The research activities of the NMR group focus on the determination of the 3D solution structures of biomolecules from NMR data. While a wide range of biological questions are addressed, the constant objective in all these studies is to establish relationships between the biomolecules NMR 3D structures and their biological functions.
Most of the time structural studies involve studying the interactions with other biological molecules (DNA-protein, protein-peptide or protein-ligand complexes), and the dynamics in the free or bound form.
Up to now, NMR has been the main technique used to develop our research projects, combined with molecular modelling and docking techniques.
I. Small cystein-rich defense proteins
Our biological research theme concentrates on small cystein-rich defense proteins. The aim is to advance our understanding of the mechanism of action at the atomic level. It will lead us to study complex systems by liquid NMR and to closely associate a series of complementary biophysical or biological techniques.
Two families of small cystein-rich defense proteins are studied : Antimicrobial proteins and entomotoxins, adopting different 3D folds and different disulfide bridges arrays. Many of these molecules are now obtained by chemical synthesis in close collaboration with A. Delmas’ group.
3 levels are explored :
Studying new molecules with therapeutic/agronomic interest
Exploring other biological functions
Understanding the mechanism of action at the atomic level
II. Methodological expertise : NMR, Modelling & Docking
In order to respond to the variety of questions addressed by our biologist collaborators, we carry on a series of more methodological NMR projects. The defense proteins projects and these methodological expertises are mutually supportive, and are inseparable.
- Folding studies
Ex : The folding dynamics of the dimeric protein HU involved in the cold response.
- Protein/ligand interactions
Ex : RKIP, a human protein of the PEBP family, which inhibits Raf kinase. It is involved in cancerous processes and acts as a metastasis suppressor.
- DNA/protein interactions
Ex : MC1 is a small architectural protein from Methanosarcina which curves and compacts the DNA of these Archaea.
- Paquet, F., Delalande, O., Goffinont, S., Culard, F., Loth, K., Asseline, U., Castaing, B., and Landon, C.
Model of a DNA-protein Complex of the Architectural Monomeric Protein MC1 from Euryarchea.
PlosOne (2014) 9, e88809
- Hervé-Grepinet, V., Meudal, H., Labas, V., Réhault-Godbert, S., Gautron, J., Berges, M., Guyot, N., Delmas, A. F., Nys, Y., and Landon, C.
3D NMR structure of hen egg gallin (chicken ovo-defensin) reveals a new variation of the beta-defensin fold.
J. Biol. Chem. (2014) 289, 7211-20
- Senille, V., Lelievre, D., Paquet, F., Garnier, N., Lamb, N., Legrand, A., Delmas, A. F., and Landon, C.
The addressing fragment of mitogaligin : first insights into functional and structural properties.
Chembiochem : a European journal of chemical biology (2013) 14, 711-720
- Derache, C., Meudal, H., Aucagne, V., Kevin, M., Cadène, M., Delmas, A. F., Lalmanach, A.-C., and Landon, C.
Initial insights into the structure-activity relationships of avian β-defensins.
J. Biol. Chem. (2012) 287, 7746-7755
- Tavel, L., Jacquillard, L., Karsisiotis, A.I., Saab, F., Jouvensal, L., Brans, A., Delmas, A., Schoentgen, F., Cadène, M., and Damblon, C.
Ligand binding studies of human PEBP1/RKIP : interaction with nucleotides and Raf-1 peptides evidenced by NMR and mass spectrometry.
PloS One (2012) 7, e36187
Blasco H., Veyrat-Durebex C., Bertrand M., Patin F., Labarthe F., Henique H., Emond P., Andres C.R., Antar C., Landon C., Nadal-Desbarats L. and Maillot F. (2016)
A multiplatform metabolomics approach to characterize plasma levels of phenylalanine and tyrosine in phenylketonuriaJournal of Inherited Metabolic Disease Reports (sous presse) Loth K., Alami S. A. I., Habès C., Garrido S., Aucagne V., Delmas A.F., Moreau, T., Zani M.-L. and Landon C. (2016)
Complete 1H, 15N and 13C assignment of trappin-2 and 1H assignment of its two domains, elafin and cementoinBiomolecular NMR Assignments (2016) 10 (1) 223-226 - doi : 10.1007/s12104-016-9671-1
Trappin-2 is a serine protease inhibitor with a very narrow inhibitory spectrum and has significant anti-microbial activities. It is a 10 kDa cationic protein composed of two distinct domains. The N-terminal domain (38 residues) named cementoin is known to be intrinsically disordered when it is not linked to the elafin. The C-terminal domain (57 residues), corresponding to elafin, is a cysteine-rich domain stabilized by four disulfide bridges and is characterized by a flat core and a flexible N-terminal part. To our knowledge, there is no structural data available on trappin-2. We report here the complete 1H, 15N and 13C resonance assignment of the recombinant trappin-2 and the 1H assignments of cementoin and elafin, under the same experimental conditions. This is the first step towards the 3D structure determination of the trappin-2.
Oukhatar, F., Meudal, H., Landon, C., Logothetis, N. K., Platas-Iglesias, C., Angelovski, G., Tóth, É. (2015)
Macrocyclic Gd(3+) Complexes with Pendant Crown Ethers Designed for Binding Zwitterionic NeurotransmittersChemistry - A European Journal (2015) 21 (31) 11226-11237 - doi : 10.1002/chem.201500542
A series of Gd(3+) complexes exhibiting a relaxometric response to zwitterionic amino acid neurotransmitters was synthesized. The design concept involves ditopic interactions 1) between a positively charged and coordinatively unsaturated Gd(3+) chelate and the carboxylate group of the neurotransmitters and 2) between an azacrown ether appended to the chelate and the amino group of the neurotransmitters. The chelates differ in the nature and length of the linker connecting the cyclen-type macrocycle that binds the Ln(3+) ion and the crown ether. The complexes are monohydrated, but they exhibit high proton relaxivities (up to 7.7 mM(-1) s(-1) at 60 MHz, 310 K) due to slow molecular tumbling. The formation of ternary complexes with neurotransmitters was monitored by (1) H relaxometric titrations of the Gd(3+) complexes and by luminescence measurements on the Eu(3+) and Tb(3+) analogues at pH 7.4. The remarkable relaxivity decrease (≈80 %) observed on neurotransmitter binding is related to the decrease in the hydration number, as evidenced by luminescence lifetime measurements on the Eu(3+) complexes. These complexes show affinity for amino acid neurotransmitters in the millimolar range, which can be suited to imaging concentrations of synaptically released neurotransmitters. They display good selectivity over non-amino acid neurotransmitters (acetylcholine, serotonin, and noradrenaline) and hydrogenphosphate, but selectivity over hydrogencarbonate was not achieved.
Backbone assignment of the three dimers of HU from Escherichia coli at 293 K : EcHUα2, EcHUβ2 and EcHUαβBiomolecular NMR Assignments (2015) 1-5 - doi : 10.1007/s12104-015-9610-6
HU is one of the major nucleoid-associated proteins involved in bacterial chromosome structure and in all DNA-dependent cellular activities. Similarly to eukaryotic histones, this small dimeric basic protein wraps DNA in a non-sequence specific manner, promoting DNA super-structures. In most bacteria, HU is a homodimeric protein encoded by a single gene. However, in enterobacteria such as Escherichia coli, the presence of two genes coding for two peptidic chains, HUα and HUβ, lead to the coexistence of three forms : two homodimers EcHUα2 and EcHUβ2, as well as a heterodimer EcHUαβ. Genetic and biochemical investigation suggest that each EcHU dimer plays a specific physiological role in bacteria. Their relative abundance depends on the environmental conditions and is driven by an essential, yet unknown, fast outstanding chain-exchange mechanism at physiological temperature. Our goal is to understand this fundamental mechanism from a structural and kinetics standpoint using NMR. For this purpose, the first steps are the assignment of each dimer in their native and intermediate states. Here, we report the backbone assignment of each HU dimers from E. coli at 293 K in their native state.
In the track of new biopesticides, four genes namely cytA, cytB, cytC and cytD encoding proteins homologous to Bacillus thuringiensis (Bt) Cyt toxins have been identified in the plant pathogenic bacteria Dickeya dadantii genome. Here we show that three Cyt-like δ-endotoxins from D. dadantii (CytA, CytB and CytC) are toxic to the pathogen of the pea aphid Acyrthosiphon pisum in terms of both mortality and growth rate. The phylogenetic analysis of the comprehensive set of Cyt toxins available in genomic databases shows that the whole family is of limited taxonomic occurrence, though in quite diverse microbial taxa. From a structure-function perspective the 3D structure of CytC and its backbone dynamics in solution have been determined by NMR. CytC adopts a cytolysin fold, structurally classified as a Cyt2-like protein. Moreover, the identification of a putative lipid binding pocket in CytC structure, which has been probably maintained in most members of the Cyt-toxin family, could support the importance of this lipid binding cavity for the mechanism of action of the whole family. This integrative approach provided significant insights into the evolutionary and functional history of D. dadantii Cyt toxins, which appears to be interesting leads for biopesticides.
Chemical shifts assignments of the archaeal MC1 protein and a strongly bent 15 base pairs DNA duplex in complexBiomolecular NMR Assignments (2015) - 9 (1) 215-217 - doi : 10.1007/s12104-014-9577-8
MC1 is the most abundant architectural protein present in Methanosarcina thermophila CHTI55 in laboratory growth conditions and is structurally unrelated to other DNA-binding proteins. MC1 functions are to shape and to protect DNA against thermal denaturation by binding to it. Therefore, MC1 has a strong affinity for any double-stranded DNA. However, it recognizes and preferentially binds to bent DNA, such as four-way junctions and negatively supercoiled DNA minicircles. Combining NMR data, electron microscopy data, biochemistry, molecular modelisation and docking approaches, we proposed recently a new type of DNA/protein complex, in which the monomeric protein MC1 binds on the concave side of a strongly bent 15 base pairs DNA. We present here the NMR chemical shifts assignments of each partner in the complex, 1H 15N MC1 protein and 1H 13C 15N bent duplex DNA, as first step towards the first experimental 3D structure of this new type of DNA/protein complex.
Meindre, F., Lelièvre, D., Loth, K., Mith, O., Aucagne, V., Berthomieu, P., Marquès, L., Delmas, A. F., Landon, C. and Paquet, F. (2014)
The Nuclear Magnetic Resonance Solution Structure of the Synthetic AhPDF1.1b Plant Defensin Evidences the Structural Feature within the γ-MotifBiochemistry (2014) 53 (49) 7745-7754 - doi : 10.1021/bi501285k
Plant defensins (PDF) are cysteine-rich peptides that are major actors in the innate immunity in plants. Besides their antifungal activity, some PDF such as Arabidopsis halleri PDF1.1b confer zinc tolerance in plants. Here we present (i) an efficient protocol for the production of AhPDF1.1b by solid-phase peptide synthesis followed by controlled oxidative folding to obtain the highly pure native form of the defensin and (ii) the three-dimensional (3D) nuclear magnetic resonance structure of AhPDF1.1b, the first 3D structure of plant defensin obtained with a synthetic peptide. Its fold is organized around the typical cysteine-stabilized α-helix ?-sheet motif and contains the ?-core motif involved in the antifungal activity of all plant defensins. On the basis of our structural analysis of AhPDF1 defensins combined with previous biological data for antifungal and zinc tolerance activities, we established the essential role of cis-Pro41 within the ?-core. In fact, the four consecutive residues (Val39-Phe40-Pro41-Ala42) are strictly conserved for plant defensins able to tolerate zinc. We hypothesized that structural and/or dynamic features of this sequence are related to the ability of the defensin to chelate zinc.
Three-dimensional NMR Structure of Hen Egg Gallin (Chicken Ovodefensin) Reveals a New Variation of the beta-Defensin FoldJournal of Biological Chemistry (2014) 289 (10) 7211-7220 - doi : 10.1074/jbc.M113.507046
Gallin is a 41-residue protein, first identified as a minor component of hen egg white and found to be antimicrobial against Escherichia coli. Gallin may participate in the protection of the embryo during its development in the egg. Its sequence is related to antimicrobial beta-defensin peptides. In the present study, gallin was chemically synthesized 1) to further investigate its antimicrobial spectrum and 2) to solve its three-dimensional NMR structure and thus gain insight into structure-function relationships, a prerequisite to understanding its mode(s) of action. Antibacterial assays confirmed that gallin was active against Escherichia coli, but no additional antibacterial activity was observed against the other Gram-positive or Gram-negative bacteria tested. The three-dimensional structure of gallin, which is the first ovodefensin structure to have been solved to date, displays a new five-stranded arrangement. The gallin three-dimensional fold contains the three-stranded antiparallel beta-sheet and the disulfide bridge array typical of vertebrate beta-defensins. Gallin can therefore be unambiguously classified as a beta-defensin. However, an additional short two-stranded beta-sheet reveals that gallin and presumably the other ovodefensins form a new structural subfamily of beta-defensins. Moreover, gallin and the other ovodefensins calculated by homology modeling exhibit atypical hydrophobic surface properties, compared with the already known vertebrate beta-defensins. These specific structural features of gallin might be related to its restricted activity against E. coli and/or to other yet unknown functions. This work provides initial understanding of a critical sequence-structure-function relationship for the ovodefensin family.
In Archaea the two major modes of DNA packaging are wrapping by histone proteins or bending by architectural non-histone proteins. To supplement our knowledge about the binding mode of the different DNA-bending proteins observed across the three domains of life, we present here the first model of a complex in which the monomeric Methanogen Chromosomal protein 1 (MC1) from Euryarchaea binds to the concave side of a strongly bent DNA. In laboratory growth conditions MC1 is the most abundant architectural protein present in Methanosarcina thermophila CHTI55. Like most proteins that strongly bend DNA, MC1 is known to bind in the minor groove. Interaction areas for MC1 and DNA were mapped by Nuclear Magnetic Resonance (NMR) data. The polarity of protein binding was determined using paramagnetic probes attached to the DNA. The first structural model of the DNA-MC1 complex we propose here was obtained by two complementary docking approaches and is in good agreement with the experimental data previously provided by electron microscopy and biochemistry. Residues essential to DNA-binding and -bending were highlighted and confirmed by site-directed mutagenesis. It was found that the Arg25 side-chain was essential to neutralize the negative charge of two phosphates that come very close in response to a dramatic curvature of the DNA.
Loth K., Gnida M., Romanuka J., Kaptein R. and Boelens R. (2013)
In non-specific lac headpiece-DNA complexes selective NMR line broadening is observed that strongly depends on length and composition of the DNA fragments. This broadening involves amide protons found in the non-specific lac-DNA structure to be interacting with the DNA phosphate backbone, and can be ascribed to DNA sliding of the protein along the DNA. This NMR exchange broadening has been used to estimate the 1D diffusion constant for sliding along non-specific DNA. The observed 1D diffusion constant of 4×10-12 cm2/s is two orders of magnitude smaller than derived from previous kinetic experiments, but falls in the range of values determined more recently using single molecule methods. This strongly supports the notion that sliding could play at most a minor role in the association kinetics of binding of lac repressor to lac operator and that other processes such as hopping and intersegment transfer contribute to facilitate the DNA recognition process.
Mitogaligin is a mitochondrion-targeting protein involved in cell death. The sequence of the protein is unrelated to that of any known pro- or antiapoptotic protein. Mitochondrial targeting is controlled by an internal sequence from residues 31 to 53, and although this sequence is essential and sufficient to provoke cell death, the precise mechanism of action at the mitochondrial membrane remains to be elucidated. Here, by focusing on the [31–53] fragment, we first assessed and confirmed its cell cytotoxicity by microinjection. Subsequently, with the aid of membrane models, we evaluated the impact of the membrane environment on the 3D structure of the peptide and on how the peptide is embedded and oriented within membranes. The fragment is well organized, even though it does not contain a canonical secondary structure, and adopts an interfacial location. Structural comparison with other membrane-interacting Trp-rich peptides demonstrated similarities with the antimicrobial peptide tritrpcidin.
Tavel, L., Jaquillard, L., Karsisiotis, A.I., Saab, F., Jouvensal, L., Brans, A., Delmas, A., Schoentgen, F., Cadene, M. and Damblon, C. (2012)
Ligand binding study of human PEBP1/RKIP : interaction with nucleotides and Raf-1 peptides evidenced by NMR and Mass SpectrometryPLoS ONE 7 (4) e36187
In the present study, we investigated human PEBP1 by NMR to determine the binding site of four different ligands : GTP, FMN, and one Raf-1 peptide in tri-phosphorylated and non-phosphorylated forms. The study was carried out by NMR in near physiological conditions, allowing for the identification of the binding site and the determination of the affinity constants KD for different ligands. Native mass spectrometry was used as an alternative method for measuring KD values. Our study demonstrates and/or confirms the binding of hPEBP1 to the four studied ligands. All of them bind to the same region centered on the conserved ligand-binding pocket of hPEBP1. Although the affinities for GTP and FMN decrease as pH, salt concentration and temperature increase from pH 6.5/NaCl 0 mM/20°C to pH 7.5/NaCl 100 mM/30°C, both ligands clearly do bind under conditions similar to what is found in cells regarding pH, salt concentration and temperature. In addition, our work confirms that residues in the vicinity of the pocket rather than those within the pocket seem to be required for interaction with Raf-1.
Numerous β-defensins have been identified in birds and the potential use of these peptides as alternatives to antibiotics has been proposed, in particular to fight antibiotic-resistant and zoonotic bacterial species. Little is known about the mechanism of antibacterial activity of avian β-defensins (AvBDs), and the present work was carried out to obtain initial insights into the involvement of structural features or specific residues in the antimicrobial activity of chicken AvBD2. Chicken AvBD2 and its enantiomeric counterpart were chemically synthesized. Peptide elongation and oxidative folding were both optimized. The similar antimicrobial activity measured for both L- and D- proteins clearly indicates that there is no chiral partner. Therefore the bacterial membrane is in all likelihood the primary target. Moreover, this work evidences that the three-dimensional fold is required for an optimal antimicrobial activity, in particular for Gram-positive bacterial strains. The three-dimensional NMR structure of chicken AvBD2 defensin displays the structural 3-stranded antiparallel β-sheet characteristic of β-defensins. The surface of the molecule does not display any amphipathic character. In light of this new structure and of the king penguin AvBD103b defensin structure, the consensus sequence of avian β-defensin′s family was analyzed. Well conserved residues were highlighted and the potential strategic role of the lysine 31 residue of AvBD2 emphasized. The synthetic AvBD2-K31A variant displayed substantial N-terminal structural modifications and a dramatic decrease in activity. Taken together, these results demonstrate the structural as well as the functional role of the critical lysine 31 residue in antimicrobial activity.
Savard, P.-Y., Daigle, R., Morin, S., Sebilo, A., Meindre, F., Laguee, P., Guertin, M., Gagne, S. M. (2011)
Structure and Dynamics of Mycobacterium tuberculosis Truncated Hemoglobin N : Insights from NMR Spectroscopy and Molecular Dynamics SimulationsBiochemistry (2011) 50 (51) 11121-11130 - doi : 10.1021/bi201059a
The potent nitric oxide dioxygenase (NOD) activity (trHbN-Fe(2+)-O(2) + (center dot)NO -> trHbN-Fe(3+)-OH(2) + NO(3)(-)) of Mycobacterium tuberculosis truncated hemoglobin N (trHbN) protects aerobic respiration from inhibition by (center dot)NO. The high activity of trHbN has been attributed in part to the presence of numerous short-lived hydrophobic cavities that allow partition and diffusion of the gaseous substrates (center dot)NO and O(2) to the active site. We investigated the relation between these cavities and the dynamics of the protein using solution NMR spectroscopy and molecular dynamics (MD). Results from both approaches indicate that the protein is mainly rigid with very limited motions of the backbone N H bond vectors on the picoseconds nanoseconds time scale, indicating that substrate diffusion and partition within trHbN may be controlled by side-chains movements. Model-free analysis also revealed the presence of slow motions (microseconds milliseconds), not observed in MD simulations, for many residues located in helices B and G including the distal heme pocket Tyr33(B10). All currently known crystal structures and molecular dynamics data of truncated hemoglobins with the so-called pre-A N-terminal extension suggest a stable alpha-helical conformation that extends in solution. Moreover, a recent study attributed a crucial role to the pre-A helix for NOD activity. However, solution NMR data clearly show that in near-physiological conditions these residues do not adopt an alpha-helical conformation and are significantly disordered and that the helical conformation seen in crystal structures is likely induced by crystal contacts. Although this lack of order for the pre-A does not disagree with an important functional role for these residues, our data show that one should not assume an helical conformation for these residues in any functional interpretation. Moreover, future molecular dynamics simulations should not use an initial alpha-helical conformation for these residues in order to avoid a bias based on an erroneous initial structure for the N-termini residues. This work constitutes the first study of a truncated hemoglobin dynamics performed by solution heteronuclear relaxation NMR spectroscopy.
An alternative flexible conformation of the E. coli HUβ(2) protein : structural, dynamics, and functional aspects.European Biophysics Journal 40 (2) 117-129
The histone-like HU protein is the major nucleoid-associated protein involved in the dynamics and structure of the bacterial chromosome. Under physiological conditions, the three possible dimeric forms of the E. coli HU protein (EcHUα₂, EcHUβ₂, and EcHUαβ) are in thermal equilibrium between two dimeric conformations (N₂ ↔ I₂) varying in their secondary structure content. High-temperature molecular dynamics simulations combined with NMR experiments provide information about structural and dynamics features at the atomic level for the N₂ to I₂ thermal transition of the EcHUβ₂ homodimer. On the basis of these data, a realistic 3D model is proposed for the major I₂ conformation of EcHUβ₂. This model is in agreement with previous experimental data.
Rautureau, G. J. P. Vovelle, F. Schoentgen, F. Decoville, M. Locker, D. Damblon C. & Jouvensal, L. (2010)
Da Silva, P., Strzepa, A., Jouvensal, L., Rahioui, I., Gressent, F. & Delmas, A.F. (2009)
Gomesin : a powerful antimicrobial peptide isolated from the Brazilian tarantula spider Acanthoscurria gomesianaIn "Animal Toxins : State of the Art. Perspectives in Health and Biotechnology." (De Lima, M. E., Pimenta, A. M. C., Martin-Eauclaire, M. F., Zingali, R., Rochat, H. Eds.) Belo Horizonte : Editora UFMG, 323-343. Rautureau, G., Jouvensal, L., Vovelle, F., Schoentgen, F., Locker, D. & Decoville, M. (2009)
Chauvin, T ; Durand, P ; Bernier, M ; Meudal, H ; Doan, BT ; Noury, F ; Badet, B ; Beloeil, JC ; Tóth, E (2008)
Detection of enzymatic activity by PARACEST MRI : a general approach to target a large variety of enzymesAngew. Chem. Int. Edit. 47 (23) 4370 -4372 Landon, C ; Barbault, F ; Legrain, M ; Guenneugues, M ; Vovelle, F (2008)
Rautureau, G ; Jouvensal, L ; Schoentgen, F ; Vovelle, F (2007)
We have studied the self-diffusion properties of butyl-methyl-imidazolium bis(trifluoromethylsulfonyl)-imide ([BMIM][TFSI]) + water system. The self-diffusion coefficients of cations, anions, and water molecules were determined by pulsed field gradient NMR. These measures were performed with increased water quantity up to saturation (from 0.3 to 30 mol %). Unexpected variations have been observed. The self-diffusion coefficient of every species increases with the quantity of water but not in the same order of magnitude. Whereas very similar evolutions are observed for the anion and cation, the increase is 25 times greater for water molecules. We interpret our data by the existence of phase separation at microscopic scale.
Loth, K., Abergel, D., Pelupessy, P., Delarue, M., Lopes, P., Ouazzani, J., Duclert-Savatier, N., Nilges, M., Bodenhausen, G. & Stoven, V (2006)
Determination of dihedral Psi angles in large proteins by combining NHN/C-alpha-H-alpha dipole/dipole cross-correlation and chemical shifts.Proteins 64, 931-939. Kieken, F ; Paquet, F ; Brule, F ; Paoletti, J ; Lancelot, G (2006)
Dimerization of genomic RNA is directly related with the event of encapsidation and maturation of the virion. The initiating sequence of the dimerization is a short autocomplementary region in the hairpin loop SL1. We describe here a new solution structure of the RNA dimerization initiation site (DIS) of HIV-1(Lai). NMR pulsed field-gradient spin-echo techniques and multidimensional heteronuclear NMR spectroscopy indicate that this structure is formed by two hairpins linked by six Watson-Crick GC base pairs. Hinges between the stems and the loops are stabilized by intra and intermolecular interactions involving the A8, A9 and A16 adenines. The coaxial alignment of the three A-type helices present in the structure is supported by previous crystallography analysis but the A8 and A9 adenines are found in a bulged in position. These data suggest the existence of an equilibrium between bulged in and bulged out conformations in solution.
Structure of the zinc finger domain encompassing residues 13-51 of the nucleocapsid protein from simian immunodeficiency virusBiochemical Journal 393 725-732 Part 3
The NCps (nucleocapsid proteins) of HIV-1 (HIV type 1), HIV-2 and SIV (simian immunodeficiency virus) are small highly basic proteins, characterized by the presence of two CCHC ZF (zinc finger) domains. NCps, closely associated with the dimeric RNA genome in the core of the virus particle, were shown to promote the specific encapsidation of the viral RNA and are implicated in reverse transcription. Solution structure of the HIV-1 NCp7 and complexes of NCp7 with RNA or DNA showed the critical relationships between the structure and its various functions. HIV-1 and HIV-2 have resulted respectively from transmissions of SIV from chimpanzees and sooty mangabeys.
Solution structures of stomoxyn and spinigerin, two insect antimicrobial peptides with an a-helical conformationBiopolymers 81 (2) 92-103
Stomoxyn and spinigerin belong to the class of linear cysteine-free insect antimicrobial peptides that kill a range of microorganisms, parasites, and some viruses but without any lytic activity against mammalian erythrocytes. Stomoxyn is localized in the gut epithelium of the nonvector stable fly that is sympatric with the trypanosome vector tsetse fly. Spinigerin is stored and secreted by hemocytes from the fungus-growing termite.
NMR studies of telomeric nucleoprotein complexes involving the Myb-like domain of the human telomeric protein TRF2Comptes Rendus Chimie 9 (3-4) 452-458
In order to study the binding of the Myb-like domain of the human telomeric protein TRF2 (Myb-TRF2) with different structural components of the t-loop model, we report NMR studies of the binding of Myb-TRF2 protein with two repeats human telomeric DNA under three conformations. Our results showed that Myb-TRF2 binds to the duplex and even to the quadruplex and the random coil G-rich strand. The solution structure of Myb-TRF2 reported here looks like Myb-TRF1 suggesting similar DNA binding mode. As a matter of fact, we have shown that its binding to the human telomeric duplex presents great similarities with this of Myb-TRF1.
Cloning, high yield over-expression, purification, and characterization of CG18594, a new PEBP/RKIP family member from Drosophila melanogasterProtein Expression and Purification 48 (1) 90-97
The phosphatidylethanolamine-binding protein (PEBP) family is widely distributed in various species, from bacteria to mammals. These proteins seem to modulate important cell mechanisms : they control heterotrimeric G-proteins, inhibit the MAP-kinase and NF kappa B signaling pathways, and also serine proteases (thrombin, neuropsin, and chymotrypsin). In order to establish structure-function relationships for this family of proteins, our study focuses on PEBPs expressed within a single organism : Drosophila melanogaster, which constitutes a model system that lends itself well to establishing links between genes’ expression and the corresponding proteins’ functions, and to studying physiological mechanisms such as development.
Accessibility of tobacco lipid transfer protein cavity revealed by N-15 NMR relaxation studies and molecular dynamics simulationsProteins-Structure Function and Bioinformatics 64 (1) 124-132
Plant LTP1 are small helical proteins stabilized by four disulfide bridges and are characterized by the presence of an internal cavity, in which various hydrophobic ligands can be inserted. Recently, we have determined the solution structure of the recombinant tobacco LTP1_1. Unexpectedly, despite a global fold very similar to the structures already known for cereal seed LTP1, its binding properties are different : Tobacco LTP1_1 is able to bind only one monoacylated lipid, whereas cereal LTP1 can bind either one or two. The 3D structure of tobacco LTP1_1 revealed the presence of a hydrophobic cluster, not observed on cereal LTP1 structures, which may hinder one of the two entrances of the cavity defined for wheat LTP1.
Da Silva, P ; Landon, C ; Industri, B ; Marais, A ; Marion, D ; Ponchet, M ; Vovelle, F (2005)
Solution structure of a tobacco lipid transfer protein exhibiting new biophysical and biological featuresProteins-Structure Function and Bioinformatics 59 (2) 356-367
Plant lipid transfer proteins are small soluble extracellular proteins that are able to bind and transfer a variety of lipids in vitro. Recently, it has been proposed that lipid transfer proteins may play a key role in plant defence mechanisms, especially during the induction of systemic acquired resistance. However, very little is known about the proteins expressed in developing plants and tissues, since almost all the biophysical and structural data available to date on lipid transfer proteins originate from proteins present in storage tissues of monocot cereal seeds. In this paper, we report the structural and functional characteristics of a lipid transfer protein (named LTP1_1) constitutively expressed in young aerial organs of Nicotiana tabacum (common tobacco).
Landon, C ; Thouzeau, C ; Labbe, H ; Bulet, P ; Vovelle, F (2004)
Recently two ß-defensins, named spheniscins, have been isolated from the stomach content of the king penguin (Aptenodytes patagonicus), which is capable of preserving food for several weeks during egg incubation (Thouzeau, C., Le Maho, Y., Froget, G., Sabatier, L., Le Bohec, C., Hoffmann, J.A., and Bulet, P. (2003) J. Biol. Chem. 278, 51053-51058). It has been proposed that, in combination with other antimicrobial peptides, spheniscins may be involved in this long term preservation of food in the bird’s stomach. To draw some structure/function features, the three-dimensional structure in aqueous solution of the most abundant spheniscin (Sphe-2) was determined by two-dimensional NMR and molecular modeling techniques. The overall fold of Sphe-2 includes a three-stranded antiparallel ß-sheet stabilized by three disulfide bridges with a pairing typical of ß-defensins. In addition, the N-terminal segment shows helical features on most structures. Sphe-2 is highly cationic, and its surface displays a hydrophobic patch.
Sequence and solution structure of cherimolacyclopeptides A and B, novel cyclooctapeptides from the seeds of Annona cherimolaTetrahedron 60 (2) 405-414
Two new cyclooctapeptides, cherimolacyclopeptide A, cyclo(Pro(1)-Gln(2)-Thr(3)-Gly(4)-Met(5)-Leu(6)-Pro(7)-Ile(8)-) (1) and the related cherimolacyclopeptide B, cyclo(Pro(1)-Gln(2)-Thr(3)-Gly(4)-Mso(5)-Leu(6)-Pro(7)-Ile(8)-) (2), have been isolated from the methanol extract of the seeds of Annona cherimola Miller. The sequences were elucidated on the basis of the MS/MS fragmentation, using a Q-TOF mass spectrometer equipped with an ESI source, chemical degradation and extensive 2D-heteronuclear NMR. The three-dimensional solution structure of cherimolacyclopeptide A (1) determined by H-1 NMR data and molecular modelling is characterised by the presence of two ß turns and a new type of ß-bulge. (C) 2003 Elsevier Ltd. All rights reserved.
Probing the hydrophobic cavity of Lipid Transfer Protein from Nicotiana tabacum through xenon-based NMR spectroscopyJournal of The American Chemical Society 126 (48) 15738-15746
The hydrophobic cavity of Lipid Transfer Protein 1 from Nicotiana tabacum is investigated in detail by NMR using xenon as a spy. The analysis of the Xe-129 chemical shifts and self-relaxation times gives evidence of protein-xenon interaction. Thermodynamics of the binding is characterized through the study of aliphatic H-1 and C-13 chemical shift variation as a function of xenon pressure. The binding constant is evaluated to 75.5 +/- 1.0 M-1 at 293 K. The location of xenon inside the cavity is deduced from SPINOE experiments.
Antimicrobial peptides are key components of the innate immune response in most multicellular organisms. These molecules are considered as one of the most innovative class of anti-infective agents that have been discovered over the last two decades, and therefore, as a source of inspiration for novel drug design. Insect cystein-rich antimicrobial peptides with the CSaß scaffold (an a-helix linked to a ß-sheet by two disulfide bridges) represent particularly attractive templates for the development of systemic agents owing to their remarkable resistance to protease degradation.
Anne, C ; Blommaert, A ; Turcaud, S ; Martin, AS ; Meudal, H ; Roques, BP (2003)
Thio-derived disulfides as potent inhibitors of botulinum neurotoxin type B : Implications for zinc interactionBioorganic & Medicinal Chemistry 11 (21) 4655-4660
Botulinum neurotoxin type B causes the inhibition of acetylcholine release at the neuromuscular junction resulting in a flaccid paralysis designated botulism. This occurs through the cleavage of synaptobrevin, an intracellular critical component of neurotransmitter exocytosis, by the zinc-metallopeptidase activity of the smallest subunit of the toxin. Blocking the proteolytic activity may present an attractive approach to treat botulism as to date there is no efficient specific drug therapy avalaible. We have therefore recently described a series of ß-amino-thiol derived pseudotripeptides able of inhibiting the toxin at low (10(-8) M) concentration. In this study, binding characteristics of the protein’s active site are explored through various structural modifications of the thiol functionality which was supposed to be a key structural constituent for effective zinc-ion chelation. Surprisingly, sulfanyl-derivatives such as symmetric disulfides were shown to be better inhibitors than their thiol-counterparts, the most potent compound displaying a K-i value of 3.4 nM. (C) 2003 Elsevier Ltd. All rights reserved.
Botulinum neurotoxins are the most potent toxins known to date. They are zinc-metalloproteases able to cleave selectively an essential component of neurotransmitter exocytosis, causing the syndrome of botulism characterized by a flaccid paralysis. There is a great interest in designing antagonists of the action of these toxins. One way is to inhibit their catalytic activity. In this study, we report the design of such inhibitors directed toward BoNT/B. A study of the S-1 subsite specificity, using several ß-amino thiols, has shown that this subsite prefers a p-carboxybenzyl moiety. The specificity of the S-1’ and S-2’ subsites was studied using two libraries of pseudotripeptides containing the S-1 synthon derived from the best ß-amino thiol tested. Finally, a selection of various non natural amino acids for the recognition of the "prime" domain led to the most potent inhibitor of BoNT/B described to date with a K-i value of 20 nM.
Solution structure of Alo-3 : A new knottin-type antifungal peptide from the insect Acrocinus longimanusBiochemistry 42 (49) 14434-14442
Insect peptides are key elements of the innate immunity against bacteria and fungi. These molecules offer remarkable properties : high efficacy, a low probability of resistance, limited toxicity, and immunogenicity. In this context, we are investigating several classes of peptides, and we have been successful in identifying biologically important classes of peptides and small molecules that will provide a stream of drug candidates for treating severe, life-threatening, hospital-acquired infections and other pathologies of high medical need.
Solution structure of termicin, an antimicrobial peptide from the termite Pseudacanthotermes spinigerProtein Science 12 (3) 438-446
The solution structure of termicin from hemocytes of the termite Pseudacanthotermes spiniger was determined by proton two-dimensional nuclear magnetic resonance spectroscopy and molecular modeling techniques. Termicin is a cysteine-rich antifungal peptide also exhibiting a weak antibacterial activity. The global fold of termicin consists of an a-helical segment (Phe4-Gln14) and a two-stranded (Phe19-Asp25 and Gln28-Phe33) antiparallel. ß-sheet forming a "cysteine stabilized aß motif" (CSaß) also found in antibacterial and antifungal defensins from insects and from plants.
PA1b, an insecticidal protein extracted from pea seeds (Pisum sativum) : H-1-2-D NMR study and molecular modelingBiochemistry 42 (41) 11915-11923
PA1b (pea albumin 1, subunit b) is a 37-amino acid cysteine-rich plant defense protein isolated from pea seeds (Pisum sativum). It induces short-term mortality in several pests, among which the cereal weevils Sitophilus sp. (Sitophilus oryzae, Sitophilus granarius, and Sitophilus zeamais) that are a major nuisance for stored cereals, all over the world.
Kieken, F ; Arnoult, E ; Barbault, F ; Paquet, F ; Huynh-Dinh, T ; Paoletti, J ; Genest, D ; Lancelot, G (2002)
HIV-1(Lai) genomic RNA : combined used of NMR and molecular dynamics simulation for studying the structure and internal dynamics of a mutated SL1 hairpinEuropean Biophysics Journal 31 (7) 521-531
The genome of all retroviruses consists of two identical copies of an RNA sequence associated in a non-covalent dimer. A region upstream from the splice donor (SL1) comprising a self-complementary sequence is responsible for the initiation of the dimerization. This region is able to dimerize in two conformations : a loop-loop complex or an extended duplex. Here, we solve by 2D NMR techniques the solution structure of a 23-nucleotide sequence corresponding to HIV-1 SL1(Lai) in which the mutation G12—>A12 is included to prevent dimerization. It is shown that this monomer adopts a stem-loop conformation with a seven base pairs stem and a nine nucleotide loop containing the G10 C11 A12 C13 G14 C15 sequence.
Toward an optimal joint recognition of the S-1 ’ subsites of endothelin converting enzyme-1 (ECE-1), angiotensin converting enzyme (ACE), and neutral endopeptidase (NEP)Journal of Medicinal Chemistry 45 (7) 1477-1486
The formation of vasoconstrictors (e.g., angiotensin II and endothelin) and the inactivation of vasodilators (e.g., bradykinin and atrial natriuretic) by membrane-bound zinc metallopeptidases are key mechanisms in the control of blood pressure and fluid homeostasis. The way in which these peptides modulate physiological functions has been intensively studied. With the aim to develop compounds that can jointly block the three metallopeptidases-neutral endopeptidase (NEP, neprilysin), angiotensin-converting enzyme (ACE), and endothelin-converting enzyme (ECE-1)-we studied the common structural specificity of the S-1’ subsites of these peptidases.
Most of the structural elements of the globular domain of murine prion protein form fibrils with predominant ß-sheet structureFebs Letters 529 (2-3) 256-260
The conversion of the cellular prion protein into the ß-sheet-rich scrapie prion protein is thought to be the key step in the pathogenesis of prion diseases. To gain insight into this structural conversion, we analyzed the intrinsic structural propensity of the amino acid sequence of the murine prion C-terminal domain. For that purpose, this globular domain was dissected into its secondary structural elements and the structural propensity of the protein fragments was determined. Our results show that all these fragments, excepted that strictly encompassing helix 1, have a very high propensity to form structured aggregates with a dominant content of ß-sheet structures. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
Lamberty, M ; Caille, A ; Landon, C ; Tassin-Moindrot, S ; Hetru, C ; Bulet, P ; Vovelle, F (2001)
Solution structures of the antifungal heliomicin and a selected variant with both antibacterial and antifungal activitiesBiochemistry 40 (40) 11995-12003
In response to an experimental infection, the lepidopteran Heliothis virescens produces an antifungal protein named heliomicin. Heliomicin displays sequence similarities with antifungal plant defensins and antibacterial or antifungal insect defensins. To gain information about the structural elements required for either antifungal or antibacterial activity, heliomicin and selected point-mutated variants were expressed in yeast as fusion proteins. The effects of mutations, defined by comparing the primary structure of heliomicin with the sequences of members of the insect defensin family, were analyzed using antibacterial and antifungal assays. One of the variants shows significant activity against Gram-positive bacteria while remaining efficient against fungi.
Androctonin is a 25-residue antibacterial peptide extracted from the hemolymph of the scorpion Androctonus australis. In order to understand the structural requirements for hairpin fold and for interactions with the bacterial membrane, we have analysed the chemical shifts and the noes of three synthetic androctonin mutants for which the disulfide bridges were selectively removed. (C) 2001 Academie des sciences/Editions scientifiques et medicales Elsevier SAS.
Magnetization transfer from laser-polarized xenon to protons located in the hydrophobic cavity of the wheat nonspecific lipid transfer proteinProtein Science 10 (4) 762-770
Nonspecific lipid transfer protein from wheat is studied by liquid-state NMR in the presence of xenon. The gas-protein interaction is indicated by the dependence of the protein proton chemical shifts on the xenon pressure and formally confirmed by the first observation of magnetization transfer from laser-polarized xenon to the protein protons. Twenty-six heteronuclear nOes have allowed the characterization of four interaction sites inside the wheat ns-LTP cavity, Their locations are in agreement with the variations of the chemical shifts under xenon pressure and with solvation simulations. The richness of the information obtained by the noble gas with a nuclear polarization multiplied by similar to 12,000 makes this approach based on dipolar cross-relaxation with laser-polarized xenon promising for probing protein hydrophobic pockets at ambient pressure.
First direct observation of magnetization transfer between laser-polarized xenon and protein protonsComptes Rendus de L Academie Des Sciences Serie Iv Physique Astrophysique 2 (2) 327-332
The conditions of observation and characterization of magnetization transfert between laser-polarized xenon 129 and protein protons are addressed. This is experimentally illustrated by its first detection obtained an the wheat non-specific lipid transfer protein. (C) 2001 Academic des sciences/Editions scientifiques et medicales Elsevier SAS.
Bellier, B ; Million, ME ; DaNascimento, S ; Meudal, H ; Kellou, S ; Maigret, B ; Garbay, C (2000)
Replacement of glycine with dicarbonyl and related moieties in analogues of the C-terminal pentapeptide of cholecystokinin : CCK2 agonists displaying a novel binding modeJournal of Medicinal Chemistry 43 (20) 3614-3623
Recent advances in the field of cholecystokinin have indicated the possible occurrence of multiple affinity states of the CCK2 receptor. Besides, numerous pharmacological experiments performed "in vitro" and "in vivo" support the eventuality of different pharmacological profiles associated to CCK2 ligands. Indeed, some agonists are essentially anxiogenic and uneffective in memory tests, whereas others are not anxiogenic and appear as able to reinforce memory. The reference compound for the latter profile is the CCK-8 analogue BC 264 (Boc-Tyr(SO3H)-gnle-mGly-Trp-(NMe)Nle-Asp-Phe-NH2).
Phosphinic derivatives as new dual enkephalin-degrading enzyme inhibitors : Synthesis, biological properties, and antinociceptive activitiesJournal of Medicinal Chemistry 43 (7) 1398-1408
The development of dual inhibitors of the two zinc metallopeptidases, neprilysin (neutral endopeptidase) and aminopeptidase N involved in the inactivation of the opioid peptides, enkephalins, represents an attractive physiological approach in the search for new analgesics devoid of the major drawbacks of morphine. Phosphinic compounds, corresponding to the general formula H3N+-CH(R-1)-P(O)(OH)-CH2-CH(R-2)-CONH-CH(R-3)-COO-, able to act as transition-state analogues and to fit the S-1, S-1’, and S-2’ subsites of both enzymes were designed. Selection of the R-1, R-2, and R-3 residues for optimal recognition of these enzymes led to the first dual competitive inhibitors with K-i values in the nanomolar range for neprilysin and aminopeptidase N. These compounds induce potent analgesic responses after intracerebroventricular or intravenous administrations in mice (hot plate test), and several of them were shown to be, at least, 10 times more potent than the previously described dual inhibitors.
The active site of drosomycin, a small insect antifungal protein, delineated by comparison with the modeled structure of Rs-AFP2, a plant antifungal proteinJournal of Peptide Research 56 (4) 231-238
Drosomycin is the first strictly antifungal protein isolated from an insect (Drosophila melanogaster). The solution structure of this 44-residue protein has been reported previously. It involves a three-stranded ß-sheet and an a-helix, the protein global fold being maintained by four disulfide bridges. Rs-AFP2 is a plant antifungal protein exhibiting 41% sequence similarity with drosomycin.