Dynamics of glyphosate-induced conformational changes of Mycobacterium tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.19) determined by hydrogen-deuterium exchange and electrospray mass spectrometry

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the reaction between shikimate 3-phosphate and phosphoenolpyruvate to form 5-enolpyruvylshikimate 3-phosphate, an intermediate in the shikimate pathway, which leads to the biosynthesis of aromatic amino acids. EPSPS exists in an open conformation in the absence of substrates and/or inhibitors and in a closed conformation when bound to the substrate and/or inhibitor. In the present report, the H/D exchange properties of EPSPS from Mycobacterium tuberculosis (Mt) were investigated for both enzyme conformations using ESI mass spectrometry and circular dichroism (CD). When the conformational changes identified by H/D exchanges were mapped on the 3-D structure, it was observed that the apoenzyme underwent extensive conformational changes due to glyphosate complexation, characterized by an increase in the content of alpha-helices from 40% to 57%, while the beta-sheet content decreased from 30% to 23%. These results indicate that the enzyme underwent a series of rearrangements of its secondary structure that were accompanied by a large decrease in solvent access to many different regions of the protein. This was attributed to the compaction of 71% of alpha-helices and 57% of beta-sheets as a consequence of glyphosate binding to the enzyme. Apparently, MtEPSPS undergoes a series of inhibitor-induced conformational changes, which seem to have caused synergistic effects in preventing solvent access to the core of molecule, especially in the cleft region. This may be part of the mechanism of inhibition of the enzyme, which is required to prevent the hydration of the substrate binding site and also to induce the cleft closure to avoid entrance of the substrates.

Investigating the effect of different positioning of lysine residues along the peptide chain of mastoparans for their secondary structures and biological activities

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Phosphate closes the solution structure of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Mycobacterium tuberculosis

The 5-enolpyruvylshikimate-3-phosphate synthase catalyses the sixth step of the shikimate pathway that is responsible for synthesizing aromatic compounds and is absent in mammals, which makes it a potential target for drugs development against microbial diseases. Here, we report the phosphate binding effects at the structure of the 5-enolpyruvyl shikimate-3-phosphate synthase from Mycobacterium tuberculosis. This enzyme is formed by two similar domains that close on each other induced by ligand binding, showing the occurrence of a large conformation change. We have monitored the phosphate binding effects using analytical ultracentrifugation, small angle X-ray scattering and, circular dichroism techniques. The low resolution results showed that the enzyme in the presence of phosphate clearly presented a more compact structure. Thermal-induced unfolding experiments followed by circular dichroism suggested that phosphate rigidified the enzyme. Summarizing, these data suggested that the phosphate itself is able to induce conformational change resulting in the closure movement in the M. tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase. (c) 2006 Elsevier B.V. All rights reserved.

How C-terminal carboxyamidation alters the biological activity of peptides from the venom of the eumenine solitary wasp

Inflammatory peptides display different types of post-transcriptional modifications, such as C-terminal amidation, that alter their biological activity. Here we describe the structural and molecular dynamics features of the mast cell degranulating peptide, eumenine mastoparan-AF (EMP-AF-NH2), found in the venom of the solitary wasp, and of its carboxyl-free C-terminal form (EMP-AF-COO-) characterized by a reduced activity. Circular dichroism indicates that both peptides switch from a random coil conformation in water to a helical structure in TFE and SDS micelles. NMR data, in 30% TFE, reveal that the two peptides fold into an alpha-helix spanning most of their length, while they differ in terms of molecular rigidity. To understand the origins of the conformational flexibility observed in the case of EMP-AF-COO-, a 5 ns MD simulation was carried out for each peptide, in an explicit water/TFE environment. The results show that the two peptides differ in an H-bond between Leu14 NH2 and the backbone carbonyl of Ile11. The loss of that H-bond in EMP-AF-COO- leads to a significant modification of its structural dynamics. In fact, as evidenced by essential dynamics analysis, while EMP-AF-NH2 exists mainly as a rigid structure, EMP-AF-COO- presents two helical stretches that fluctuate in some sort of independent fashion. We conclude that the diverse biological activity of the two peptides is not simply due to the reduction of the net positive charge, as generally suggested, but also to a structural perturbation of the amphipathic alpha-helix that affects their ability to perturb the cell membrane.

Expression and purification of human respiratory syncytial virus recombinant fusion protein

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Refolding and purification of bothropstoxin-1, a Lys49 - Phospholipase A(2) homologue, expressed as inclusion bodies in Escherichia coli

Hydrolysis of phospholipids by Group II phospholipase A(2) enzymes involves a nucleophilic attack on the sn-2 ester bond by the His48 residue and stabilization of the reaction intermediate by a Ca2+ ion cofactor bound to the Asp49 residue in the protein active site region, Bothropstoxin-I (BthTX-I) is a PLA, variant present in the venom of the snake Bothrops jararacussu which shows a Asp49 to Lys substitution and which lacks hydrolytic activity yet damages artificial membranes by a noncatalytic Ca2+-independent mechanism. In order to better characterize this unusual mechanism of membrane damage, we have established an expression system for BthTX-I in Escherichia coli. The DNA-coding sequence for BthTX-I was subcloned into the vector pET11-d, and the BthTX-I was expressed as inclusion bodies in E, coli BL21(DE3). The native BthTX-I contains seven disulfide bonds, and a straightforward protocol has been developed to refold the recombinant protein at high protein concentration in the presence of surfactants using a size-exclusion chromatography matrix. After refolding, recovery yields of 2.5% (corresponding to 4-5 mg of refolded recombinant BthTX-I per liter of bacterial culture) were routinely obtained. After refolding, identical fluorescent and circular dichroism spectra were obtained for the recombinant BthTX-I compared to those of the native protein. Furthermore, the native and refolded recombinant protein demonstrated identical membrane-damaging properties as evaluated by measuring the release of an entrapped fluorescent marker from liposomes, (C) 2001 Academic Press.

A stability transition at mildly acidic pH in the alpha-hemolysin (alpha-toxin) from Staphylococcus aureus

The effects of mildly acidic conditions on the free energy of unfolding (Delta G(u)(buff)) of the pore-forming alpha-hemolysin (alpha HL) from Staphylococcus aureus were assessed between pH 5.0 and 7.5 by measuring intrinsic tryptophan fluorescence, circular dichroism and elution time in size exclusion chromatography during urea denaturation, Decreasing the pH from 7.0 to 5.0 reduced the calculated Delta G(u)(buff) from 8.9 to 4.2 kcal moI(-1), which correlates with an increased rate of pore formation previously observed over the same pH range, It is proposed that the lowered surface pH of biological membranes reduces the stability of alpha HL thereby modulating the rate of pore formation. (C) 1999 Federation of European Biochemical Societies.

Activation of Ca2+-independent membrane-damaging activity in Lys49-phospholipase A(2) promoted by amphiphilic molecules

Association of class-II phospholipase A(2) (PLA(2)) with aggregated phospholipid substrate results in elevated levels of the Ca2+-dependent hydrolytic activity. The Asp49 residue participates in coordination of the Ca2+ ion cofactor, however, in Lys49-PLA(2) homologues (Lys49-PLA(2)S), substitution of the Asp49 by Lys results in loss of Ca2+ binding and lack of detectable phospholipid hydrolysis. Nevertheless, Lys49-PLA2S cause Ca2+-independent damage of liposome membranes. Bothropstoxin-I is a homodimeric Lys49-PLA(2) from the venom of Bothrops jararacussu, and in fluorescent marker release and dynamic light scattering experiments with DPPC liposomes we demonstrate activation of the Ca2+-independent membrane damaging activity by similar to4 molecules of sodium dodecyl sulphate (SDS) per protein monomer. Activation is accomparlied by significant changes in the intrinsic tryptophan fluorescence emission (ITFE) and near UV circular dichroism (UVCD) spectra of the protein. Subsequent binding of 7-10 SDS molecules results in further alterations in the ITFE and far UVCD spectra. Reduction in the rate of N-bromosuccinimide modification of Trp77 at the dimer interface suggests that initial binding of SDS to this region accompanies the activation of the membrane damaging activity. 1-anilinonaphthalene-8-sulphonic acid binding studies indicate that subsequent SDS binding to the active site is concomitant with the second structural transition. These results provide insights in the structural basis of amphiphile/protein coupling in class-II PLA(2)s. (C) 2004 Published by Elsevier B.V.

A micelle nucleation model for the interaction of dodecyl sulphate with Lys49-phospholipases A(2)

Bothropstoxin-I (BthTx-I) is a Lys49-PLA(2) from the venom of Bothrops jararacussu that lacks detectable catalytic activity, yet causes rapid Ca2+-independent membrane damage. With the aim of understanding the interaction between BthTx-I and amphiphilic molecules, we have studied the interaction of sodium dodecyl sulphate (SDS) with the protein. Circular dichroism and attenuated total reflection Fourier-transform infrared spectra of BthTx-I reveal changes in the alpha-helical organization of the protein at an SDS/BthTx-I molar ratio of 20-25. At SDS/BthTx-I ratios of 40-45 the alpha-helices return to a native-like conformation, although fluorescence emission anisotropy measurements of 2-amino-N-hexadecyl-benzamide (AHBA) demonstrate that the total SDS is below the critical micelle concentration when this transition occurs. These results may be interpreted as the result of SDS accumulation by the BthTx-I homodimer and the formation of a pre-micelle SDS/BthTx-I complex, which may subsequently be released from the protein surface as a free micelle. Similar changes in the alpha-helical organization of BthTx-I were observed in the presence of dipalmitoylphosphatidylcholine liposomes, suggesting that protein structure transitions coupled to organization changes of bound amphiphiles may play a role in the Ca2+-independent membrane damage by Lys49-PLA(2)s. (c) 2006 Elsevier B.V. All rights reserved.

Structural studies of prephenate dehydratase from Mycobacterium tuberculosis H37Rv by SAXS, ultracentrifugation, and computational analysis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Selectivity in the mechanism of action of antimicrobial mastoparan peptide Polybia-MP1

Many potent antimicrobial peptides also present hemolytic activity, an undesired collateral effect for the therapeutic application. Unlike other mastoparan peptides, Polybia-MP1 (IDWKKLLDAAKQIL), obtained from the venom of the social wasp Polybia paulista, is highly selective of bacterial cells. The study of its mechanism of action demonstrated that it permeates vesicles at a greater rate of leakage on the anionic over the zwitterionic, impaired by the presence of cholesterol or cardiolipin; its lytic activity is characterized by a threshold peptide to lipid molar ratio that depends on the phospholipid composition of the vesicles. At these particular threshold concentrations, the apparent average pore number is distinctive between anionic and zwitterionic vesicles, suggesting that pores are similarly formed depending on the ionic character of the bilayer. To prospect the molecular reasons for the strengthened selectivity in Polybia-MP1 and its absence in Mastoparan-X, MD simulations were carried out. Both peptides presented amphipathic alpha-helical structures, as previously observed in Circular Dichroism spectra, with important differences in the extension and stability of the helix; their backbone solvation analysis also indicate a different profile, suggesting that the selectivity of Polybia-MP1 is a consequence of the distribution of the charged and polar residues along the peptide helix, and on how the solvent molecules orient themselves according to these electrostatic interactions. We suggest that the lack of hemolytic activity of Polybia-MP1 is due to the presence and position of Asp residues that enable the equilibrium of electrostatic interactions and favor the preference for the more hydrophilic environment.

Influence of N-Terminus Modifications on the Biological Activity, Membrane Interaction, and Secondary Structure of the Antimicrobial Peptide Hylin-a1

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Labaditin, a cyclic peptide with rich biotechnological potential: preliminary toxicological studies and structural changes in water and lipid membrane environment

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Mechanism of Action and Relationship Between Structure and Biological Activity of Ctx-Ha: A New Ceratotoxin-like Peptide from Hypsiboas albopunctatus

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Further monoterpene chromane esters from Peperomia obtusifolia: VCD determination of the absolute configuration of a new diastereomeric mixture

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)