Teresa E. Lehmann

Listeria monocytogenes exopolysaccharide: origin, structure, biosynthetic machinery and c-di-GMP-dependent regulation

Elevated levels of the second messenger c‐di‐GMP activate biosynthesis of an unknown exopolysaccharide (EPS) in the food‐borne pathogen Listeria monocytogenes. This EPS strongly protects cells against disinfectants and desiccation, indicating its potential significance for listerial persistence in the environment and for food safety. We analyzed the potential phylogenetic origin of this EPS, determined its complete structure, characterized genes involved in its biosynthesis and hydrolysis and identified diguanylate cyclases activating its synthesis. Phylogenetic analysis of EPS biosynthesis proteins suggests that they have evolved within monoderms. Scanning electron microscopy revealed that L. monocytogenes EPS is cell surface‐bound. Secreted carbohydrates represent exclusively cell‐wall debris. Based on carbohydrate composition, linkage and NMR analysis, the structure of the purified EPS is identified as a β‐1,4‐linked N‐acetylmannosamine chain decorated with terminal α‐1,6‐linked galactose. All genes of the pssA‐E operon are required for EPS production and so is a separately located pssZ gene. We show that PssZ has an EPS‐specific glycosylhydrolase activity. Exogenously added PssZ prevents EPS‐mediated cell aggregation and disperses preformed aggregates, whereas an E72Q mutant in the presumed catalytic residue is much less active. The diguanylate cyclases DgcA and DgcB, whose genes are located next to pssZ, are primarily responsible for c‐di‐GMP‐dependent EPS production.

Effects of enzymatically inactive recombinant botulinum neurotoxin type A at the mouse neuromuscular junctions.

Botulinum neurotoxin A (BoNT/A) is used clinically to treat several neurological and metabolic diseases. However, the mechanisms that underlie the clinical use of the toxin remain still to be elusive. BoNT/A inhibits acetylcholine (ACh) release at the motor nerve terminals (MNT) and causes neuroparalysis. The toxic effects of BoNT/A at the MNT occur in sub-pico molar range, and it is invaluable to determine the half-life and the persistence of catalytic activity of the toxin to develop therapeutics against BoNT/A intoxication. However, the use of extremely low concentrations of BoNT/A in cellular, or animal models due to high toxicity makes it difficult to determine new cellular mechanisms and binding or interacting partners of BoNT/A. In order to address this, a catalytically deactivated, non-toxic version of BoNT/A, designated as DrBoNT/A, was characterized. DrBoNT/A lacks endoprotease activity (SNAP-25 cleavage) at concentrations as high as 46,875-fold, compared to wild-type BoNT/A. Unlike BoNT/A injection (3.2 pg), injection of the recombinant product (150 ng or 3.2 pg) into mouse hind limbs failed to cause neuroparalysis as exhibited by the lack of inhibition of toe spread reflex (ability of the mouse to spread its hindlimb toes), and inhibit ACh release at the MNT. The in vitro experiments also demonstrate that DrBoNT/A uptake (at concentrations equivalent to BoNT/A), internalization and localization at the MNT remained unaltered. In addition, modeling studies support that DrBoNT/A lacked the zinc binding ability, and the ability to directly participate in the hydrolysis of SNAP-25 substrate. Collectively, we demonstrate that DrBoNT/A is non-toxic to the MNT and can be used as a surrogate tool to understand the mechanism by which BoNT/A modulates signal transduction mechanisms.

Novel Curcumin Derivative CNB-001 Mitigates Obesity-Associated Insulin Resistance

Type 2 diabetes is growing at epidemic proportions, and pharmacological interventions are being actively sought. This study examined the effect of a novel neuroprotective curcuminoid, CNB-001 [4-((1E)-2-(5-(4-hydroxy-3-methoxystyryl-)-1-phenyl-1H-pyrazoyl-3-yl)vinyl)-2-methoxy-phenol], on glucose intolerance and insulin signaling in high-fat diet (HFD)–fed mice. C57BL6 mice (5–6 weeks old) were randomly assigned to receive either a HFD (45% fat) or a low-fat diet (LFD, 10% fat) for 24 weeks, together with CNB-001 (40 mg/kg i.p. per day). Glucose tolerance test revealed that the area under the curve of postchallenge glucose concentration was elevated on HF-feeding, which was attenuated by CNB-001. CNB-001 attenuated body weight gain, serum triglycerides, and IL-6, and augmented insulin signaling [elevated phosphoprotein kinase B (p-Akt), and phosphoinsulin receptor (p-IR)β, lowered endoplasmic reticulum (ER) stress, protein–tyrosine phosphatase 1B (PTP1B)] and glucose uptake in gastrocnemius muscle of HFD-fed mice. Respiratory quotient, measured using a metabolic chamber, was elevated in HFD-fed mice, which was unaltered by CNB-001, although CNB-001 treatment resulted in higher energy expenditure. In cultured myotubes, CNB-001 reversed palmitate-induced impairment of insulin signaling and glucose uptake. Docking studies suggest a potential interaction between CNB-001 and PTP1B. Taken together, CNB-001 alleviates obesity-induced glucose intolerance and represents a potential candidate for further development as an antidiabetic agent.

Possible structural role of the disaccharide unit in Fe-bleomycin before and after oxygen activation

Our previous investigation of the solution structure of Fe(II)-bleomycin pointed toward the carbamoyl group in the mannose moiety or a water molecule as possible alternative axial ligands to the metal center in this metallo-bleomycin. The possibility of a solvent molecule occupying the apical position trans to the primary amine has not been ruled out yet. In order to explore this possibility even further, the coordination chemistry of azide-bound Fe(II)-bleomycin was investigated with the use of NMR applied to paramagnetic molecules. Fe(II)- and apo-bleomycin were also re-visited. Comparison of the NMR results for both Fe(II)-bound molecules obtained in the present study strongly suggests that the carbamoyl oxygen is ligated to Fe(II), and it is released from coordination upon azide binding. This event is suggested based on the diminished paramagnetic character exhibited by the carbohydrate moiety in Fe(II)-azide-bleomycin when compared with its parent metal complex. A possible structural role for the glucopyranose fragment, which changes throughout the process that starts with metallo-bleomycin formation and ends with DNA binding, is discussed. The study of the coordination of azide by Fe(II)-bleomycin through NMR has not been reported previously. Unlike magnetic CD data, NMR offers a residue-by-residue account of the possible structural changes that take place in Fe(II)-bleomycin after azide binding.

Chelation of Ca2+ ions by a peptide from the repeat region of the Plasmodium falciparum circumsporozoite protein

BackgroundElegant efforts towards the determination of the structural tendencies of peptides derived from the Plasmodium falciparum circumsporozoite protein allowed the proposal of a left-handed helical conformation for this protein. The use of circular dichroism and Fourier-transformed infrared spectroscopy applied to various peptides derived from this protein, indicated that they bind Ca2+ ions in helical environments. The essential role of calcium in cell function and biological mechanisms is well known. It influences the development of several stages of the P. falciparum parasite. However, there is very little knowledge regarding calcium coordination to circumsporozoite proteins. In the present investigation the chelation of Ca2+ by the (NANPNVDP)3NANP peptide, which contains the first seven 4-amino-acid blocks of the repeat region of the P. falciparum circumsporozoite protein, is tested with the use of circular dichroism and nuclear magnetic resonance spectroscopies. Spectroscopy-based solution conformations of the Ca-bound peptide are also determined.MethodsNMR spectroscopy and circular dichroism were used to test Ca2+ coordination by the peptide (NANPNVDP)3NANP. Solution conformations for the Ca-bound peptide were determined through molecular dynamics calculations.ResultsThe NMR spectra collected for (NANPNVDP)3NANP indicate that the signals generated by some of the amino acids located at its C-terminal end are shifted from their original positions upon Ca2+ addition. The solution conformations determined for the Ca-bound peptide indicate that the metal ion can be either six- or seven-coordinate.ConclusionsThe investigation described herein strongly supports the coordination of Ca2+ ions to some of the amino acids located at the C-terminus of the peptide (NANPNVDP)3NANP. The solution conformations determined for the Ca-bound congener of this peptide display many structural features associated to Ca-binding proteins.

T1BT* structural study of an anti-plasmodial peptide through NMR and molecular dynamics

BackgroundT1BT* is a peptide construct containing the T1 and B epitopes located in the 5’ minor repeat and the 3’ major repeat of the central repeat region of the Plasmodium falciparum circumsporozoite protein (CSP), respectively, and the universal T* epitope located in the C-terminus of the same protein. This peptide construct, with B = (NANP)3, has been found to elicit antisporozoite antibodies and gamma-interferon-screening T-cell responses in inbred strains of mice and in outbred nonhuman primates. On the other hand, NMR and CD spectroscopies have identified the peptide B’ = (NPNA)3 as the structural unit of the major repeat in the CSP, rather than the more commonly quoted NANP. With the goal of assessing the structural impact of the NPNA cadence on a proven anti-plasmodial peptide, the solution structures of T1BT* and T1B’T* were determined in this work.MethodsNMR spectroscopy and molecular dynamics calculations were used to determine the solution structures of T1BT* and T1B’T*. These structures were compared to determine the main differences and similarities between them.ResultsBoth peptides exhibit radically different structures, with the T1B’T* showing strong helical tendencies. NMR and CD data, in conjunction with molecular modelling, provide additional information about the topologies of T1BT* and T1B’T*. Knowing the peptide structures required to elicit the proper immunogenic response can help in the design of more effective, conformationally defined malaria vaccine candidates. If peptides derived from the CSP are required to have helical structures to interact efficiently with their corresponding antibodies, a vaccine based on the T1B’T* construct should show higher efficiency as a pre-erythrocyte vaccine that would prevent infection of hepatocytes by sporozoites.

Coordination chemistry and solution structure of Fe(II)-peplomycin. Two possible coordination geometries.

The solution structure of Fe(II)-peplomycin was determined from NMR data collected for this molecule. As found previously for Fe(II)- and Co(II)-bound bleomycin; the coordination sphere of the metal is composed of the primary and secondary amines in β-aminoalanine, the pyrimidine and imidazole rings in the pyrimidinylpropionamide, and β-hydroxyhistidine moieties, respectively, the amine nitrogen in β-hydroxyhistidine, and either the carbamoyl group in mannose or a solvent molecule. The two most discussed coordination geometries for the aforementioned ligands in metallo-bleomycins have been tested against the NMR data generated for Fe(II)-peplomycin. The interpretation of the experimental evidence obtained through molecular dynamics indicates that both geometries are equally likely in solution for this compound in the absence of DNA, but arguments are offered to explain why one of these geometries is preferred in the presence of DNA.

Solution structure of Fe(II)–azide–bleomycin derived from NMR data: transition from Fe(II)–bleomycin to Fe(II)–azide–bleomycin as derived from NMR data and structural calculations

The coordination cage of the metal center in Fe(II)–bleomycin has been proposed to consist of the secondary amines in β-aminoalanine, the pyrimidinylpropionamide and imidazole rings, and the amide nitrogen in β-hydroxyhistidine as equatorial ligands, and the primary amine in β-aminoalanine and either the carbamoyl group in mannose or a solvent molecule occupying the axial sites. With the aim of supporting or not supporting coordination of a water molecule to the metal center in Fe(II)–bleomycin, the solution structure of Fe(II)–azide–bleomycin has been derived from NMR data. The structural changes that occur in Fe(II)–bleomycin upon azide binding have been monitored by comparing the experimental results with those obtained from the calculated structures for both bleomycin adducts. The results of this investigation strongly support a model of Fe(II)–bleomycin with six endogenous ligands as the most likely structure held in solution by this metallobleomycin in the absence of DNA.

Contributions of NMR to the Understanding of the Coordination Chemistry and DNA Interactions of Metallo-Bleomycins

Bleomycins are a family of glycopeptide antibiotics that have the ability to bind and degrade DNA when bound to key metal ions, which is believed to be responsible for their antitumor activity. Knowledge of the structures of metallo-bleomycins is vital to further characterize their mechanism of action. To this end, numerous structural studies on metallo-bleomycins have been conducted. NMR spectroscopy has had a key role in most of these studies, and has led to very important findings involving the coordination chemistry of metallo-bleomycins, and the details of many metallo-bleomycin-DNA spatial correlations for this important drug. This paper reviews the most important contributions of NMR to the bleomycin field.

NMR study of peplomycin in aqueous solution. Assignment of resonances by means of two-dimensional spectroscopy.

1H-NMR spectra of peplomycin (PEP) recorded at 400 and, for the first time, 900 MHz at 2 °C were examined. All the spin systems in the PEP molecule were identified through 2D NMR spectroscopy. The use of NMR spectroscopy allowed the unambiguous assignment of 62 protons, generating 47 non-exchangeable and 15 exchangeable signals. The analysis of the signals observed in 2D-NOE spectra indicates that PEP exhibits an extended conformation at 2 °C. A comparison between the solution conformation of apo-PEP and the solution structure of HOO-Co(III)-PEP indicates that the overall structure of apo-PEP is extended in solution, but exhibiting a conformation of the bithiazole (B)-sulfonium (S) unit similar to that of HOO-Co(III)-PEP. The present investigation represents the initial stage of an NMR study of the solution conformation and dynamics of PEP, its derivatives, its metal complexes and the interactions of metallo-PEPs with their target DNA.