to be maintained or restored. This paper reports analyses conducted on a Hohner Clavinet D6 and proposes a computational model to faithfully reproduce the Clavinet sound in real time, from tone generation to the emulation of the electronic components. The string excitation signal model is physically inspired and represents a cheap solution in terms of both computational resources and especially memory requirements (compared, e.g., to sample playback systems). Pickups and amplifier models have been implemented which enhance the natural character of the sound with respect to previous work. A model has been implemented on a real-time software platform, Pure Data, capable of a 10-voice polyphony with low latency on an embedded device. Finally, subjective listening tests conducted using the current model are compared to previous tests showing slightly improved results

Two Structures of a Thiazolinyl Imine Reductase from Yersinia enterocolitica Provide Insight into Catalysis and Binding to the Nonribosomal Peptide Synthetase Module of HMWP1

The thiazolinyl imine reductase from Yersinia enterocolitica (Irp3) catalyzes the NADPH-dependent reduction of a thiazoline ring in an intermediate for the formation of the siderophore yersiniabactin. Two structures of Irp3 were determined in the apo- (1.85 Å) and NADP+-bound (2.31 Å) forms. Irp3 shows structural homology to sugar oxidoreductases such as glucose-fructose oxidoreductase and 1,5-anhydro-D-fructose reductase, as well as to biliverdin reductase. A homology model of the thiazolinyl imine reductase from Pseudomonas aeruginosa (PchG) was generated. Extensive loop insertions are observed in the C-terminal domain that are unique to Irp3 and PchG and not found in the structural homologs that recognize small molecular substrates. These loops are hypothesized to be important for binding of the nonribosomal peptide synthetase modules (found in HMWP1 and PchF, respectively) to which the substrate of the reductase is covalently attached. A catalytic mechanism of proton donation from a general acid (either histidine-101 or tyrosine-128) and hydride donation from C4 of nicotinamide of the NADPH cofactor is proposed for reduction of the carbon-nitrogen double bond of the thiazoline

A Topical Microbicide Gel Formulation of CCR5 Antagonist Maraviroc Prevents HIV-1 Vaginal Transmission in Humanized RAG-hu Mice

For prevention of HIV infection many currently licensed anti-HIV drugs and new ones in the pipeline show potential as topically applied microbicides. While macaque models have been the gold standard for in vivo microbicide testing, they are expensive and sufficient numbers are not available. Therefore, a small animal model that facilitates rapid evaluation of potential candidates for their preliminary efficacy is urgently needed in the microbicide field. We previously demonstrated that RAG-hu humanized mouse model permits HIV-1 mucosal transmission via both vaginal and rectal routes and that oral pre-exposure chemo-prophylactic strategies could be tested in this system. Here in these proof-of-concept studies, we extended this system for topical microbicide testing using HIV-1 as the challenge virus. Maraviroc, a clinically approved CCR5 inhibitor drug for HIV treatment, was formulated as a microbicide gel at 5 mM concentration in 2.2% hydroxyl ethyl cellulose. Female RAG-hu mice were challenged vaginally with HIV-1 an hour after intravaginal application of the maraviroc gel. Our results showed that maraviroc gel treated mice were fully protected against vaginal HIV-1 challenge in contrast to placebo gel treated mice which all became infected. These findings highlight the utility of the humanized mouse models for microbicide testing and, together with the recent data from macaque studies, suggest that maraviroc is a promising candidate for future microbicide clinical trials in the field

Multifaceted SlyD from Helicobacter pylori: implication in [NiFe] hydrogenase maturation

SlyD belongs to the FK506-binding protein (FKBP) family with both peptidylprolyl isomerase (PPIase) and chaperone activities, and is considered to be a ubiquitous cytosolic protein-folding facilitator in bacteria. It possesses a histidine- and cysteine-rich C-terminus binding to selected divalent metal ions (e.g., Ni2+, Zn2+), which is important for its involvement in the maturation processes of metalloenzymes. We have determined the solution structure of C-terminus-truncated SlyD from Helicobacter pylori (HpSlyDΔC). HpSlyDΔC folds into two well-separated, orientation-independent domains: the PPIase-active FKBP domain and the chaperone-active insert-in-flap (IF) domain. The FKBP domain consists of a four-stranded antiparallel β-sheet with an α-helix on one side, whereas the IF domain folds into a four-stranded antiparallel β-sheet accompanied by a short α-helix. Intact H. pylori SlyD binds both Ni2+ and Zn2+, with dissociation constants of 2.74 and 3.79 μM respectively. Intriguingly, binding of Ni2+ instead of Zn2+ induces protein conformational changes around the active sites of the FKBP domain, implicating a regulatory role of nickel. The twin-arginine translocation (Tat) signal peptide from the small subunit of [NiFe] hydrogenase (HydA) binds the protein at the IF domain. Nickel binding and the recognition of the Tat signal peptide by the protein suggest that SlyD participates in [NiFe] hydrogenase maturation processes

Structural Analysis of Papain-Like NlpC/P60 Superfamily Enzymes with a Circularly Permuted Topology Reveals Potential Lipid Binding Sites

NlpC/P60 superfamily papain-like enzymes play important roles in all kingdoms of life. Two members of this superfamily, LRAT-like and YaeF/YiiX-like families, were predicted to contain a catalytic domain that is circularly permuted such that the catalytic cysteine is located near the C-terminus, instead of at the N-terminus. These permuted enzymes are widespread in virus, pathogenic bacteria, and eukaryotes. We determined the crystal structure of a member of the YaeF/YiiX-like family from Bacillus cereus in complex with lysine. The structure, which adopts a ligand-induced, “closed” conformation, confirms the circular permutation of catalytic residues. A comparative analysis of other related protein structures within the NlpC/P60 superfamily is presented. Permutated NlpC/P60 enzymes contain a similar conserved core and arrangement of catalytic residues, including a Cys/His-containing triad and an additional conserved tyrosine. More surprisingly, permuted enzymes have a hydrophobic S1 binding pocket that is distinct from previously characterized enzymes in the family, indicative of novel substrate specificity. Further analysis of a structural homolog, YiiX (PDB 2if6) identified a fatty acid in the conserved hydrophobic pocket, thus providing additional insights into possible function of these novel enzymes

Oral Pre-Exposure Prophylaxis by Anti-Retrovirals Raltegravir and Maraviroc Protects against HIV-1 Vaginal Transmission in a Humanized Mouse Model

Sexual HIV-1 transmission by vaginal route is the most predominant mode of viral transmission, resulting in millions of new infections every year. In the absence of an effective vaccine, there is an urgent need to develop other alternative methods of pre-exposure prophylaxis (PrEP). Many novel drugs that are currently approved for clinical use also show great potential to prevent viral sexual transmission when administered systemically. A small animal model that permits rapid preclinical evaluation of potential candidates for their systemic PrEP efficacy will greatly enhance progress in this area of investigation. We have previously shown that RAG-hu humanized mouse model permits HIV-1 mucosal transmission via both vaginal and rectal routes and displays CD4 T cell loss typical to that seen in the human. Thus far systemic PrEP studies have been primarily limited to RT inhibitors exemplified by tenofovir and emtricitabine. In these proof-of-concept studies we evaluated two new classes of clinically approved drugs with different modes of action namely, an integrase inhibitor raltegravir and a CCR5 inhibitor maraviroc as potential systemically administered chemo-prophylactics. Our results showed that oral administration of either of these drugs fully protects against vaginal HIV-1 challenge in the RAG-hu mouse model. Based on these results both these drugs show great promise for further development as orally administered PrEPs