Inhibiting Type VI Secretion System Activity with a Biomimetic Peptide Designed To Target the Baseplate Wedge Complex.

Human health is threatened by bacterial infections that are increasingly resistant to multiple drugs. A recently emerged strategy consists of disarming pathogenic bacteria by targeting and blocking their virulence factors. The type VI secretion system (T6SS) is a widespread secretion nanomachine encoded and employed by pathogenic strains to establish their virulence process during host invasion. Given the conservation of T6SS in several human bacterial pathogens, the discovery of an effective broad-spectrum T6SS virulence blocker represents an attractive target for development of antivirulence therapies. Here, we identified and validated a protein-protein interaction interface, TssK-TssG, as a key factor in the assembly of the T6SS baseplate (BP) complex in the pathogen enteroaggregative Escherichia coli (EAEC). In silico and biochemical studies revealed that the determinants of the interface are broadly conserved among pathogenic species, suggesting a role for this interface as a target for T6SS inhibition. Based on the high-resolution structure of the TssKFGE wedge complex, we rationally designed a biomimetic cyclic peptide (BCP) that blocks the assembly of the EAEC BP complex and inhibits the function of T6SS in bacterial cultures. Our BCP is the first compound completely designed from prior structural knowledge with anti-T6SS activity that can be used as a model to target human pathogens. IMPORTANCE New therapeutic options are urgently needed to fight drug-resistant and life-threatening infections. In contrast to antibiotics that inhibit the growth pathways of bacteria, the antivirulence strategy is a promising approach to disarm pathogens by interfering with bacterial virulence factors without exerting evolutionary pressure. The type VI secretion system (T6SS) is used by many pathogens, including members of the antibiotic-resistant ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), to establish their virulence during the invasion of the human host. Although the T6SS is undoubtedly involved in pathogenesis, strategies targeting this virulence factor are crucially lacking. Here, we used a combination of genetics, microbiology, biochemical, biophysics, and bioinformatics approaches to rationally design a biomimetic peptide that interferes with T6SS assembly and functioning. This study represents a novel proof of concept for an antivirulence strategy which aims to interfere with the assembly of the T6SS.

Pharmacological inhibition of syntenin PDZ2 domain impairs breast cancer cell activities and exosome loading with syndecan and EpCAM cargo.

Exosomes support cell-to-cell communication in physiology and disease, including cancer. We currently lack tools, such as small chemicals, capable of modifying exosome composition and activity in a specific manner. Building on our previous understanding of how syntenin, and its PDZ partner syndecan (SDC), impact on exosome composition we optimized a small chemical compound targeting the PDZ2 domain of syntenin. In vitro , in tests on MCF-7 breast carcinoma cells, this compound is non-toxic and impairs cell proliferation, migration and primary sphere formation. It does not affect the size or the number of secreted particles, yet it decreases the amounts of exosomal syntenin, ALIX and SDC4 while leaving other exosomal markers unaffected. Interestingly, it also blocks the sorting of EpCAM, a bona fide target used for carcinoma exosome immunocapture. Our study highlights the first characterization of a small pharmacological inhibitor of the syntenin-exosomal pathway, of potential interest for exosome research and oncology.

Conotoxins and structural biology: a prospective paradigm for drug discovery.

Understanding the interactions between activating or antagonizing ligands and their cognate receptors at a molecular level offers promise for the development of pharmacological therapeutics for CNS disorders. The discovery of novel molecules that are capable of discriminating between the varied molecular subunits or isoforms of ion channels should provide a more detailed understanding of the pathophysiology of many CNS disorders. Abundant natural sources of pharmacologically active agents that demonstrate this refined selectivity and specificity are found in the animal toxins of venomous species including: snakes, spiders and the marine snail of the genus Conus. The uniquely fascinating combinatorial ability of the marine snail, genus Conus to modify the pharmacological properties of these neurotoxins or conopeptides within its venom is depicted throughout this review. The myriad of posttranslational modifications and disulfide bonded architectures that have been identified in the conopeptides, are described with an emphasis on the unique pharmacological properties and receptor target specificities that have been ascribed to each of these modifications. The ability of NMR spectroscopy to provide three-dimensional structural information within the interaction interface for both the ligand and target protein following complex formation and its application to conopeptide drug discovery are discussed. Similarly, the strength of merging NMR spectroscopy data with ab initio "restrained soft-docking" for rational pharmacophore design and the identification of lead compounds from in silico library screens will also be discussed. The initial phases of this stratagem are illustrated using two toxin antagonists and the recently determined structure of the KcsA potassium channel. These data exemplify the utility of this approach in elucidating important molecular interfaces of specific toxin-receptor/ion channel complexes, which can be further exploited in drug discovery initiatives.

A novel approach for assessing macromolecular complexes combining soft-docking calculations with NMR data.

We present a novel and efficient approach for assessing protein-protein complex formation, which combines ab initio docking calculations performed with the protein docking algorithm BiGGER and chemical shift perturbation data collected with heteronuclear single quantum coherence (HSQC) or TROSY nuclear magnetic resonance (NMR) spectroscopy. This method, termed "restrained soft-docking," is validated for several known protein complexes. These data demonstrate that restrained soft-docking extends the size limitations of NMR spectroscopy and provides an alternative method for investigating macromolecular protein complexes that requires less experimental time, effort, and resources. The potential utility of this novel NMR and simulated docking approach in current structural genomic initiatives is discussed.

Alteration of a single tryptophan residue of the cellulose-binding domain blocks secretion of the Erwinia chrysanthemi Cel5 cellulase (ex-EGZ) via the type II system.

Cel5 (formerly known as endoglucanase Z) of Erwinia chrysanthemi is secreted by the Out type II pathway. Previous studies have shown that the catalytic domain (CD), linker region (LR) and cellulose-binding domain (CBD) each contain information needed for secretion. The aim of this work was to further investigate the secretion-related information present in the CBD(Cel5). Firstly(, )deleting a surface-exposed flexible loop had no effect on secretion. This indicated that some structural freedom is tolerated by the type II system. Secondly, mutation of a single tryptophan residue, previously shown to be important for binding to cellulose, i.e. Trp43, was found also to impair secretion. This indicated that the flat cellulose-binding surface of CBD(Cel5 )contains secretion-related information. Thirdly, CBD(Cel5) was substituted by the CBD(EGG) of Alteromonas haloplanctis endoglucanase G, yielding a hybrid protein CD(Cel5)-LR(Cel5)-CBD(EGG) that exhibited 90 % identity with Cel5, including the Trp43 residue. The hybrid protein was not secreted. This indicated that the Trp43 residue is necessary but not sufficient for secretion. Here we propose a model in which the secretion of Cel5 involves a transient intramolecular interaction between the cellulose-binding surface of CBD(Cel5) and a region close to the entry into the active site in CD(Cel5). Once secreted, the protein may then open out to allow the cellulose-binding surface of CBD(Cel5 )to interact with the surface of the cellulose substrate. An implication of this model is that protein molecules fold to a specific secretion-competent conformation prior to secretion that is different from the folding state of the secreted species.

Structural model of the Fe-hydrogenase/cytochrome c553 complex combining transverse relaxation-optimized spectroscopy experiments and soft docking calculations.

Fe-hydrogenase is a 54-kDa iron-sulfur enzyme essential for hydrogen cycling in sulfate-reducing bacteria. The x-ray structure of Desulfovibrio desulfuricans Fe-hydrogenase has recently been solved, but structural information on the recognition of its redox partners is essential to understand the structure-function relationships of the enzyme. In the present work, we have obtained a structural model of the complex of Fe-hydrogenase with its redox partner, the cytochrome c(553), combining docking calculations and NMR experiments. The putative models of the complex demonstrate that the small subunit of the hydrogenase has an important role in the complex formation with the redox partner; 50% of the interacting site on the hydrogenase involves the small subunit. The closest contact between the redox centers is observed between Cys-38, a ligand of the distal cluster of the hydrogenase and Cys-10, a ligand of the heme in the cytochrome. The electron pathway from the distal cluster of the Fe-hydrogenase to the heme of cytochrome c(553) was investigated using the software Greenpath and indicates that the observed cysteine/cysteine contact has an essential role. The spatial arrangement of the residues on the interface of the complex is very similar to that already described in the ferredoxin-cytochrome c(553) complex, which therefore, is a very good model for the interacting domain of the Fe-hydrogenase-cytochrome c(553).

Heteronuclear NMR and soft docking: an experimental approach for a structural model of the cytochrome c553-ferredoxin complex.

The combination of docking algorithms with NMR data has been developed extensively for the studies of protein-ligand interactions. However, to extend this development for the studies of protein-protein interactions, the intermolecular NOE constraints, which are needed, are more difficult to access. In the present work, we describe a new approach that combines an ab initio docking calculation and the mapping of an interaction site using chemical shift variation analysis. The cytochrome c553-ferredoxin complex is used as a model of numerous electron-transfer complexes. The 15N-labeling of both molecules has been obtained, and the mapping of the interacting site on each partner, respectively, has been done using HSQC experiments. 1H and 15N chemical shift analysis defines the area of both molecules involved in the recognition interface. Models of the complex were generated by an ab initio docking software, the BiGGER program (bimolecular complex generation with global evaluation and ranking). This program generates a population of protein-protein docked geometries ranked by a scoring function, combining relevant stabilization parameters such as geometric complementarity surfaces, electrostatic interactions, desolvation energy, and pairwise affinities of amino acid side chains. We have implemented a new module that includes experimental input (here, NMR mapping of the interacting site) as a filter to select the accurate models. Final structures were energy minimized using the X-PLOR software and then analyzed. The best solution has an interface area (1037.4 A2) falling close to the range of generally observed recognition interfaces, with a distance of 10.0 A between the redox centers.

Mapping the cytochrome c553 interacting site using 1H and 15N NMR.

Cytochrome c553 is the electron transfer partner of formate dehydrogenase and of [Fel-hydrogenase, two metalloenzymes essential in the metabolism of sulfate reducing bacteria. These two enzymes contain a 'ferredoxin-like' domain which presents 30% identity with Desulfovibrio desulfuricans Norway ferredoxin 1. This was chosen as a model for the 'ferredoxin-like' domain involved in the electron transfer reaction with cytochrome c553. ID NMR titration of complex formation gave us the stoichiometry (1:1) and the dissociation constant of the complex (Kd approximately 3x10(-6) M). 2D heteronuclear NMR experiments were performed to analyze the 1H and 15N chemical shift variations that are induced by the protein-protein recognition. This is the first mapping of the interaction site on a c-type cytochrome, using heteronuclear NMR.

15N-labelling and preliminary heteronuclear NMR study of Desulfovibrio vulgaris Hildenborough cytochrome c553.

When using heteronuclear NMR, 15N-labelling is necessary for structural analysis, dynamic studies and determination of complex formation. The problems that arise with isotopic labelling of metalloproteins are due to their complex maturation process, which involves a large number of factors. Cytochromes c are poorly expressed in Escherichia coli and the overexpression that is necessary for 15N-labelling, requires an investigation of the expression host and special attention to growth conditions. We have succeeded in the heterologous expression and the complete and uniform isotopic 15N-labelling of the cytochrome c553 from Desulfovibrio vulgaris Hildenborough, in a sulphate-reducing bacterium, D. desulfuricans G200, by using a growth medium combining 15N-ammonium chloride and 15N-Celtone. These conditions allowed us to obtain approximately 0.8 mg x L-1 of pure labelled cytochrome c553. 1H and 15N-assignments for both the oxidized and the reduced states of cytochrome c553 were obtained from two-dimensional heteronuclear experiments. Pseudocontact effects due to the haem Fe3+ have been analysed for the first time through 15N and 1H chemical shifts in a c-type cytochrome.