Bacterial conjugation is one of the main mechanisms for horizontal gene transfer. It constitutes a key element in the dissemination of antibiotic resistance and virulence genes to human pathogenic bacteria. DNA transfer is mediated by a membrane-associated macromolecular machinery called Type IV secretion system (T4SS). T4SSs are involved not only in bacterial conjugation but also in the transport of virulence factors by pathogenic bacteria. Thus, the search for specific inhibitors of different T4SS components opens a novel approach to restrict plasmid dissemination. This review highlights recent biochemical and structural findings that shed new light on the molecular mechanisms of DNA and protein transport by T4SS. Based on these data, a model for pilus biogenesis and substrate transfer in conjugative systems is proposed. This model provides a renewed view of the mechanism that might help to envisage new strategies to curb the threating expansion of antibiotic resistance.
Madanin-1 and chimadanin are two small cysteine-free thrombin inhibitors that facilitate blood feeding in the tick Haemaphysalis longicornis. Here, we report a post-translational modification-tyrosine sulfation-of these two proteins that is critical for potent anti-thrombotic and anticoagulant activity. Inhibitors produced in baculovirus-infected insect cells displayed heterogeneous sulfation of two tyrosine residues within each of the proteins. One-pot ligation-desulfurization chemistry enabled access to homogeneous samples of all possible sulfated variants of the proteins. Tyrosine sulfation of madanin-1 and chimadanin proved crucial for thrombin inhibitory activity, with the doubly sulfated variants three orders of magnitude more potent than the unmodified inhibitors. The three-dimensional structure of madanin-1 in complex with thrombin revealed a unique mode of inhibition, with the sulfated tyrosine residues binding to the basic exosite II of the protease. The importance of tyrosine sulfation within this family of thrombin inhibitors, together with their unique binding mode, paves the way for the development of anti-thrombotic drug leads based on these privileged scaffolds.
Here, we describe the development of a highly efficient one-pot ligationdeselenization technology at aspartate and glutamate that enables the synthesis of polypeptides and proteins on unprecedented timescales. The power of the methodology is showcased through the rapid assembly of three thrombininhibiting tick-derived proteins as well as the synthesis of the 21 kDa homodimeric selenoprotein K. This work lays the foundation for the facile synthesis of a range of bioactive polypeptides and proteins in the future. HIGHLIGHTSRapid one-pot ligationdeselenization at b-selenoaspartate and g-selenoglutamate Methodology enables chemical protein synthesis on unprecedented timescales Synthesis of Selenoprotein K through chemoselective deselenization of b-selenoaspartate Synthesis, purification, and quantification of thrombin inhibitory proteins in 3 hr Mitchell et al., Chem 2, 703-715 May 11, 2017 ª 2017 Elsevier Inc. http://dx. SUMMARYPeptide ligation chemistry has revolutionized protein science by facilitating access to synthetic proteins. Here, we describe the development of additive-free ligation-deselenization chemistry at b-selenoaspartate and g-selenoglutamate that enables the generation of native polypeptide products on unprecedented timescales. The deselenization step is chemoselective in the presence of unprotected selenocysteine, which is highlighted in the synthesis of selenoprotein K. The power of the methodology is also showcased through the synthesis of three tick-derived thrombin-inhibiting proteins, each of which were assembled, purified, and isolated for biological assays within a few hours. The methodology described here should serve as a powerful means of accessing synthetic proteins, including therapeutic leads, in the future.
Summary Bacterial conjugation is the main mechanism responsible for the dissemination of antibiotic resistance genes. Hence, the search for specific conjugation inhibitors is paramount in the fight against the spread of these genes. In this pursuit, unsaturated fatty acids have been found to specifically inhibit bacterial conjugation. Despite the growing interest on these compounds, their mode of action and their specific target remain unknown. Here, we identified TrwD, a Type IV secretion traffic ATPase, as the molecular target for fatty acid-mediated inhibition of conjugation. Moreover, 2-alkynoic fatty acids, which are also potent inhibitors of bacterial conjugation, are also powerful inhibitors of the ATPase activity of TrwD. Characterization of the kinetic parameters of ATPase inhibition has led us to identify the catalytic mechanism by which fatty acids exert their activity. These results open a new avenue for the rational design of inhibitors of bacterial conjugation in the fight against the dissemination of antibiotic resistance genes.
Pilus biogenesis and substrate transport by type IV secretion systems require energy, which is provided by three molecular motors localized at the base of the secretion channel. One of these motors, VirB11, belongs to the superfamily of traffic ATPases, which includes members of the type II secretion system and the type IV pilus and archaeal flagellar assembly apparatus. Here, we report the functional interactions between TrwD, the VirB11 homolog of the conjugative plasmid R388, and TrwK and TrwB, the motors involved in pilus biogenesis and DNA transport, respectively. Although these interactions remained standing upon replacement of the traffic ATPase by a homolog from a phylogenetically related conjugative system, namely, TraG of plasmid pKM101, this homolog could not replace the TrwD function for DNA transfer. This result suggests that VirB11 works as a switch between pilus biogenesis and DNA transport and reinforces a mechanistic model in which VirB11 proteins act as traffic ATPases by regulating both events in type IV secretion systems. Type IV secretion systems (T4SSs) export proteins and virulence effectors to other bacteria and eukaryotic cells (1-3). They are also responsible for genetic exchange between bacteria during conjugation (4, 5). T4SSs are large macromolecular assemblies formed by 12 different protein subunits, named VirB1 to VirB11 and VirD4, following the Agrobacterium tumefaciens T4SS nomenclature. Three of these proteins (VirD4, VirB4, and VirB11) are hexameric ATPases (6-8) that provide the energy for substrate transport and T4SS biogenesis.VirB11 proteins are traffic ATPases that belong to a large AAA ϩ secretion protein superfamily, which also includes proteins of type II secretion systems and the type IV pilus biogenesis and archaeal flagellar assembly machineries (9). All of them are soluble, hexameric proteins located at the cytoplasmic side of the inner membrane. The monomer is characterized by the presence of an N-terminal domain (NTD) and a C-terminal domain (CTD), which are connected by a flexible linker that plays a key role in the catalytic cycle (10-12). Comparison of the crystal structures of VirB11 from Brucella suis (13) and its homolog in Helicobacter pylori, HP0525 (10,14), revealed that this linker is responsible for a large domain swap of the NTD over the CTD without affecting the hexameric assembly (13).VirB11 was reported to assist VirB4 during pilus biogenesis by dislocating pilin subunits from the inner membrane to the periplasmic space, thus promoting pilus polymerization (15). VirB4 proteins are the largest and most conserved components of T4SSs. TrwK, the VirB4 homolog of plasmid R388, consists of a hexameric double ring with a barrel-shaped structure (16). The atomic structure of the C-terminal domain of the VirB4 homolog in Thermoanaerobacter pseudethanolicus was recently obtained (17), revealing a striking structural similarity with TrwB, the VirD4 homolog of the R388 plasmid. TrwB was extensively characterized as a DNA-dependent ATPase (6, 18), playing an ess...
The anophelins are small protein thrombin inhibitors that are produced in the salivary glands of the Anopheles mosquito to fulfill a vital role in blood feeding. A bioinformatic analysis of anophelin sequences revealed the presence of conserved tyrosine residues in an acidic environment that were predicted to be post-translationally sulfated in vivo. To test this prediction, insect cell expression of two anophelin proteins, from Anopheles albimanus and Anopheles gambiae, was performed, followed by analysis by mass spectrometry, which showed heterogeneous sulfation at the predicted sites. Homogeneously sulfated variants of the two proteins were subsequently generated by chemical synthesis via a one-pot ligation–desulfurization strategy. Tyrosine sulfation of the anophelins was shown to significantly enhance the thrombin inhibitory activity, with a doubly sulfated variant of the anophelin from A. albimanus exhibiting a 100-fold increase in potency compared with the unmodified homologue. Sulfated anophelins were also shown to exhibit potent in vivo anticoagulant and antithrombotic activity.
Hematophagous organisms produce a suite of salivary proteins which interact with the host’s coagulation machinery to facilitate the acquisition and digestion of a bloodmeal. Many of these biomolecules inhibit the central blood-clotting serine proteinase thrombin that is also the target of several clinically approved anticoagulants. Here a bioinformatics approach is used to identify seven tick proteins with putative thrombin inhibitory activity that we predict to be posttranslationally sulfated at two conserved tyrosine residues. To corroborate the biological role of these molecules and investigate the effects of amino acid sequence and sulfation modifications on thrombin inhibition and anticoagulant activity, a library of 34 homogeneously sulfated protein variants were rapidly assembled using one-pot diselenide-selenoester ligation (DSL)-deselenization chemistry. Downstream functional characterization validated the thrombin-directed activity of all target molecules and revealed that posttranslational sulfation of specific tyrosine residues crucially modulates potency. Importantly, access to this homogeneously modified protein library not only enabled the determination of key structure–activity relationships and the identification of potent anticoagulant leads, but also revealed subtleties in the mechanism of thrombin inhibition, between and within the families, that would be impossible to predict from the amino acid sequence alone. The synthetic platform described here therefore serves as a highly valuable tool for the generation and thorough characterization of libraries of related peptide and/or protein molecules (with or without modifications) for the identification of lead candidates for medicinal chemistry programs.
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