Biomedical subjects

Mariella Tegoni

Publications and source records attributed to Mariella Tegoni.

At least 19 recordsLinked to original sources

PubMed2006

The membrane bound bacterial lipocalin Blc is a functional dimer with binding preference for lysophospholipids.

Lipocalins, a widespread multifunctional family of small proteins (15-25kDa) have been first described in eukaryotes and more recently in Gram-negative bacteria. Bacterial lipocalins belonging to class I are outer membrane lipoproteins, among which Blc from E. coli is the better studied. Blc is expressed under conditions of starvation and high osmolarity, conditions known to exert stress on the cell envelope. The structure of Blc that we have previously solved (V. Campanacci, D. Nurizzo, S. Spinelli, C. Valencia, M. Tegoni, C. Cambillau, FEBS Lett. 562 (2004) 183-188.) suggested its possible role in binding fatty acids or phospholipids. Both physiological and structural data on Blc, therefore, point to a role in storage or transport of lipids necessary for membrane maintenance. In order to further document this hypothesis for Blc function, we have performed binding studies using fluorescence quenching experiments. Our results indicate that dimeric Blc binds fatty acids and phospholipids in a micromolar K(d) range. The crystal structure of Blc with vaccenic acid, an unsaturated C18 fatty acid, reveals that the binding site spans across the Blc dimer, opposite to its membrane anchored face. An exposed unfilled pocket seemingly suited to bind a polar group attached to the fatty acid prompted us to investigate lyso-phospholipids, which were found to bind in a nanomolar K(d) range. We discuss these findings in terms of a potential role for Blc in the metabolism of lysophospholipids generated in the bacterial outer membrane.

Amino Acid Sequence↗

PubMed2006

Modular structure of the receptor binding proteins of Lactococcus lactis phages. The RBP structure of the temperate phage TP901-1.

Lactococcus lactis is a gram-positive bacterium widely used by the dairy industry. Several industrial L. lactis strains are sensitive to various distinct bacteriophages. Most of them belong to the Siphoviridae family and comprise several species, among which the 936 and P335 are prominent. Members of these two phage species recognize their hosts through the interaction of their receptor-binding protein (RBP) with external cell wall saccharidices of the host, the "receptors." We report here the 1.65 A resolution crystal structure of the RBP from phage TP901-1, a member of the P335 species. This RBP of 163 amino acids is a homotrimer comprising three domains: a helical N terminus, an interlaced beta-prism, and a beta-barrel, the head domain (residues 64-163), which binds a glycerol molecule. Fluorescence quenching experiments indicated that the RBP exhibits high affinity for glycerol, muramyl-dipeptide, and other saccharides in solution. The structural comparison of this RBP with that of lactococcal phage p2 RBP, a member of the 936 species (Spinelli, S., Desmyter, A., Verrips, C. T., de Haard, J. W., Moineau, S., and Cambillau, C. (2006) Nat. Struct. Mol. Biol. 13, 85-89) suggests a large extent of modularity in RBPs of lactococcal phages.

Amino Acid Sequence↗

PubMed2006

Receptor-binding protein of Lactococcus lactis phages: identification and characterization of the saccharide receptor-binding site.

Phage p2, a member of the lactococcal 936 phage species, infects Lactococcus lactis strains by binding initially to specific carbohydrate receptors using its receptor-binding protein (RBP). The structures of p2 RBP, a homotrimeric protein composed of three domains, and of its complex with a neutralizing llama VH domain (VHH5) have been determined (S. Spinelli, A. Desmyter, C. T. Verrips, H. J. de Haard, S. Moineau, and C. Cambillau, Nat. Struct. Mol. Biol. 13:85-89, 2006). Here, we show that VHH5 was able to neutralize 12 of 50 lactococcal phages belonging to the 936 species. Moreover, escape phage mutants no longer neutralized by VHH5 were isolated from 11 of these phages. All of the mutations (but one) cluster in the RBP/VHH5 interaction surface that delineates the receptor-binding area. A glycerol molecule, observed in the 1.7-A resolution structure of RBP, was found to bind tightly (Kd= 0.26 microM) in a crevice located in this area. Other saccharides bind RBP with comparable high affinity. These data prove the saccharidic nature of the bacterial receptor recognized by phage p2 and identify the position of its binding site in the RBP head domain.

Amino Acid Sequence↗

PubMed2006

Crystal structure of the receptor-binding protein head domain from Lactococcus lactis phage bIL170.

Lactococcus lactis, a gram-positive bacterium widely used by the dairy industry, is subject to lytic phage infections. In the first step of infection, phages recognize the host saccharidic receptor using their receptor binding protein (RBP). Here, we report the 2.30-A-resolution crystal structure of the RBP head domain from phage bIL170. The structure of the head monomer is remarkably close to those of other lactococcal phages, p2 and TP901-1, despite any sequence identity with them. The knowledge of the three-dimensional structures of three RBPs gives a better insight into the module exchanges which have occurred among phages.

Amino Acid Sequence↗

PubMed2004

Crystal structure and kinetics identify Escherichia coli YdcW gene product as a medium-chain aldehyde dehydrogenase.

In the context of a medium-scaled structural genomics program aiming at solving the structures of as many as possible bacterial unknown open reading frame products from Escherichia coli (Y prefix), we have solved the structure of YdcW at 2.1A resolution, using molecular replacement. According to its sequence identity, YdcW has been classified into the betaine aldehyde dehydrogenases family (EC 1.2.1.8), catalysing the oxidation of betaine aldehyde into glycine betaine. The structure of YdcW resembles that of other aldehyde dehydrogenases: it is tetrameric and binds a NADH molecule in each monomer. The NADH molecules, bound in the active site by soaking, are revealed to be in the "hydrolysis position". Activities experiments demonstrate that YdcW is more active on medium-chains aldehyde than on betaine aldehyde. However, soaking of betaine into YdcW crystals revealed its presence in one of the subunits, in two positions, a putative resting position and a hydride transfer ready position. Analysis of kinetics data and of the active site shape suggest an optimum binding of n-alkyl aldehydes up to seven to eight carbon atoms, possibly followed by a bulky cyclic or aromatic group.

Aldehyde Dehydrogenase↗

PubMed2004

Structure of Escherichia coli YhdH, a putative quinone oxidoreductase.

As part of a structural genomics project on bacterial gene products of unknown function, the crystal structures of YhdH, a putative quinone oxidoreductase, and its complex with NADP have been determined at 2.25 and 2.6 A resolution, respectively. The overall fold of YhdH is very similar to that of alcohol dehydrogenases and quinone reductases despite its low sequence identity. The absence of any Zn ion indicates that YdhH is a putative quinone oxidoreductase. YhdH forms a homodimer, with each subunit composed of two domains: a catalytic domain and a coenzyme-binding domain. NADP is bound in a deep cleft formed between the two domains. Large conformational changes occur upon NADP binding, with the two domains closing up to each other and narrowing the NADP-binding cleft. Comparisons of the YdhH active site with those of the quinone oxidoreductases from Escherichia coli and Thermus thermophilus made it possible to identify essential conserved residues as being Asn41, Asp43, Asp64 and Arg318. The active-site size is very narrow and unless an induced fit occurs is accessible only to reagents the size of benzoquinone.

Amino Acid Sequence↗

PubMed2004

Crystal structure of E.coli alcohol dehydrogenase YqhD: evidence of a covalently modified NADP coenzyme.

In the course of a structural genomics program aiming at solving the structures of Escherichia coli open reading frame (ORF) products of unknown function, we have determined the structure of YqhD at 2.0A resolution using the single wavelength anomalous diffraction method at the Pt edge. The crystal structure of YqhD reveals that it is an NADP-dependent dehydrogenase, a result confirmed by activity measurements with several alcohols. The current interpretation of our findings is that YqhD is an alcohol dehydrogenase (ADH) with preference for alcohols longer than C(3). YqhD is a dimer of 2x387 residues, each monomer being composed of two domains, a Rossmann-type fold and an alpha-helical domain. The crystals contain two dimers in the asymmetric unit. While one of the dimers contains a cofactor in both subunits, only one of the subunits in the second dimer contains it, making it possible to compare bound and unbound active sites. The active site contains a Zn atom, as verified by EXAFS on the crystals. The electron density maps of NADP revealed modifications of the nicotinamide ring by oxygen atoms at positions 5 and 6. Further analysis by electrospray mass spectrometry and comparison with the mass spectra of NADP and NADPH revealed the nature of the modification and the incorporation of two hydroxyl moieties at the 5 and 6 position in the nicotinamide ring, yielding NADPH(OH)(2). These modifications might be due to oxygen stress on an enzyme, which would functionally work under anaerobic conditions.

Alcohol Dehydrogenase↗

PubMed2004

Plant stress proteins of the thaumatin-like family discovered in animals.

Thaumatin-like proteins (TLPs) are polypeptides of about 200 residues synthesized by plants in response to fungal infection. In addition to the exceptionally strong sweet taste exhibited by some members, they are also reported to be endowed with endo-beta-1,3-glucanase activity and alpha-amylase inhibiting properties. However, the detailed mechanism of their antifungal action is not completely understood. So far, TLPs have only been described in plants, with several members of the family expressed in the same species. Here, for the first time in animals, we report the identification of two genes encoding members of the thaumatin-like proteins family in the desert locust Schistocerca gregaria and show their expression in different parts of the body. Southern blot and Western blot experiments revealed the presence of orthologous genes and their expression products in the related species Locusta migratoria. A search through the available genomes yielded similar sequences in the nematode Caenorhabditis but not in Drosophila and other insects. A three-dimensional model of S. gregaria TLP suggests a glucanase function. As in plants, TLPs could play a defense role in insects against pathogens.

Amino Acid Sequence↗

PubMed2004

Crystal structure and reactivity of YbdL from Escherichia coli identify a methionine aminotransferase function.

The ybdL gene of Escherichia coli codes for a protein of unknown function. Sequence analysis showed moderate homology to several vitamin B(6) dependent enzymes, suggesting that it may bind pyridoxal-5'-phosphate. The structure analysis of YbdL to 2.35 A resolution by protein crystallography verifies that it is a PLP dependent enzyme of fold type I, the typical aspartate aminotransferase fold. The active site contains a bound pyridoxal-5'-phosphate, covalently attached to the conserved active site lysine residue Lys236. The pattern of conserved amino acids in the putative substrate binding pocket of the enzyme reveals that it is most closely related to a hyperthermophilic aromatic residue aminotransferase from the archeon Pyrococcus horikoshii. Activity tests with 10 amino acids as amino-donors reveal, however, a preference for Met, followed by His and Phe, results which can be rationalized by modelization studies.

Amino Acid Sequence↗

PubMed2004

Domain swapping of a llama VHH domain builds a crystal-wide beta-sheet structure.

Among mammals, camelids have a unique immunological system since they produce functional antibodies devoid of light chains and CH1 domains. To bind antigens, whether they are proteins or haptens, camelids use the single domain VH from their heavy chain (VHH). We report here on such a llama VHH domain (VHH-R9) which was raised against a hapten, the RR6 red dye. This VHH possesses the shortest complementarity determining region 3 (CDR3) among all the known VHH sequences and nevertheless binds RR6 efficiently with a K(d) value of 83 nM. However, the crystal structure of VHH-R9 exhibits a striking feature: its CDR3 and its last beta-strand (beta9) do not follow the immunoglobulin VH domain fold, but instead extend out of the VHH molecular boundary and associate with a symmetry-related molecule. The two monomers thus form a domain-swapped dimer which establishes further contacts with symmetry-related molecules and build a crystal-wide beta-sheet structure. The driving force of the dimer formation is probably the strain induced by the short CDR3 together with the cleavage of the first seven residues.

Amino Acid Sequence↗

PubMed2004

The crystal structure of the Escherichia coli lipocalin Blc suggests a possible role in phospholipid binding.

Lipocalins form a large multifunctional family of small proteins (15-25 kDa) first discovered in eukaryotes. More recently, several types of bacterial lipocalins have been reported, among which Blc from Escherichia coli is an outer membrane lipoprotein. As part of our structural genomics effort on proteins from E. coli, we have expressed, crystallized and solved the structure of Blc at 1.8 A resolution using remote SAD with xenon. The structure of Blc, the first of a bacterial lipocalin, exhibits a classical fold formed by a beta-barrel and a alpha-helix similar to that of the moth bilin binding protein. Its empty and open cavity, however, is too narrow to accommodate bilin, while the alkyl chains of two fatty acids or of a phospholipid could be readily modeled inside the cavity. Blc was reported to be expressed under stress conditions such as starvation or high osmolarity, during which the cell envelope suffers and requires maintenance. These data, together with our structural interpretation, suggest a role for Blc in storage or transport of lipids necessary for membrane repair or maintenance.

Amino Acid Sequence↗

PubMed2004

Structural aspects of sexual attraction and chemical communication in insects.

In the animal kingdom, the relationship between individuals and the environment is often modulated by chemical communication. In the olfaction of insects, small antennary proteins--such as odorant- and pheromone-binding proteins (OBPs and PBPs, respectively) and chemosensory proteins (CSP)--have been proposed to carry their ligand from the air-fluid interface to the olfactory receptors. Binding experiments and recent structural studies of three PBPs, an OBP and a CSP have illustrated their versatility and ability to accommodate ligands of different shapes and chemical structures. The role of these proteins--as simple transporters or receptor triggers--is still a matter of debate, but some recent data seem to support the latter.

Amino Acid Sequence↗

PubMed2004

Crystal structures of bovine odorant-binding protein in complex with odorant molecules.

The structure of bovine odorant-binding protein (bOBP) revealed a striking feature of a dimer formed by domain swapping [Tegoni, M., Ramoni, R., Bignetti, E., Spinelli, S. & Cambillau, C. (1996) Nat. Struct. Biol.3, 863-867; Bianchet, M.A., Bains, G., Pelosi, P., Pevsner, J., Snyder, S.H., Monaco, H.L. & Amzel, L.M. (1996) Nat. Struct. Biol.3, 934-939] and the presence of a naturally occuring ligand [Ramoni, R., Vincent, F., Grolli, S., Conti, V., Malosse, C., Boyer, F.D., Nagnan-Le Meillour, P., Spinelli, S., Cambillau, C. & Tegoni, M. (2001) J. Biol. Chem.276, 7150-7155]. These features led us to investigate the binding of odorant molecules with bOBP in solution and in the crystal. The behavior of odorant molecules in bOBP resembles that observed with porcine OBP (pOBP), although the latter is monomeric and devoid of ligand when purified. The odorant molecules presented K(d) values with bOBP in the micromolar range. Most of the X-ray structures revealed that odorant molecules interact with a common set of residues forming the cavity wall and do not exhibit specific interactions. Depending on the ligand and on the monomer (A or B), a single residue--Phe89--presents alternate conformations and might control cross-talking between the subunits. Crystal data on both pOBP and bOBP, in contrast with binding and spectroscopic studies on rat OBP in solution, reveal an absence of significant conformational changes involving protein loops or backbone. Thus, the role of OBP in signal triggering remains unresolved.

Aldehydes↗

PubMed2003

Sulfur single-wavelength anomalous diffraction crystal structure of a pheromone-binding protein from the honeybee Apis mellifera L.

Pheromone binding proteins (PBPs) are small helical proteins ( approximately 13-17 kDa) present in several sensory organs from moth and other insect species. They are involved in the transport of pheromones from the sensillar lymph to the olfactory receptors. We report here the crystal structure of a PBP (Amel-ASP1) originating from the honey-bee (Apis mellifera) antennae and expressed as recombinant protein in the yeast Pichia pastoris. Crystals of Amel-ASP1 were obtained at pH 5.5 using the nano-drops technique of crystallization with a novel optimization procedure, and the structure was solved initially with the single-wavelength anomalous diffraction technique using sulfur anomalous dispersion. The structure of Amel-ASP1 has been refined at 1.6-A resolution. Its fold is roughly similar to that of other PBP/odorant binding proteins, presenting six helices and three disulfide bridges. Contrary to the PBPs from Bombyx mori (Sandler, B. H., Nikonova, L., Leal, W. S., and Clardy, J. (2000) Chem. Biol. 7, 143-151) and Leucophea maderae (Lartigue, A., Gruez, A., Spinelli, S., Riviere, S., Brossut, R., Tegoni, M., and Cambillau, C. (2003) J. Biol. Chem. 278, 30213-30218), the extended C terminus folds into the protein and forms a wall of the internal hydrophobic cavity. Its backbone groups establish two hydrogen bonds with a serendipitous ligand, n-butyl-benzene-sulfonamide, an additive used in plastics. This mode of binding might, however, mimic that used by one of the pheromonal blend components and illustrates the binding versatility of PBPs.

Animals↗

PubMed2003

The crystal structure of a cockroach pheromone-binding protein suggests a new ligand binding and release mechanism.

Pheromone-binding proteins (PBPs) are small helical proteins found in sensorial organs, particularly in the antennae, of moth and other insect species. They were proposed to solubilize and carry the hydrophobic pheromonal compounds through the antennal lymph to receptors, participating thus in the peri-receptor events of signal transduction. The x-ray structure of Bombyx mori PBP (BmorPBP), from male antennae, revealed a six-helix fold forming a cavity that contains the pheromone bombykol. We have identified a PBP (LmaPBP) from the cockroach Leucophaea maderae in the antennae of the females, the gender attracted by pheromones in this species. Here we report the crystal structure of LmaPBP alone or in complex with a fluorescent reporter (amino-naphthalen sulfonate, ANS) or with a component of the pheromonal blend, 3-hydroxy-butan-2-one. Both compounds bind in the internal cavity of LmaPBP, which is more hydrophilic than BmorPBP cavity. LmaPBP structure ends just after the sixth helix (helix F). BmorPBP structure extends beyond the sixth helix with a stretch of residues elongated at neutral pH and folding as a seventh internalized helix at low pH. These differences between LmaPBP and BmorPBP structures suggest that different binding and release mechanism may be adapted to the hydrophilicity or hydrophobicity of the pheromonal ligand.

Amino Acid Sequence↗

PubMed2003

Crystallization and preliminary crystallographic study of a pheromone-binding protein from the cockroach Leucophaea maderae.

Pheromone-binding proteins (PBPs) are small helical proteins (13-18 kDa) present in various sensory organs of moths and other insect species. An antennal protein from the cockroach Leucophaea maderae (LmaPBP) has been found to share all the hallmarks of the PBP family and is expressed specifically in the female adult antennae, the gender that perceives the sex pheromone. Here, the crystallization of LmaPBP expressed as a recombinant protein in Escherichia coli periplasm is reported. Crystals of LmaPBP were obtained by the sitting-drop vapour-diffusion method using a nanodrop-dispensing robot. The protein crystallizes in two different crystal forms. Form 1 belongs to space group P1, with unit-cell parameters a = 43.2, b = 45.1, c = 45.7 A, alpha = 118.6, beta = 93.0, gamma = 106.9 degrees. With two molecules in the asymmetric unit, V(M) is 2.7 A(3) Da(-1) and the solvent content is 47%. A complete data set has been collected at 1.6 A resolution on beamline ID14-2 (ESRF, Grenoble). Form 2 was obtained in the presence of the pheromone (3-hydroxy-butan-2-one) and belongs to space group P2(1), with unit-cell parameters a = 38.2, b = 62.2, c = 45.1 A, beta = 93.0 degrees. With two molecules in the asymmetric unit, V(M) is 2.0 A(3) Da(-1) and the solvent content is 39%. A complete data set has been collected at 1.7 A resolution on beamline BM14 (ESRF, Grenoble). SeMet expression has been performed with a view to solving the structure by MAD data collection using the Se absorption edge.

Amino Acid Sequence↗

PubMed2003

Optimization of crystals from nanodrops: crystallization and preliminary crystallographic study of a pheromone-binding protein from the honeybee Apis mellifera L.

Pheromone-binding proteins (PBPs) are small helical proteins ( approximately 13-17 kDa) present in various sensory organs from moths and other insect species. They are involved in the transport of pheromones from the sensillar lymph to the olfactory receptors. Here, crystals of a PBP (Amel-ASP1) originating from honeybee (Apis mellifera L.) antennae and expressed as recombinant protein using the yeast Pichia pastoris are reported. Crystals of Amel-ASP1 have been obtained by the sitting-drop vapour-diffusion method using a nanodrop-dispensing robot under the following conditions: 200 nl of 40 mg ml(-1) protein solution in 10 mM Tris, 25 mM NaCl pH 8.0 was mixed with 100 nl of well solution containing 0.15 M sodium citrate, 1.5 M ammonium sulfate pH 5.5. The protein crystallizes in space group C222(1), with unit-cell parameters a = 74.8, b = 85.8, c = 50.2 A. With one molecule in the asymmetric unit, V(M) is 3.05 A(3) Da(-1) and the solvent content is 60%. A complete data set has been collected at 1.6 A resolution on beamline ID14-2 (ESRF, Grenoble). The nanodrop crystallization technique used with a novel optimization procedure made it possible to consume small amounts of protein and to obtain a unique crystal per nanodrop, suitable directly for data collection in-house or at a synchrotron-radiation source.

Amino Acid Sequence↗

PubMed2003

A pheromone-binding protein from the cockroach Leucophaea maderae: cloning, expression and pheromone binding.

Odorant-binding proteins (OBPs) are thought to transport volatile compounds from air to their receptors through the sensillary lymph. In this protein family, the subgroup of pheromone-binding proteins (PBPs) is specifically tuned to the perception of the sexual pheromone. To date, the description of OBPs has been restricted to Endopterygota and Paraneoptera. Their expression in Orthopteroid has been hypothesized, but no evidence of OBP has been produced in this assemblage to date. In the present study, we describe the first OBP from a Dictyopteran insect that belongs to the cockroach Leucophaea maderae. The PBP of L. maderae (PBPLma) shares all the hallmarks of the OBP family and is expressed specifically in the female adult antennae, the sex that perceives the sexual pheromone. The affinity of the recombinant PBPLma produced in the Escherichia coli periplasm for the pheromonal compounds has been tested by displacement of a fluorophore, 8-anilino-1-naphtalenesulphonic acid (ANS). Our results suggest that two chemically close compounds of the pheromonal blend (3-hydroxy-butan-2-one and butane-2,3-diol) are capable of displacing ANS, whereas two other pheromone components (E-2-octenoic acid and senecioic acid) and other alkyl volatile compounds are not capable of displacing ANS, indicating a certain filtering of binding, which can be correlated with the putative function.

Amino Acid Sequence↗