A review: what is the spermosphere and how can it be studied?

The spermosphere is the zone surrounding seeds where interactions between the soil, microbial communities and germinating seeds take place. The concept of the spermosphere is usually only applied during germination sensu stricto. Despite the transient nature of this very small zone of soil around the germinating seed, the microbial activities which occur there may have long-lasting impacts on plants. The spermosphere is indirectly characterized by either (i) seed exudates, which could be inhibitors or stimulators of micro-organism growth or (ii) the composition of the microbiome on and around the germinating seeds. The microbial communities present in the spermosphere directly reflect that of the germination medium or are host-dependent and influenced quantitatively and qualitatively by host exudates. Despite its strong impact on the future development of plants, the spermosphere remains little studied. This can be explained by the technical difficulties related to characterizing this concept due to its short duration, small size and biomass, and the number and complexity of the interactions that take place. However, recent technical methods, such as metabolite profiling, combining phenotypic methods with DNA- and RNA-based methods, could be used to investigate seed exudates, microbial communities and their interactions with the soil environment.

Structural investigation of an exopolysaccharide substituted with a lactyl ether group produced by Raoultella terrigena Ez-555-6 isolated in the Chernobyl exclusion zone.

Raoultella terrigena strain Ez-555-6, isolated from a root nodule of Medicago sativa harvested in the Chernobyl exclusion zone, produces a non-referenced high-molecular-mass exopolysaccharide (EPS). The structure of this EPS was determined using a combination approach including monosaccharide composition (GLC-FID, HPAEC-PAD), determination of glycosylation sites (GLC-EIMS) and 1D/2D NMR ((1)H, (13)C) and ESIMS (HR, MS/MS) studies of oligosaccharides obtained from mild acid hydrolysis. The EPS was found to be a charged pentasaccharide with a repeating unit composed of D-galactose, D-glucose, D-mannose and D-glucuronic acid (1:2:1:1). Lactic acid and O-acetyl substituents were localized on galactose and glucose residues, respectively, as presented in the following structure:

Extended glutamate activates metabotropic receptor types 1, 2 and 4: selective features at mGluR4 binding site.

To get an insight into the bioactive conformation of glutamic acid and its topological environment at the mGluR4 binding site, a pharmacophore model was constructed using molecular modeling. Agonists of known activities were used to run the Apex-3D program or to validate the resulting model. An extended glutamate conformer, two selective hydrophilic sites and bulk tolerance regions are disclosed. Selective features of mGluR1, mGluR2 and mGluR4 are discussed.

Agonist selectivity of mGluR1 and mGluR2 metabotropic receptors: a different environment but similar recognition of an extended glutamate conformation.

To investigate the structural requirements for selective activation or blockade of metabotropic glutamate receptors, we developed a pharmacophore model for group I (mGluR1) and group II (mGluR2) agonists. The Apex-3D program was used with a training set of known active, inactive, and/or selective compounds with a wide structural diversity. The pharmacophore models were then validated by testing a set of additional known agonists. We also used competitive antagonist superpositions in order to define more precisely the topology of the mGluR1 and mGluR2 agonists' recognition site. Both models account for the activity of most potent compounds and show that the selectivity between mGluR1 and mGluR2 subtypes may be due to excluded volumes and additional binding sites, while the relative spatial position of functional groups (NH2, alpha- and gamma-CO2H) remains very similar. On both models glutamate lies in an extended form. An additional binding site is disclosed on mGluR1, while this region would be forbidden on mGluR2. This new site combines a closed and an open model for mGluR1 and accounts for the increased affinity of quisqualic acid. The models show another large hydrophobic region which is tolerated for mGluR2 and restricted for mGluR1.

A three-dimensional construction of the active site (region 507-749) of human neutral endopeptidase (EC.3.4.24.11).

A three-dimensional model of the 507-749 region of neutral endopeptidase-24.11 (NEP; E.C.3.4.24.11) was constructed integrating the results of secondary structure predictions and sequence homologies with the bacterial endopeptidase thermolysin. Additional data were extracted from the structure of two other metalloproteases, astacin and stromelysin. The resulting model accounts for the main biological properties of NEP and has been used to describe the environment close to the zinc atom defining the catalytic site. The analysis of several thiol inhibitors, complexed in the model active site, revealed the presence of a large hydrophobic pocket at the S1' subsite level. This is supported by the nature of the constitutive amino acids. The computed energies of bound inhibitors correspond with the relative affinities of the stereoisomers of benzofused macrocycle derivatives of thiorphan. The model could be used to facilitate the design of new NEP inhibitors, as illustrated in the paper.

Structure of the human glycogen-associated protein phosphatase 1 regulatory subunit hGM: homology modeling revealed an (alpha/beta)8-barrel-like fold in the multidomain protein.

Protein phosphatase 1 (PP1) is widely distributed among tissues and species and acts as a regulator of many important cellular processes. By targeting the catalytic part of PP1 (PP1C) toward particular loci and substrates, regulatory subunits constitute key elements conferring specificity to the holoenzyme. Here, we report the identification of an (alpha/beta)8-barrel-like structure within the N-ter stretch of the human PP1 regulatory subunit hGM, which is part of the family of diverse proteins associated with glycogen metabolism. Protein homology modeling gave rise to a three-dimensional (3D) model for the 381 N-ter residue stretch of hGM, based on sequence similarity with Streptomyces olivochromogenes xylose isomerase, identified by using FASTA. The alignment was subsequently extended by using hydrophobic cluster analysis. The homology-derived model includes the putative glycogen binding area located within the 142-230 domain of hGM as well as a structural characterization of the PP1C interacting domain (segment 51-67). Refinement of the latter by molecular dynamics afforded a topology that is in agreement with previous X-ray studies (Egloff et al., 1997). Finite difference Poisson-Boltzmann calculations performed on the interacting domains of PP1C and hGM confirm the complementarity of the local electrostatic potentials of the two partners. This work highlights the presence of a conserved fold among distant species (mammalian, Caenorhabditis elegans, yeast) and, thus, emphasizes the involvement of PP1 in crucial basic cellular functions.

Structure, biological functions and inhibition of the HIV-1 proteins Vpr and NCp7.

The Gag-encoded nucleocapsid protein NCp7 (72 amino acids) from HIV-1, the regulatory protein, Vpr (96 amino acids) and numerous derivatives have been synthesized by solid phase method and their structures determined by 2D NMR. In NCp7, the two highly folded zinc fingers of the Cx2Cx4Hx4C type are in close spacial proximity and the replacement of H by C in the first zinc finger or P by L in the short interdigital domain led to structural modifications evidenced by NMR. In vivo, these point mutations induced a complete loss of viral infectivity by interrupting critical step(s) of the retroviral life cycle. To account for these findings, a model of the complex between NCp7 and d (ACGCC) has been proposed from NMR data, showing the intercalation of Trp37 in the oligonucleotide. This model could also explain the role of NCp7 in the formation of viral particles and agrees with the modifications in morphology of the virions containing mutations in the NCp7 zinc fingers. Vpr is essentially constituted by two long helical domains at its N- and C-terminals and the side chains of Leu60 and Leu67 participate in a leucine-zipper mode of intramolecular interaction. The results obtained have been used to try to develop new antiviral agents inhibiting NCp7 functions and thus possibly devoid of the resistance effects found with inhibitors of HIV enzymes (reverse transcriptase and protease).

1H NMR structure and biological studies of the His23-->Cys mutant nucleocapsid protein of HIV-1 indicate that the conformation of the first zinc finger is critical for virus infectivity.

The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 (HIV-1), which has key functions in the virus life cycle, possesses two zinc fingers of the CX2CX4HX4C type characterized by three successive loops containing a tetrahedrally coordinated zinc atom. The replacement of any cysteine by a serine in either finger has been shown to result in the production of noninfectious viruses, probably by impairing the biological functions of NCp7. In order to more precisely elucidate the structural role of the zinc finger motif, His23 was replaced by Cys in the proximal finger of the peptide (13-64)NCp7 which retains NCp7 activities in vitro. The peptide Cys23(13-64)NCp7 was synthesized by solid phase and studied by 2D 1H NMR and molecular modeling. The His to Cys modification causes important structural modifications of the N-terminal zinc finger which impair the spatial proximity of the two zinc fingers as shown by the disappearance of several interresidue NOEs. The side chains of Val13, Lys14, Phe16, Thr24, Ala25, Trp37, Gln45, and Met46, which are thought to be involved in nucleic acid recognition, are no longer found clustered in the Cys23(13-64)NCp7 mutant as they are in the wild-type NCp7 structure. In vitro, Cys23(13-64)NCp7 is unable to tightly interact with the viral RNA or replication primer tRNA(Lys,3). The Cys23(NCp7) mutation was introduced into an infectious HIV-1 molecular clone, and virions produced upon DNA transfection into cells were analyzed for their viral protein and RNA compositions as well as for their infectivity. Results show that, while the Cys23(NCp7) mutation does not impair virion production, viruses contain a low amount of degraded viral RNA and are not infectious. These findings suggest that a bona fide conformation of the HIV-1 NCp7 is critical for the packaging of viral RNA, its stability in virions, and virus infectivity.

Spatial proximity of the HIV-1 nucleocapsid protein zinc fingers investigated by time-resolved fluorescence and fluorescence resonance energy transfer.

The three-dimensional structure of peptides encompassing the two zinc-saturated finger motifs of the nucleocapsid protein NCp7 of HIV-1 has been reported by several groups. Whereas the folded structures of the finger motifs were in good agreement, discrepancies existed concerning their spatial relationship since the fingers were found either close to each other [Morellet, N., Jullian, N., De Rocquigny, H., Maigret, B., Darlix, J. L., & Roques, B. P. (1992) Embo J. 11, 3059-3065] or independently folded [Omichinski, J. G., Clore, G. M., Sakaguchi, K., Appella, E., & Gronenborn, A. M. (1991) FEBS Lett. 292, 25-30, Summers, M. F., Henderson, L. E., Chance, M. R., Bess, J. W., Jr., South, T. L., Blake, P. R., Sagi, I., Perez-Alvarado, G., Sowder, R.C., III, Hare, D.R., & Arthur, L. O. (1992) Protein Sci. 1, 563-574]. As in the interacting finger model, Phe16 in the NH2-terminal finger and Trp37 in the COOH-terminal finger were found to be spatially close, the fluorescence properties of the aromatic residues at positions 16 and 37 in the wild-type and two conservatively substituted (12-53) NCp7 peptides were investigated and compared with those of three negative control derivatives where the finger motifs were not in close contact. Direct distance measurements by Tyr-Trp fluorescence resonance energy transfer of the former derivatives yielded a 7-12 A interchromophore distance range which is clearly inconsistent with the 12.5-18 A range measured for the negative controls and thus a random orientation of the zinc finger motifs.(ABSTRACT TRUNCATED AT 250 WORDS)

New dual inhibitors of neutral endopeptidase and angiotensin-converting enzyme: rational design, bioavailability, and pharmacological responses in experimental hypertension.

In the treatment of cardiovascular diseases, it could be of therapeutic interest to associate the hypotensive effects resulting from the inhibition of angiotensin II formation, ensured by endothelial angiotensin-converting enzyme (ACE), with the diuretic and natriuretic responses due to the protection of the endogenous atrial natriuretic peptide (ANP) from inactivation by epithelial neutral endopeptidase (NEP). However, an investigation of this hypothesis requires an orally active compound able to jointly inhibit ACE and NEP. Dual inhibitors have therefore been designed by a rational approach, based on the characteristics of the active sites of both enzymes, which belong to the same family of zinc metallopeptidases, and on the structures of their most potent and selective inhibitors. As both NEP and ACE contain a large S'1-S'2 domain able to accommodate aromatic residues, the cyclic ACE inhibitor 3-(mercaptomethyl)-3,4,5,6-tetrahydro-2-oxo-1H-1-benzazocine-1-ace tic acid was selected as a template. Various aliphatic constraints were introduced on the benzyl moiety of the potent NEP inhibitor N-[2-(mercaptomethyl)-3-phenylpropanoyl]-L-tyrosine (IC50 NEP = 2 nM, IC50 ACE = 25 nM) to improve the fit between the computed most stable conformers of these molecules and the ACE template. New dual inhibitors, of general formula, N-[2(R,S)-(mercaptomethyl)-3(R,S)-phenylbutanoyl]-L-amino acid with IC50 values in the nanomolar range for both enzymes were generated by this approach. The separation of the four stereoisomers using chiral amines and the stereoselective synthesis of the 2-(mercaptomethyl)-3-phenylbutanoyl moiety showed that inhibitors with the 2S,3R configuration are the most potent on both NEP and ACE. The "in vivo" potency of various prodrugs of these inhibitors to inhibit ACE activity in lung and NEP activity in kidney was measured after oral administration in mice. From this pharmacokinetical study the most potent dual inhibitor RB 105 (N-[(2S,3R)-2-(mercaptomethyl)-3-phenylbutanoyl-L-alanine (compound 44c) (KI NEP 1.7 nM, KI ACE 4.5 nM) and its most efficient in vivo prodrug mixanpril, [N-[(2S,3R)-2-[(benzoylthio)methyl]-3-phenylbutanoyl]-L-alan ine (compound 18) (ED50 NEP approximately 1 mg/kg, ED50 ACE approximately 7 mg/kg) were selected. Competition experiments with a tritiated inhibitor of ACE or NEP bound to mouse lung and kidney membranes respectively showed that mixanpril has a long duration of action (> 8 h). As expected, after i.v. administration in the spontaneously hypertensive rat (SHR), RB 105 decreased blood pressure and increased diuresis and natriuresis.(ABSTRACT TRUNCATED AT 400 WORDS)

Three-dimensional 1H NMR structure of the nucleocapsid protein NCp10 of Moloney murine leukemia virus.

The nucleocapsid protein of Moloney murine leukemia virus (NCp10) is a 56-amino acid protein which contains one zinc finger of the CysX2CysX4HisX4Cys form, a highly conserved motif present in most retroviruses and retroelements. At pH > or = 5, NCp10 binds one zinc atom and the complexation induces a folding of the CysX2CysX4HEsX4Cys box, similar to that observed for the zinc-binding domains of HIV-1 NC protein. The three-dimensional structure of NCp10 has been determined in aqueous solution by 600 MHz 1H NMR spectroscopy. The proton resonances could be almost completely assigned by means of phase-sensitive double-quantum-filtered COSY, TOCSY and NOESY techniques. NOESY spectra yielded 597 relevant structural constraints, which were used as input for distance geometry calculations with DIANA. Further refinement was performed by minimization with the program AMBER, which was modified by introducing a zinc force field. The solution structure is characterized by a well-defined central zinc finger (rmsd of 0.747 +/- 0.209 A for backbone atoms and 1.709 +/- 0.187 A when all atoms are considered), surrounded by flexible N- and C-terminal domains. The Tyr28, Trp35, Lys37, Lys41 and Lys42 residues, which are essential for activity, lie on the same face of the zinc finger, forming a bulge structure probably involved in viral RNA binding. The significance of these structural characteristics for the various biological functions of the protein is discussed, taking into account the results obtained with various mutants.

Solution structure of GAP SH3 domain by 1H NMR and spatial arrangement of essential Ras signaling-involved sequence.

Src homology 3 (SH3) domains are found in numerous cytoplasmic proteins involved in intracellular signal transduction. We used 2-D 1H NMR to determine the structure of the SH3 domain of the guanosine triphosphatase-activating protein (GAP), an essential component of the Ras signaling pathway. The structure of the GAP SH3 domain (275-350) was found to be a compact beta-barrel made of six antiparallel beta-strands arranged in two roughly perpendicular beta-sheets with the acidic residues located at the surface of the protein. The Trp317, Trp319, Thr321 and Leu323 residues belonging to the sequence (317-326), which was shown to be essential for Ras signaling, formed two nearby lipophilic bulges followed by a hydrophilic domain (Arg324-Asp326). These structural data could be used to characterize the still unidentified downstream components of GAP, which are involved in Ras signaling, and to rationally design inhibitors of this pathway.

Conformational behaviour of the active and inactive forms of the nucleocapsid NCp7 of HIV-1 studied by 1H NMR.

The nucleocapsid protein NCp7 of the human immunodeficiency virus type I (HIV-1) is a 72 amino acid peptide containing two zinc fingers of the type CX2CX4HX4C linked by a short basic sequence 29RAPRKKG35. NCp7 was shown to activate in vitro both viral RNA dimerization and replication primer tRNA(Lys,3) annealing to the initiation site of reverse transcription. In order to clarify the possible structural role of the zinc fingers in the various functions of NCp7, complete sequence specific 1H NMR assignment of the entire protein was achieved by two-dimensional NMR experiments. Moreover, to characterize the role of the peptide linker in NCp7 folding, a synthetic analogue with an inversion of Pro31 configuration was studied by NMR and fluorescence techniques. Several long range NOEs implying amino acid protons from the folded zinc fingers and the spacer, such as Ala25 and Trp37, Phe16 and Trp37, Arg32 and Trp37, Lys33 and Trp37, Cys18 and Lys33 disappeared in the D-Pro31 (12-53)NCp7, confirming the spatial proximity of the two CCHC boxes observed in the (13-51)NCp7. This was also confirmed by iodide fluorescence quenching experiments. The N and C-terminal parts of NCp7 displayed a large flexibility except for two short sequences Tyr56 to Gly58 and Tyr64 to Gly66, which seemed to oscillate between random-coil and helical conformations. The biological relevance of the structural characteristics of NCp7 was studied in vitro and in vivo. Substitution of Pro31 by D-Pro31 in the active (13-64)NCp7 peptide led to a severe reduction of dimerization in vitro. Moreover, site-directed mutagenesis substituting Leu for Pro31 resulted in the formation of non-infectious and immature viral particles. These results suggest that the spatial proximity of the zinc fingers induced by the peptide linker, plays a critical role in encapsidation of genomic RNA and morphogenesis of HIV-1 infectious particles.

Structural and dynamic characterization of the aromatic amino acids of the human immunodeficiency virus type I nucleocapsid protein zinc fingers and their involvement in heterologous tRNA(Phe) binding: a steady-state and time-resolved fluorescence study.

The steady-state and time-resolved fluorescence properties of two zinc-saturated 18-residue synthetic peptides with the amino acid sequence of the NH2-terminal (NCp7 13-30 F16W, where the naturally occurring Phe was replaced by a Trp residue) and the COOH-terminal (NCp7 34-51) zinc finger domains of human immunodeficiency virus type I nucleocapsid protein were investigated. Fluorescence intensity decay of both Trp 16 and Trp 37 residues suggested the existence of two fully solvent-exposed ground-state classes governed by a C = 2.2 equilibrium constant. The lifetimes of Trp 16 classes differed from those of Trp 37 essentially because of differences in nonradiative rate constants. Arrhenius plots of the temperature-dependent nonradiative rate constants suggested that the fluorescence quenchers involved in both classes and in both peptides were different and the collisional rate of these quenchers with the indole ring was very low, probably because of the highly constrained peptide chain conformation. The nature of the ground-state classes was discussed in relation to 1H nuclear magnetic resonance data. Using Trp fluorescence to monitor the interaction of both peptides with tRNA(Phe) we found that a stacking between the indole ring of both Trp residues and the bases of tRNA(Phe) occurred. This stacking constituted the main driving force of the interaction and modified the tRNA(Phe) conformation. Moreover, the binding of both fingers to tRNA(Phe) was noncooperative with similar site size (3 nucleotide residues/peptide), but the affinity of the NH2-terminal finger domain (K = 1.3 (+/- 0.2) 10(5) M-1) in low ionic strength buffer was one order of magnitude larger than the COOH-terminal one due to additional electrostatic interactions involving Lys 14 and/or Arg 29 residues.

Influence of the N- and C-terminal chains on the zinc-binding and conformational properties of the central zinc-finger structure of Moloney murine leukaemia virus nucleocapsid protein: a steady-state and time-resolved fluorescence study.

The nucleocapsid protein NCp10 of the Moloney murine leukaemia virus is a small basic protein characterized by a central Cys26-X2-Cys29-X4-His34-X4-Cys39 zinc-finger domain. Mutants with deletion of either the N- or C-terminal chain (or both) surrounding the central zinc-finger domain were synthesized by a solid-phase approach in order to evaluate the influence of these lateral chains on zinc binding and conformational properties of NCp10. For this purpose, the steady-state and time-resolved fluorescence properties of the single Trp-35 residue of the various NCp10 derivatives were analyzed. The binding properties of the various derivatives suggest that the central zinc-finger domain affinity for zinc is not modified by the N-terminal chain and is only slightly (about one order of magnitude) increased by the C-terminal chain leading to a Kapp of (1.2 +/- 0.2).10(14) M-1 for the whole NCp10. Concerning the conformation of the NCp10 derivatives, fluorescence data are in agreement with structureless polypeptide chains in the absence of zinc. In contrast, in the presence of zinc, the fluorescence intensity decays are in agreement with a unique conformation of the finger motif backbone and a distribution of the Trp-indole moiety into two classes with different local environments. Decay-associated spectra, fluorescence quenching by acrylamide and anisotropy decay data further suggest that the Trp-indole moiety of both classes was highly exposed to solvent and had a high degree of rotational freedom. Finally, in contrast to the C-terminal chain, the N-terminal chain modifies the local environment and the accessibility to external quenchers of both Trp-35 classes, suggesting that it was folded in the vicinity of the Trp-35 residue.

Determination of the structure of the nucleocapsid protein NCp7 from the human immunodeficiency virus type 1 by 1H NMR.

The retroviral gag nucleocapsid protein NCp7 (72 amino acids) of HIV-1 (LAV strain), which contains two successive zinc fingers of the Cys-X2-Cys-X4-His-X4-Cys form linked by a stretch of basic residues, promotes viral RNA dimerization and encapsidation and activates annealing of the primer tRNA to the initiation site of reverse transcription. The structure of NCp7 and other shorter fragments was studied by 600 MHz 1H nuclear magnetic resonance (NMR) in aqueous solution to account for its various biological properties. Complete sequence specific 1H NMR assignments of the 13-51 residues of NCp7 encompassing the two zinc fingers was achieved by two-dimensional NMR experiments and the three-dimensional structure of (13-51)NCp7 was deduced from DIANA calculations, using nuclear Overhauser effects as constraints. The structure of the zinc complexed form of NCp7 is characterized by a kink at the Pro31 level in the basic Arg29-Ala-Pro-Arg-Lys-Lys-Gly35 RNA binding linker leading to a proximity of the Lys14-Cys18 to the Gly35-Cys39 sequences, which belong to the folded proximal and distal zinc fingers, respectively. Accordingly, the aromatic residues Phe16 and Trp37 were found to be spatially close. The Lys33 and Lys34 side-chains involved in viral RNA dimerization were solvent exposed. The N- and C-terminal sequences of NCp7 behave as flexible independent domains. The proposed structure of NCp7 might be used to rationally design new anti-viral agents aimed at inhibiting its functions.