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.

Clinical and mutational investigations of tyrosinemia type II in Northern Tunisia: identification and structural characterization of two novel TAT mutations.

Tyrosinemia type II or Richner-Hanhart Syndrome (RHS) is an autosomal recessive disorder characterized by keratitis, palmoplantar keratosis, mental retardation, and elevated blood tyrosine levels. The disease is due to a deficiency of hepatic cytosolic tyrosine aminotransferase (TATc), an enzyme involved in the tyrosine catabolic pathway. Because of the high rate of consanguinity this disorder seems to be relatively common among the Arab and Mediterranean populations. RHS is characterized by inter and intrafamilial phenotypic variability. A large spectrum of mutations within TATc gene has been shown to be responsible for RHS. In the present study, we report the clinical features and the molecular investigation of RHS in three unrelated consanguineous Tunisian families including 7 patients with confirmed biochemical diagnosis of tyrosinemia type II. Mutation analyses were performed and two novel missense mutations were identified (C151Y) and (L273P) within exon 5 and exon 8, respectively. The 3D-structural characterization of these mutations provides evidence of defective folding of the mutant proteins, and likely alteration of the enzymatic activity. Phenotype variability was observed even among individuals sharing the same pathogenic mutation.

The three-dimensional structure of the HRDC domain and implications for the Werner and Bloom syndrome proteins.

BACKGROUND: The HRDC (helicase and RNaseD C-terminal) domain is found at the C terminus of many RecQ helicases, including the human Werner and Bloom syndrome proteins. RecQ helicases have been shown to unwind DNA in an ATP-dependent manner. However, the specific functional roles of these proteins in DNA recombination and replication are not known. An HRDC domain exists in both of the human RecQ homologues that are implicated in human disease and may have an important role in their function. RESULTS: We have determined the three-dimensional structure of the HRDC domain in the Saccharomyces cerevisiae RecQ helicase Sgs1p by nuclear magnetic resonance (NMR) spectroscopy. The structure resembles auxiliary domains in bacterial DNA helicases and other proteins that interact with nucleic acids. We show that a positively charged region on the surface of the Sgs1p HRDC domain can interact with DNA. Structural similarities to bacterial DNA helicases suggest that the HRDC domain functions as an auxiliary domain in RecQ helicases. Homology models of the Werner and Bloom HRDC domains show different surface properties when compared with Sgs1p. CONCLUSIONS: The HRDC domain represents a structural scaffold that resembles auxiliary domains in proteins that are involved in nucleic acid metabolism. In Sgs1p, the HRDC domain could modulate the helicase function via auxiliary contacts to DNA. However, in the Werner and Bloom syndrome helicases the HRDC domain may have a role in their functional differences by mediating diverse molecular interactions.

The three-dimensional structure of a type I module from titin: a prototype of intracellular fibronectin type III domains.

BACKGROUND: Titin is a huge protein ( approximately 3 MDa) that is present in the contractile unit (sarcomere) of striated muscle and has a key role in muscle assembly and elasticity. Titin is mainly composed of two types of module (type I and II). Type I modules are found exclusively in the region of titin localised in the A band, where they are arranged in a super-repeat pattern that correlates with the ultrastructure of the thick filament. No structure of a titin type I module has been reported so far. RESULTS: We have determined the structure of a representative type I module, A71, using nuclear magnetic resonance (NMR) spectroscopy. The structure has the predicted fibronectin type III fold. Titin-specific conserved residues are either located at the putative module-module interfaces or along one side of the protein surface. Several proline residues that contribute to two stretches in a polyproline II helix conformation are solvent-exposed and line up as a continuous ribbon extending over more than two-thirds of the module surface. Homology models of the type I module N-terminal to A71 (A70) and the double module A70-A71 were used to discuss possible intermodule interactions and their role in module-module orientation. CONCLUSIONS: As residues at the module-module interfaces are highly conserved, we speculate that similar interactions govern all of the interfaces between type I modules in titin. This conservation would lead to a regular multiple array of similar surface structures. Such an arrangement would allow arrays of contiguous type I modules to expose multiple proline stretches in a highly regular way and these may act as binding sites for other thick filament proteins.

The structure of a novel insecticidal neurotoxin, omega-atracotoxin-HV1, from the venom of an Australian funnel web spider.

A family of potent insecticidal toxins has recently been isolated from the venom of Australian funnel web spiders. Among these is the 37-residue peptide omega-atracotoxin-HV1 (omega-ACTX-HV1) from Hadronyche versuta. We have chemically synthesized and folded omega-ACTX-HV1, shown that it is neurotoxic, ascertained its disulphide bonding pattern, and determined its three-dimensional solution structure using NMR spectroscopy. The structure consists of a solvent-accessible beta-hairpin protruding from a disulphide-bonded globular core comprising four beta-turns. The three intramolecular disulphide bonds from a cystine knot motif similar to that seen in several other neurotoxic peptides. Despite limited sequence identity, omega-ACTX-HV1 displays significant structural homology with the omega-agatoxins and omega-conotoxins, both of which are vertebrate calcium channel antagonists; however, in contrast with these toxins, we show that omega-ACTX-HV1 inhibits insect, but not mammalian, voltage-gated calcium channel currents.

High resolution NMR solution structure of the leucine zipper domain of the c-Jun homodimer.

The solution structure of the c-Jun leucine zipper domain has been determined to high resolution using a new calculation protocol designed to handle highly ambiguous sets of interproton distance restraints. The domain comprises a coiled coil of parallel alpha-helices in which most of the hydrophobic residues are buried at the highly symmetrical dimer interface; this interface extends over 10 helical turns and is the most elongated protein domain solved to date using NMR methods. The backbone fold is very similar to that seen in crystal structures of the GCN4 and Jun-Fos leucine zippers; however, in contrast with these crystal structures, the Jun leucine zipper dimer appears to be devoid of favorable intermolecular electrostatic interactions. A polar asparagine residue, located at the dimer interface, forms the sole point of asymmetry in the structure; furthermore, the side chain of this residue is disordered due to motional averaging. This residue, which is highly conserved in the leucine zipper family of transcription factors, provides a destabilizing influence that is likely to facilitate the rapid exchange of zipper strands in vivo.

The leucine zippers of the HLH-LZ proteins Max and c-Myc preferentially form heterodimers.

c-Myc and Max are members of a subfamily of the helix-loop-helix transcription-regulating proteins. Their function is mediated by switches in the dimerization partners; c-Myc does not homodimerize in vivo but competes with Mad, another member of the subfamily, to form heterodimers with Max, leading to either activation or repression of transcription. Max is also able to form homodimers. In an attempt to identify which regions of the proteins carry the information to determine specific recognition of the dimerization partner, we have investigated the dimerization properties of synthetic peptides corresponding to the leucine zipper sequence of Max and c-Myc using circular dichroism and nuclear magnetic resonance techniques. We show that the heterodimer is obtained readily by simply mixing the peptides and that at neutral pH it is more stable than the homodimer of the Max leucine zipper. We have shown in a previous paper [Muhle-Goll, C. et al. (1994) Biochemistry 33, 11296-11306] that the leucine zipper of c-Myc does not form stable homodimers under these conditions. Thus, the leucine zipper regions of these two proteins by themselves display the same behavior as the entire proteins. However, even the heterodimer is less stable than dimers of leucine zippers of the basic leucine zipper family such as GCN4 and Fos-Jun. The specificity of the interaction between different monomers can be explained by polar interactions. We investigate the structural role of the polar and charged residues in the hydrophobic interface by molecular-modeling studies.

Calculation of protein structures with ambiguous distance restraints. Automated assignment of ambiguous NOE crosspeaks and disulphide connectivities.

The distances derived from nuclear Overhauser effect (NOE) spectra are usually converted into three-dimensional structures by computer algorithms loosely termed distance geometry. To a varying degree, these methods require that the distance data is unambiguously assigned to pairs of atoms. Typically, however, there are many NOE crosspeaks that cannot be assigned without some knowledge of the structure. These crosspeaks have to be assigned in an iterative manner, using preliminary structures calculated from the unambiguous crosspeaks. In this paper, I present an alternative to this iterative approach. The ambiguity of an NOE crosspeak is correctly described in terms of the distances between all pairs of protons that may be involved. A simple restraining term is defined in terms of "ambiguous" distance restraints that can allow all possible assignments. A new minimization procedure based on simulated annealing is described that is capable of using highly ambiguous data for ab initio structure calculations. In particular, it is feasible to specify the restraint list directly in terms of the proton chemical shift assignment and the NOE peak table, without having assigned NOE crosspeaks to proton pairs. While the primary aim of this paper is determining the global fold of proteins from NMR data, similar strategies can be used for other types of ambiguous distance data. The application to one example, disulphide bridges with unknown connectivity, is described. Model NOE data were generated from the X-ray crystal structure of a small protein with known chemical shift assignments. Varying degrees of ambiguity in the data were assumed. The method obtained the correct polypeptide fold even when all distance restraints were ambiguous. Thus, the new approach may facilitate structure calculations with data derived from very overlapped spectra. It is also a step towards automating the calculation of structures from NMR data. This could prove especially valuable for data derived from three- and four-dimensional experiments. The approach may also prove useful for model building studies and tertiary structure prediction.

The dimerization stability of the HLH-LZ transcription protein family is modulated by the leucine zippers: a CD and NMR study of TFEB and c-Myc.

In the HLH-LZ protein family, the helix-loop-helix DNA-binding dimerization domain is followed in the sequence by a leucine zipper motif. The precise function of this second dimerization domain is still unclear, since the HLH motif of a subset of this family has been shown to be necessary and sufficient for dimerization. However, deletion and mutagenesis studies of the leucine zipper in various HLH-LZ proteins have shown a clear influence of this motif on homo- and heterodimerization. In this paper, we present a structural characterization of synthetic peptides encompassing the leucine zipper sequences of c-Myc and TFEB, using circular dichroism, analytical ultracentrifugation, and nuclear magnetic resonance. We show that the different ability of the synthetic leucine zippers of c-Myc and TFEB to homodimerize at neutral pH reflects the different dimerization properties reported for the entire proteins. The TFEB protein is known to form homodimers. c-Myc, on the other hand, does not homodimerize in vivo, but is mostly found in heterodimeric complexes with Max, another protein of the HLH-LZ family. Accordingly, our results show that the TFEB peptide homodimerizes at neutral pH whereas the Myc peptide dimerizes to a comparable amount only at acidic pH and high ionic strength. Both synthetic peptides are far less stable than leucine zippers of the b-ZIP family. The relative stability of the two leucine zippers and the factors which stabilize the dimer formation are discussed.

A standardized test for the identification and characterization of melanins using electron paramagnetic resonance (EPR) spectroscopy.

Melanins are complex, incompletely understood polymeric pigments that historically have been difficult to investigate with common chemical, histochemical, and physicochemical techniques. Because these pigments uniquely contain a stable population of organic free radicals, electron paramagnetic resonance (EPR) spectroscopy is a particularly effective method for studying them, and a set of qualitative EPR criteria has been established for their identification. However, a number of practical problems have arisen in applying these criteria to identify and characterize unknown pigments in relatively scarce pathological specimens, indicating that a standardized approach is needed. As reported here, a standardized EPR test for melanin based on the EPR criteria has been developed, guided by the requirements that it be sensitive, accurate, simple, and easy to interpret. It has been evaluated using the well-characterized synthetic melanin prepared by alkaline autooxidation of 5,6-dihydroxyphenylalanine (Dopa) and initially applied to the identification and characterization of an unknown pigment purified from an unusual malignant lung tumor.

Conformation of secretin in dimethyl sulfoxide solution. NMR studies and restrained molecular dynamics.

The solution conformation of the 27-residue polypeptide hormone secretin in dimethyl sulfoxide has been determined on the basis of 1H-NMR measurements. The experimental data set used in the structure determination consisted of 98 nuclear-Overhauser-enhancement-derived interproton and dihedral angle restraints from coupling constants. The NH-NH and H alpha-NH NOEs were determined from build-up rates, while the remaining distances were classified in a qualitative manner. The structure calculations consisted of two phases. First, dynamical simulated annealing calculations were carried out to find conformations of the peptide which satisfy NOE and phi dihedral restraints. The convergence of ten calculated structures was good except for those regions of the molecule where NOE data were not unambiguous. From the calculated set another initial structure was built which was again minimized in several 5-ps calculations now employing the full empirical energy function. The resulting structures of secretin reveal conformationally well-defined regions, but not a single uniform secondary structure. The structure is different from the calculated structure from trifluoroethanol/water measurements.

Determination of the three-dimensional solution structure of the C-terminal domain of cellobiohydrolase I from Trichoderma reesei. A study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing.

The solution structure of a synthetic 36-residue polypeptide comprising the C-terminal cellulose binding domain of cellobiohydrolase I (CT-CBH I) from Trichoderma reesei was investigated by nuclear magnetic resonance (NMR) spectroscopy. The 1H NMR spectrum was completely assigned in a sequential manner by two-dimensional NMR techniques. A large number of stereospecific assignments for beta-methylene protons, as well as ranges for the phi, psi, and chi 1 torsion angles, were obtained on the basis of sequential and intraresidue nuclear Overhauser enhancement (NOE) and coupling constant data in combination with a conformational data base search. The structure calculations were carried out in an iterative manner by using the hybrid distance geometry-dynamical simulated annealing method. This involved computing a series of initial structures from a subset of the experimental data in order to resolve ambiguities in the assignments of some NOE cross-peaks arising from chemical shift degeneracy. Additionally, this permitted us to extend the stereospecific assignments to the alpha-methylene protons of glycine using information on phi torsion angles derived from the initial structure calculations. The final experimental data set consisted of 554 interproton distance restraints, 24 restraints for 12 hydrogen bonds, and 33 phi, 24 psi, and 25 chi 1 torsion angle restraints. CT-CBH I has two disulfide bridges whose pairing was previously unknown. Analysis of structures calculated with all three possible combinations of disulfide bonds, as well as without disulfide bonds, indicated that the correct disulfide bridge pairing was 8-25 and 19-35. Forty-one structures were computed with the 8-25 and 19-35 disulfide bridges, and the average atomic rms difference between the individual structures and the mean structure obtained by averaging their coordinates was 0.33 +/- 0.04 A for the backbone atoms and 0.52 +/- 0.06 A for all atoms. The protein has a wedgelike shape with an amphiphilic character, one face being predominantly hydrophilic and the other mainly hydrophobic. The principal element of secondary structure is made up of an irregular triple-stranded antiparallel beta-sheet composed of residues 5-9 (beta 1), 24-28 (beta 2), and 33-36 (beta 3) in which strand beta 3 is hydrogen bonded to the other two strands.(ABSTRACT TRUNCATED AT 400 WORDS)

Three-dimensional structure of acyl carrier protein in solution determined by nuclear magnetic resonance and the combined use of dynamical simulated annealing and distance geometry.

The solution conformation of acyl carrier protein from Escherichia coli (77 residues) has been determined on the basis of 423 interproton-distance restraints and 32 hydrogen-bonding restraints derived from NMR measurements. A total of nine structures were computed using a hybrid approach combining metric matrix distance geometry and dynamic simulated annealing. The polypeptide fold is well defined with an average backbone atomic root-mean-square difference of 0.20 +/- 0.03 nm between the final nine converged structures and the mean structure obtained by averaging their coordinates. The principal structural motif is composed of three helices: 1 (residues 3-12), 2 (residues 37-47) and 4 (residues 65-75) which line a hydrophobic cavity. Helices 2 and 4 are approximately parallel to each other and anti-parallel at an angle of approximately equal to 150 degrees to helix 1. The smaller helix 3 (residues 56-63) is at an angle of approximately equal to 100 degrees to helix 4.

Determination of three-dimensional structures of proteins from interproton distance data by hybrid distance geometry-dynamical simulated annealing calculations.

A new hybrid distance space-real space method for determining three-dimensional structures of proteins on the basis of interproton distance restraints is presented. It involves the following steps: (i) the approximate polypeptide fold is obtained by generating a set of substructures comprising only a small subset of atoms by projection from multi-dimensional distance space into three-dimensional cartesian coordinate space using a procedure known as 'embedding'; (ii) all remaining atoms are then added by best fitting extended amino acids one residue at a time to the substructures; (iii) the resulting structures are used as the starting point for real space dynamical simulated annealing calculations. The latter involve heating the system to a high temperature followed by slow cooling in order to overcome potential barriers along the pathway towards the global minimum region. This is carried out by solving Newton's equations of motion. Unlike conventional restrained molecular dynamics, however, the non-bonded interactions are represented by a simple van der Waals repulsion term. The method is illustrated by calculations on crambin (46 residues) and the globular domain of histone H5 (79 residues). It is shown that the hybrid method is more efficient computationally and samples a larger region of conformational space consistent with the experimental data than full metric matrix distance geometry calculations alone, particularly for large systems.

Determination of the backbone conformation of secretin by restrained molecular dynamics on the basis of interproton distance data.

The backbone conformation of the 27-residue polypeptide hormone secretin has been investigated using nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations under conditions where it adopts a fully ordered structure (40% v/v trifluoroethanol). The basis for the restrained molecular dynamics calculations consists of 52 nuclear-Overhauser-enhancement-derived interproton distance restraints involving the NH, C alpha H and C beta H protons. It is shown that convergence to similar extended structures is achieved starting from four different initial structures, namely an alpha helix, a mixed alpha/beta structure, a beta strand and a polyproline helix. The converged structures are made up of short N- and C-terminal strand-like regions and a central region comprising two irregular helices connected by a 'half-turn'.

The polypeptide fold of the globular domain of histone H5 in solution. A study using nuclear magnetic resonance, distance geometry and restrained molecular dynamics.

The polypeptide fold of the 79-residue globular domain of chicken histone H5 (GH5) in solution has been determined by the combined use of distance geometry and restrained molecular dynamics calculations. The structure determination is based on 307 approximate interproton distance restraints derived from n.m.r. measurements. The structure is composed of a core made up of residues 3-18, 23-34, 37-60 and 71-79, and two loops comprising residues 19-22 and 61-70. The structure of the core is well defined with an average backbone atomic r.m.s. difference of 2.3 +/- 0.3 A between the final eight converged restrained dynamics structures and the mean structure obtained by averaging their coordinates best fitted to the core residues. The two loops are also well defined locally but their orientation with respect to the core could not be determined as no long range ([i-j[ greater than 5) proton-proton contacts could be observed between the loop and core residues in the two-dimensional nuclear Overhauser enhancement spectra. The structure of the core is dominated by three helices and has a similar fold to the C-terminal DNA binding domain of the cAMP receptor protein.

A comparison of the restrained molecular dynamics and distance geometry methods for determining three-dimensional structures of proteins on the basis of interproton distances.

A direct comparison of the metric matrix distance geometry and restrained molecular dynamics methods for determining three-dimensional structures of proteins on the basis of interproton distances is presented using crambin as a model system. It is shown that both methods reproduce the overall features of the secondary and tertiary structure (shape and polypeptide fold). The region of conformational space sampled by the converged structures generated by the two methods is similar in size, and in both cases the converged structures are distributed about mean structures which are closer to the X-ray structure than any of the individual structures. The restrained molecular dynamics structures are superior to those obtained from distance geometry as regards local backbone conformation, side chain positions and non-bonding energies.

Studies on human uterine cervix and rat uterus using S-, X- and Q-band electron-spin-resonance spectroscopy.

In previous studies we have reported on the detection of a strong e.s.r. signal in samples of normal human cervix; the signal is much reduced or absent in samples of invasive cancer of the cervix. In order to identify the species responsible for the strong signal, we have used X-, S- and Q-band e.s.r. spectroscopy. The major signal that is detectable in ground-up samples of cervix preserved at -196 degrees C has features consistent with the presence of a peroxy free radical. Good agreement with the experimental findings was obtained by computer simulation, using values for the g-tensor of gx = 2.002, gy = 2.005 and gz = 2.036. The peroxy radical is produced on grinding the normal cervix samples to a powder under liquid N2, and appears to be formed by modification of a pre-existing oxygen-containing complex. Control experiments eliminated the possibility that the strong signals seen in frozen powders prepared from normal cervix were artefacts only of the grinding procedure. Experiments with rats in vivo and with cervix samples in vitro are consistent with the conclusion that the peroxy radical is formed by disturbing the cyclo-oxygenase system that is involved in prostaglandin synthesis.

Identification by electron spin resonance of free radicals formed during the oxidation of 4-hydroxyanisole catalyzed by tyrosinase.

We have observed the formation of free radicals during the oxidation of the melanocytotoxic agent 4-hydroxyanisole with the enzyme tyrosinase as a catalyst. The first free radical to form is identified as the 4-methoxy-1,2-benzosemiquinone radical anion. The peak concentration of this radical increases with tyrosinase concentration; a minimum concentration of 50 micrograms/ml of tyrosinase was needed to observe this radical. The peak concentration of this radical is independent of 4-hydroxyanisole concentration. This radical is produced by reverse dismutation of the primary product, 4-methoxy-1,2-benzoquinone and 4-methoxycatechol produced indirectly.