Structure and dynamic association of an assembly platform subcomplex of the bacterial type II secretion system.

Type II secretion systems (T2SSs) allow diderm bacteria to secrete hydrolytic enzymes, adhesins, or toxins important for growth and virulence. To promote secretion of folded proteins, T2SSs assemble periplasmic filaments called pseudopili or endopili at an inner membrane subcomplex, the assembly platform (AP). Here, we combined biophysical approaches, nuclear magnetic resonance (NMR) and X-ray crystallography, to study the Klebsiella AP components PulL and PulM. We determined the structure and associations of their periplasmic domains and describe the structure of the heterodimer formed by their ferredoxin-like domains. We show how structural complementarity and plasticity favor their association during the secretion process. Cysteine scanning and crosslinking data provided additional constraints to build a structural model of the PulL-PulM assembly in the cellular context. Our structural and functional insights, together with the relative cellular abundance of its components, support the role of AP as a dynamic hub that orchestrates pilus polymerization.

Enzymatic and Molecular Characterization of Anti-Leishmania Molecules That Differently Target Leishmania and Mammalian eIF4A Proteins, LieIF4A and eIF4AMus.

Previous investigations of the Leishmania infantum eIF4A-like protein (LieIF4A) as a potential drug target delivered cholestanol derivatives inhibitors. Here, we investigated the mode of action of cholesterol derivatives as a novel scaffold structure of LieIF4A inhibitors on the RNA-dependent ATPase activity of LieIF4A and its mammalian ortholog (eIF4AI). We compared their biochemical effects on RNA-dependent ATPase activities of both proteins and investigated if rocaglamide, a known inhibitor of eIF4A, could affect LieIF4A as well. Kinetic measurements were conducted at different concentrations of ATP, of the compound and in the presence of saturating whole yeast RNA concentrations. Kinetic analyses showed different ATP binding affinities for the two enzymes as well as different sensitivities to 7-α-aminocholesterol and rocaglamide. The 7-α-aminocholesterol inhibited LieIF4A with a higher binding affinity relative to cholestanol analogs. Cholesterol, another tested sterol, had no effect on the ATPase activity of LieIF4A or eIF4AI. The 7-α-aminocholesterol demonstrated an anti-Leishmania activity on L. infantum promastigotes. Additionally, docking simulations explained the importance of the double bond between C5 and C6 in 7-α-aminocholesterol and the amino group in the C7 position. In conclusion, Leishmania and mammalian eIF4A proteins appeared to interact differently with effectors, thus making LieIF4A a potential drug against leishmaniases.

Secondary structure and 1H, 15 N & 13C resonance assignments of the periplasmic domain of OutG, major pseudopilin from Dickeya dadantii type II secretion system.

The ability to interact and adapt to the surrounding environment is vital for bacteria that colonise various niches and organisms. One strategy developed by Gram-negative bacteria is to secrete exoprotein substrates via the type II secretion system (T2SS). The T2SS is a proteinaceous complex spanning the bacterial envelope that translocates folded proteins such as toxins and enzymes from the periplasm to the extracellular milieu. In the T2SS, a cytoplasmic ATPase elongates in the periplasm the pseudopilus, a non-covalent polymer composed of protein subunits named pseudopilins, and anchored in the inner membrane by a transmembrane helix. The pseudopilus polymerisation is coupled to the secretion of substrates. The T2SS of Dickeya dadantii secretes more than 15 substrates, essentially plant cell wall degrading enzymes. In D. dadantii, the major pseudopilin or the major subunit of the pseudopilus is called OutG. To better understand the mechanism of secretion of these numerous substrates via the pseudopilus, we have been studying the structure of OutG by NMR. Here, as the first part of this study, we report the 1H, 15N and 13C backbone and sidechain chemical shift assignment of the periplasmic domain of OutG and its NMR derived secondary structure.

Sequence-specific DNA binding activity of the cross-brace zinc finger motif of the piggyBac transposase.

The piggyBac transposase (PB) is distinguished by its activity and utility in genome engineering, especially in humans where it has highly promising therapeutic potential. Little is known, however, about the structure-function relationships of the different domains of PB. Here, we demonstrate in vitro and in vivo that its C-terminal Cysteine-Rich Domain (CRD) is essential for DNA breakage, joining and transposition and that it binds to specific DNA sequences in the left and right transposon ends, and to an additional unexpectedly internal site at the left end. Using NMR, we show that the CRD adopts the specific fold of the cross-brace zinc finger protein family. We determine the interaction interfaces between the CRD and its target, the 5'-TGCGT-3'/3'-ACGCA-5' motifs found in the left, left internal and right transposon ends, and use NMR results to propose docking models for the complex, which are consistent with our site-directed mutagenesis data. Our results provide support for a model of the PB/DNA interactions in the context of the transpososome, which will be useful for the rational design of PB mutants with increased activity.

Identification of novel leishmanicidal molecules by virtual and biochemical screenings targeting Leishmania eukaryotic translation initiation factor 4A.

Leishmaniases are neglected parasitic diseases in spite of the major burden they inflict on public health. The identification of novel drugs and targets constitutes a research priority. For that purpose we used Leishmania infantum initiation factor 4A (LieIF), an essential translation initiation factor that belongs to the DEAD-box proteins family, as a potential drug target. We modeled its structure and identified two potential binding sites. A virtual screening of a diverse chemical library was performed for both sites. The results were analyzed with an in-house version of the Self-Organizing Maps algorithm combined with multiple filters, which led to the selection of 305 molecules. Effects of these molecules on the ATPase activity of LieIF permitted the identification of a promising hit (208) having a half maximal inhibitory concentration (IC50) of 150 ± 15 µM for 1 µM of protein. Ten chemical analogues of compound 208 were identified and two additional inhibitors were selected (20 and 48). These compounds inhibited the mammalian eIF4I with IC50 values within the same range. All three hits affected the viability of the extra-cellular form of L. infantum parasites with IC50 values at low micromolar concentrations. These molecules showed non-significant toxicity toward THP-1 macrophages. Furthermore, their anti-leishmanial activity was validated with experimental assays on L. infantum intramacrophage amastigotes showing IC50 values lower than 4.2 µM. Selected compounds exhibited selectivity indexes between 19 to 38, which reflects their potential as promising anti-Leishmania molecules.

Structural Characterization of Whirlin Reveals an Unexpected and Dynamic Supramodule Conformation of Its PDZ Tandem.

Hearing relies on the transduction of sound-evoked vibrations into electric signals, occurring in the stereocilia bundle of hair cells. The bundle is organized in a staircase pattern formed by rows of packed stereocilia. This architecture is pivotal to transduction and involves a network of scaffolding proteins with hitherto uncharacterized features. Key interactions in this network are mediated by PDZ domains. Here, we describe the architecture of the first two PDZ domains of whirlin, a protein involved in these assemblies and associated with congenital deaf-blindness. C-terminal hairpin extensions of the PDZ domains mediate the transient supramodular assembly, which improves the binding capacity of the first domain. We determined a detailed structural model of the closed conformation of the PDZ tandem and characterized its equilibrium with an ensemble of open conformations. The structural and dynamic behavior of this PDZ tandem provides key insights into the regulatory mechanisms involved in the hearing machinery.

Nicotine reverses hypofrontality in animal models of addiction and schizophrenia.

The prefrontal cortex (PFC) underlies higher cognitive processes that are modulated by nicotinic acetylcholine receptor (nAChR) activation by cholinergic inputs. PFC spontaneous default activity is altered in neuropsychiatric disorders, including schizophrenia-a disorder that can be accompanied by heavy smoking. Recently, genome-wide association studies (GWAS) identified single-nucleotide polymorphisms (SNPs) in the human CHRNA5 gene, encoding the α5 nAChR subunit, that increase the risks for both smoking and schizophrenia. Mice with altered nAChR gene function exhibit PFC-dependent behavioral deficits, but it is unknown how the corresponding human polymorphisms alter the cellular and circuit mechanisms underlying behavior. Here we show that mice expressing a human α5 SNP exhibit neurocognitive behavioral deficits in social interaction and sensorimotor gating tasks. Two-photon calcium imaging in awake mouse models showed that nicotine can differentially influence PFC pyramidal cell activity by nAChR modulation of layer II/III hierarchical inhibitory circuits. In α5-SNP-expressing and α5-knockout mice, lower activity of vasoactive intestinal polypeptide (VIP) interneurons resulted in an increased somatostatin (SOM) interneuron inhibitory drive over layer II/III pyramidal neurons. The decreased activity observed in α5-SNP-expressing mice resembles the hypofrontality observed in patients with psychiatric disorders, including schizophrenia and addiction. Chronic nicotine administration reversed this hypofrontality, suggesting that administration of nicotine may represent a therapeutic strategy for the treatment of schizophrenia, and a physiological basis for the tendency of patients with schizophrenia to self-medicate by smoking.

In Silico prediction of the molecular basis of ClTx and AaCTx interaction with matrix metalloproteinase-2 (MMP-2) to inhibit glioma cell invasion.

Glioblastoma is the deadliest type of brain cancer. Treatment could target the Matrix metalloproteinase-2 (MMP-2), which is known to be involved in the invasion process of glioblastoma cells. But current available inhibitors are not selective to MMP-2 due to their interaction with the catalytic binding site, which is highly conserved in all MMPs structures. Interestingly, members of the chloride channel blocker scorpion toxins, such as chlorotoxin (ClTx) and AaCTx, inhibit glioblastoma cell invasion and show a promising therapeutic potential. Indeed, it has been shown that CITx inhibits selectively MMP-2 and was also able to cross the blood brain and tissue barriers. Although ClTx and AaCTx show high sequence similarity, AaCTx is ten times less active than ClTx. By using molecular modeling, molecular dynamics and MM-PB(GB)SA free energy estimation, we present the first computational study reporting the interaction mode of ClTx/AaCTx with MMP-2. We found that the two peptides probably act on an exosite of MMP-2 comprising mainly residues from the collagen binding domain, a feature that could be exploited to enhance the selectivity toward MMP-2. van der Waals and hydrophobic forces are the primary mediators of this interaction. The N- and C-termini of the two peptides harbor the key residues of the interaction spread across a conserved amino acid patch. In particular, F6 contributes mostly to the binding free energy in ClTx. We also suggest that the lack of the C-terminal arginine and the residues P10 and R24, might be responsible for altering the activity of AaCTx toward glioblastoma cells compared to ClTx.

Improved large-scale prediction of growth inhibition patterns using the NCI60 cancer cell line panel.

MOTIVATION: Recent large-scale omics initiatives have catalogued the somatic alterations of cancer cell line panels along with their pharmacological response to hundreds of compounds. In this study, we have explored these data to advance computational approaches that enable more effective and targeted use of current and future anticancer therapeutics. RESULTS: We modelled the 50% growth inhibition bioassay end-point (GI50) of 17,142 compounds screened against 59 cancer cell lines from the NCI60 panel (941,831 data-points, matrix 93.08% complete) by integrating the chemical and biological (cell line) information. We determine that the protein, gene transcript and miRNA abundance provide the highest predictive signal when modelling the GI50 endpoint, which significantly outperformed the DNA copy-number variation or exome sequencing data (Tukey's Honestly Significant Difference, P <0.05). We demonstrate that, within the limits of the data, our approach exhibits the ability to both interpolate and extrapolate compound bioactivities to new cell lines and tissues and, although to a lesser extent, to dissimilar compounds. Moreover, our approach outperforms previous models generated on the GDSC dataset. Finally, we determine that in the cases investigated in more detail, the predicted drug-pathway associations and growth inhibition patterns are mostly consistent with the experimental data, which also suggests the possibility of identifying genomic markers of drug sensitivity for novel compounds on novel cell lines. CONTACT: terez@pasteur.fr; ab454@ac.cam.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Neisseria meningitidis Type IV Pili Composed of Sequence Invariable Pilins Are Masked by Multisite Glycosylation.

The ability of pathogens to cause disease depends on their aptitude to escape the immune system. Type IV pili are extracellular filamentous virulence factors composed of pilin monomers and frequently expressed by bacterial pathogens. As such they are major targets for the host immune system. In the human pathogen Neisseria meningitidis, strains expressing class I pilins contain a genetic recombination system that promotes variation of the pilin sequence and is thought to aid immune escape. However, numerous hypervirulent clinical isolates express class II pilins that lack this property. This raises the question of how they evade immunity targeting type IV pili. As glycosylation is a possible source of antigenic variation it was investigated using top-down mass spectrometry to provide the highest molecular precision on the modified proteins. Unlike class I pilins that carry a single glycan, we found that class II pilins display up to 5 glycosylation sites per monomer on the pilus surface. Swapping of pilin class and genetic background shows that the pilin primary structure determines multisite glycosylation while the genetic background determines the nature of the glycans. Absence of glycosylation in class II pilins affects pilus biogenesis or enhances pilus-dependent aggregation in a strain specific fashion highlighting the extensive functional impact of multisite glycosylation. Finally, molecular modeling shows that glycans cover the surface of class II pilins and strongly decrease antibody access to the polypeptide chain. This strongly supports a model where strains expressing class II pilins evade the immune system by changing their sugar structure rather than pilin primary structure. Overall these results show that sequence invariable class II pilins are cloaked in glycans with extensive functional and immunological consequences.

Identification of binding sites and favorable ligand binding moieties by virtual screening and self-organizing map analysis.

BACKGROUND: Identifying druggable cavities on a protein surface is a crucial step in structure based drug design. The cavities have to present suitable size and shape, as well as appropriate chemical complementarity with ligands. RESULTS: We present a novel cavity prediction method that analyzes results of virtual screening of specific ligands or fragment libraries by means of Self-Organizing Maps. We demonstrate the method with two thoroughly studied proteins where it successfully identified their active sites (AS) and relevant secondary binding sites (BS). Moreover, known active ligands mapped the AS better than inactive ones. Interestingly, docking a naive fragment library brought even more insight. We then systematically applied the method to the 102 targets from the DUD-E database, where it showed a 90% identification rate of the AS among the first three consensual clusters of the SOM, and in 82% of the cases as the first one. Further analysis by chemical decomposition of the fragments improved BS prediction. Chemical substructures that are representative of the active ligands preferentially mapped in the AS. CONCLUSION: The new approach provides valuable information both on relevant BSs and on chemical features promoting bioactivity.

Computational reverse-engineering of a spider-venom derived peptide active against Plasmodium falciparum SUB1.

BACKGROUND: Psalmopeotoxin I (PcFK1), a protein of 33 aminoacids derived from the venom of the spider Psalmopoeus Cambridgei, is able to inhibit the growth of Plasmodium falciparum malaria parasites with an IC50 in the low micromolar range. PcFK1 was proposed to act as an ion channel inhibitor, although experimental validation of this mechanism is lacking. The surface loops of PcFK1 have some sequence similarity with the parasite protein sequences cleaved by PfSUB1, a subtilisin-like protease essential for egress of Plasmodium falciparum merozoites and invasion into erythrocytes. As PfSUB1 has emerged as an interesting drug target, we explored the hypothesis that PcFK1 targeted PfSUB1 enzymatic activity. FINDINGS: Molecular modeling and docking calculations showed that one loop could interact with the binding site of PfSUB1. The calculated free energy of binding averaged -5.01 kcal/mol, corresponding to a predicted low-medium micromolar constant of inhibition. PcFK1 inhibited the enzymatic activity of the recombinant PfSUB1 enzyme and the in vitro P. falciparum culture in a range compatible with our bioinformatics analysis. Using contact analysis and free energy decomposition we propose that residues A14 and Q15 are important in the interaction with PfSUB1. CONCLUSIONS: Our computational reverse engineering supported the hypothesis that PcFK1 targeted PfSUB1, and this was confirmed by experimental evidence showing that PcFK1 inhibits PfSUB1 enzymatic activity. This outlines the usefulness of advanced bioinformatics tools to predict the function of a protein structure. The structural features of PcFK1 represent an interesting protein scaffold for future protein engineering.

Posttranslational modification of pili upon cell contact triggers N. meningitidis dissemination.

The Gram-negative bacterium Neisseria meningitidis asymptomatically colonizes the throat of 10 to 30% of the human population, but throat colonization can also act as the port of entry to the blood (septicemia) and then the brain (meningitis). Colonization is mediated by filamentous organelles referred to as type IV pili, which allow the formation of bacterial aggregates associated with host cells. We found that proliferation of N. meningitidis in contact with host cells increased the transcription of a bacterial gene encoding a transferase that adds phosphoglycerol onto type IV pili. This unusual posttranslational modification specifically released type IV pili-dependent contacts between bacteria. In turn, this regulated detachment process allowed propagation of the bacterium to new colonization sites and also migration across the epithelium, a prerequisite for dissemination and invasive disease.

A role for specific collagen motifs during wound healing and inflammatory response of fibroblasts in the teleost fish gilthead seabream.

Specific sites and sequences in collagen to which cells can attach, either directly or through protein intermediaries, were identified using Toolkits of 63-amino acid triple-helical peptides and specific shorter GXX'GEX″ motifs, which have different intrinsic affinity for integrins that mediate cell adhesion and migration. We have previously reported that collagen type I (COL-I) was able to prime in vitro the respiratory burst and induce a specific set of immune- and extracellular matrix-related molecules in phagocytes of the teleost fish gilthead seabream (Sparus aurata L.). It was also suggested that COL-I would provide an intermediate signal during the early inflammatory response in gilthead seabream. Since fibroblasts are highly involved in the initiation of wound repair and regeneration processes, in the present study SAF-1 cells (gilthead seabream fibroblasts) were used to identify the binding motifs in collagen by end-point and real-time cell adhesion assays using the collagen peptides and Toolkits. We identified the collagen motifs involved in the early magnesium-dependent adhesion of these cells. Furthermore, we found that peptides containing the GFOGER and GLOGEN motifs (where O is hydroxyproline) present high affinity for SAF-1 adhesion, expressed as both cell number and surface covering, while in cell suspensions, these motifs were also able to induce the expression of the genes encoding the proinflammatory molecules interleukin-1ß and cyclooxygenase-2. These data suggest that specific collagen motifs are involved in the regulation of the inflammatory and healing responses of teleost fish.

Modeling pilus structures from sparse data.

Bacterial Type II secretion systems (T2SS) and type IV pili (T4P) biogenesis machineries share the ability to assemble thin filaments from pilin protein subunits in the plasma membrane. Here we describe in detail the calculation strategy that served to determine a detailed atomic model of the T2SS pilus from Klebsiella oxytoca (Campos et al., PNAS 2010). The strategy is based on molecular modeling with generalized distance restraints and experimental validation (salt bridge charge inversion; double cysteine substitution and crosslinking). It does not require directly fitting structures into an envelope obtained from electron microscopy, but relies on lower resolution information, in particular the symmetry parameters of the helix forming the pilus. We validate the strategy with T4P where either a higher resolution structure is available (for the gonococcal (GC) pilus from Neisseria gonorrhoeae), or where we can compare our results to additional experimental data (for Vibrio cholerae TCP). The models are of sufficient precision to compare the architecture of the different pili in detail.

Detailed structural and assembly model of the type II secretion pilus from sparse data.

Many gram-negative bacteria secrete specific proteins via the type II secretion systems (T2SS). These complex machineries share with the related archaeal flagella and type IV pilus (T4P) biogenesis systems the ability to assemble thin, flexible filaments composed of small, initially inner membrane-localized proteins called "pilins." In the T2SS from Klebsiella oxytoca, periplasmic pseudopili that are essential for pullulanase (PulA) secretion extend beyond the bacterial surface and form pili when the major pilin PulG is overproduced. Here, we describe the detailed, experimentally validated structure of the PulG pilus generated from crystallographic and electron microscopy data by a molecular modeling approach. Two intermolecular salt bridges crucial for function were demonstrated using single and complementary charge inversions. Double-cysteine substitutions in the transmembrane segment of PulG led to position-specific cross-linking of protomers in assembled pili. These biochemical data provided information on residue distances in the filament that were used to derive a refined model of the T2SS pilus at pseudoatomic resolution. PulG is organized as a right-handed helix of subunits, consistent with protomer organization in gonococcal T4P. The conserved character of residues involved in key hydrophobic and electrostatic interactions within the major pseudopilin family supports the general relevance of this model for T2SS pseudopilus structure.

ATP conformations and ion binding modes in the active site of anthrax edema factor: a computational analysis.

The Edema Factor (EF), one of the virulence factors of anthrax, is an adenylyl cyclase that promotes the overproduction of cyclic-AMP (cAMP) from ATP, and therefore perturbs cell signaling. Crystallographic structures of EF bound to ATP analogs and reaction products, cyclic-AMP, and Pyrophosphate (PPi), revealed different substrate conformations and catalytic-cation binding modes, one or two cations being observed in the active site. To shed light into the biological significance of these crystallographic structures, the energetics, geometry, and dynamics of the active site are analyzed using molecular dynamics simulations. The ATP conformation observed in the one-metal-ion structure allows stronger interactions with the catalytic ion, and ATP is more restrained than in the structure containing two Mg(2+) ions. Therefore, we propose that the conformation observed in the one-ion crystal structure is a more probable starting point for the reaction. The simulations also suggest that a C3'-endo sugar pucker facilitates nucleophilic attack. Additionally, the two-cation binding mode restrains the mobility of the reaction products, and thus their tendency to dissociate.

Bayesian estimation of Karplus parameters and torsion angles from three-bond scalar couplings constants.

We apply Bayesian inference to analyze three-bond scalar coupling constants in an objective and consistent way. The Karplus curve and a Gaussian error law are used to model scalar coupling measurements. By applying Bayes' theorem, we obtain a probability distribution for all unknowns, i.e., the torsion angles, the Karplus parameters, and the standard deviation of the Gaussian. We infer all these unknowns from scalar coupling data using Markov chain Monte Carlo sampling and analytically derive a probability distribution that only involves the torsion angles.

Inferential structure determination.

Macromolecular structures calculated from nuclear magnetic resonance data are not fully determined by experimental data but depend on subjective choices in data treatment and parameter settings. This makes it difficult to objectively judge the precision of the structures. We used Bayesian inference to derive a probability distribution that represents the unknown structure and its precision. This probability distribution also determines additional unknowns, such as theory parameters, that previously had to be chosen empirically. We implemented this approach by using Markov chain Monte Carlo techniques. Our method provides an objective figure of merit and improves structural quality.

Conservation of the biochemical properties of IncA from Chlamydia trachomatis and Chlamydia caviae: oligomerization of IncA mediates interaction between facing membranes.

The developmental cycle of Chlamydiaceae occurs in a membrane compartment called an inclusion. IncA is a member of a family of proteins synthesized and secreted onto the inclusion membrane by bacteria. IncA proteins from different species of Chlamydiaceae show little sequence similarity. We report that the biochemical properties of Chlamydia trachomatis and Chlamydia caviae are conserved. Both proteins self-associate to form multimers. When artificially expressed by the host cell, they localize to the endoplasmic reticulum. Strikingly, heterologous expression of IncA in the endoplasmic reticulum completely inhibits concomitant inclusion development. Using truncated forms of IncA from C. caviae, we show that expression of the C-terminal cytoplasmic domain of the protein at the surface of the endoplasmic reticulum is sufficient to disrupt the bacterial developmental cycle. On the other hand, development of a C. trachomatis strain that does not express IncA is not inhibited by artificial IncA expression, showing that the disruptive effect observed with the wild-type strain requires direct interactions between IncA molecules at the inclusion and on the endoplasmic reticulum. Finally, we modeled IncA tetramers in parallel four helix bundles based on the structure of the SNARE complex, a conserved structure involved in membrane fusion in eukaryotic cells. Both C. trachomatis and C. caviae IncA tetramers were highly stable in this model. In conclusion, we show that the property of IncA proteins to assemble into multimeric structures is conserved between chlamydial species, and we propose that these proteins may have co-evolved with the SNARE machinery for a role in membrane fusion.