Refined three-dimensional solution structure of insect defensin A.

BACKGROUND: Insect defensin A is a basic 4 kDa protein secreted by Phormia terranovae larvae in response to bacterial challenges or injuries. Previous biological tests suggest that the bacterial cytoplasmic membrane is the target of defensin A. The structural study of this protein is the first step towards establishing a structure-activity relationship and forms the basis for understanding its antibiotic activity at the molecular level. RESULTS: We describe a refined model of the three-dimensional structure of defensin A derived from an extensive analysis of 786 inter-proton nuclear Overhauser effects. The backbone fold involves an N-terminal loop and an alpha-helical fragment followed by an antiparallel beta-structure. The helix and the beta-structure are connected by two of the three disulphide bridges present in defensin A, forming a so-called 'cysteine-stabilized alpha beta' (CS alpha beta) motif. The N-terminal loop, which is locally well defined, can occupy different positions with respect to the other moieties of the molecule. CONCLUSIONS: The CS alpha beta motif, which forms the core of the defensin A structure, appears to be a common organization for several families of small proteins with toxic properties. The distribution of amino acid side chains in the protein structure creates several hydrophobic or hydrophilic patches. This leads us to propose that the initial step in the action of positively charged defensin A molecules with cytoplasmic membranes may involve interactions with acidic phospholipids.

Magnetization transfer from laser-polarized xenon to protons located in the hydrophobic cavity of the wheat nonspecific lipid transfer protein.

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 approximately 12,000 makes this approach based on dipolar cross-relaxation with laser-polarized xenon promising for probing protein hydrophobic pockets at ambient pressure.

Solution structure and lipid binding of a nonspecific lipid transfer protein extracted from maize seeds.

The three-dimensional solution structure of a nonspecific lipid transfer protein extracted from maize seeds determined by 1H NMR spectroscopy is described. This cationic protein consists of 93 amino acid residues. Its structure was determined from 1,091 NOE-derived distance restraints, including 929 interresidue connectivities and 197 dihedral restraints (phi, psi, chi 1) derived from NOEs and 3J coupling constants. The global fold involving four helical fragments connected by three loops and a C-terminal tail without regular secondary structures is stabilized by four disulfide bridges. The most striking feature of this structure is the existence of an internal hydrophobic cavity running through the whole molecule. The global fold of this protein, very similar to that of a previously described lipid transfer protein extracted from wheat seeds (Gincel E et al., 1994, Eur J Biochem 226:413-422) constitutes a new architecture for alpha-class proteins. 1H NMR and fluorescence studies show that this protein forms well-defined complexes in aqueous solution with lysophosphatidylcholine. Dissociation constants, Kd, of 1.9 +/- 0.6 x 10(-6) M and > 10(-3) M were obtained with lyso-C16 and -C12, respectively. A structure model for a lipid-protein complex is proposed in which the aliphatic chain of the phospholipid is inserted in the internal cavity and the polar head interacts with the charged side chains located at one end of this cavity. Our model for the lipid-protein complex is qualitatively very similar to the recently published crystal structure (Shin DH et al., 1995, Structure 3:189-199).

Comparison of solution structure of free and complexed lac operator by molecular modelling with NMR constraints.

The structure difference between the free operator of the lac system d(GCTCACAAT).d(ATTGTGAGC) and the same operator complexed to the headpiece of the lac repressor has been investigated by 2-D-1H NMR spectroscopy in conjunction with molecular modelling in internal coordinates (JUMNA). The free and complexed operator adopt both a right-handed B helical conformation, but a more detailed analysis of the conformational parameters using the Curves program shows striking differences in the groove geometries, the rises, the twists and the total bending.

Progress in multidimensional NMR investigations of peptide and protein 3-D structures in solution. From structure to functional aspects.

2-D and 3-D NMR techniques were used to investigate the conformations in solution of several peptides and proteins for which crystalline structures are not available yet. Insect defensin A is a small (40 aa) antibiotic protein exhibiting a characteristic 'loop-helix-beta-sheet' structure. A striking analogy was found with charybdotoxin, a scorpion toxin in which a CSH (cysteine stabilized alpha-helix) motif is also present. Wheat phospholipid transfer protein (PLTP) (90 aa) has a 3-D structure resulting from the packing of four helices and of a C-terminal less well-defined fragment. Preliminary results show that PLTP forms a complex with lyso-PC and that such an interaction results in a conformational change affecting principally the C-terminal half of the protein. A last example is given with surfactin, a lipopeptide biosurfactant from bacterial origin. Its protonated form shows a very compact structure in which the two acidic residues located on the top of a 'horse saddle' topology face each other, whereas the ionized form could adopt a more extended conformation. A common property of these compounds is their capacity to interact with lipids. The present structural data open the way for a further establishment of structure-activity relationships.

Molecular modelling study of the netropsin complexation with a nucleic acid triple helix.

A detailed molecular mechanical study has been made on the complexes of netropsin with the double stranded oligonucleotide (dA)12.(dT)12 and with the triple helix (dA)12.(dT)12.(dT)12. The complexes were built using computer graphics and energy refined using JUMNA program. In agreement with circular dichroism experiments we have shown that 3 netropsins can bind the minor grooves of the triple helix and of the double helix. The groove geometry in the duplex and in the triplex is very similar. However a detailed analysis of the energetic terms shows, in agreement with thermal denaturation studies, that the affinity of netropsin toward the double helices is larger than towards triple helices.

Computer simulation of aqueous biomolecular systems.

Computer simulation techniques are increasingly being used to predict structural and thermodynamic properties of large heterogeneous macromolecule and solvent assemblies. We discuss, with examples from our own studies, some problems we and others have experienced in using these techniques, which were originally devised for simple liquids. In particular, we consider the problems which arise from the large size and heterogeneity of macromolecule water systems, comparisons with experimental data and equilibrium and sampling procedures.

Distortions of the DNA double helix induced by 1,3-trans-diamminedichloroplatinum(II)-intrastrand cross-link: an internal coordinate molecular modeling study.

A trans-diamminedichloroplatinum(II) (trans-DDP) intrastrand adduct within the sequence d(TCTG*TG*TC).d(GACACAGA) (where G* represents a platinated guanine) is modeled on the basis of qualitative experimental data concerning global unwinding and curvature as well as information on base pairing. Modeling is performed using the internal coordinate JUMNA program, specific to nucleic acids, and modified to include the possibility of covalently bound ligands. Calibration of the energy functions representing the Pt-N7 bond with guanine is described. The platinum atom and the platinum-nitrogen bonds are parameterized for use in the Hückel Del Re method to calculate monopoles at each atom. These monopoles are consistent with the Flex force field included in Jumna. By developing an appropriate minimization protocol we are able to generate stable, distorted three-dimensional structures compatible with the experimental data and including an unusually high global unwinding. No a priori geometric assumptions are made in generating these structures.

Androctonin, a novel antimicrobial peptide from scorpion Androctonus australis: solution structure and molecular dynamics simulations in the presence of a lipid monolayer.

Androctonin is a highly cationic antimicrobial peptide from scorpion exhibiting a broad spectrum of activities against bacteria and fungi. It contains 25 amino acids including four cysteine residues forming two disulfide bridges. We report here on the determination of its solution structure by conventional two-dimensional (2D) 1H-NMR spectroscopy and molecular modelling using distance geometry and molecular dynamics methods. The structure of androctonin involves a well-defined highly twisted anti-parallel beta-sheet with strands connected by a more variable positively charged turn. A comparison with the structure of tachyplesin I (horseshoe crab) reveals that the amphiphilic character of the protein surface of this homologous peptide is not observed in androctonin. We have undertaken a 200-ps molecular dynamics simulation study on a system including one androctonin molecule and a monolayer of DMPG (1,2-dimyristoylphosphatidylglycerol) lipids. On the basis of this simulation, the first steps of the membrane permeabilization process are discussed.

Sequence dependent hydration of DNA.

The transitions between the different helical conformations of DNA depend on the base sequence and the ambient conditions such as humidity and counter-ion concentration. In this study energy minimization techniques have been used to locate water molecule sites around nucleotides especially those which form hydrogen bonds between two or more nucleotide atoms and thus form solvent mediated bridges. We have studied several sequences and find that those which are known not to exist in the low hydration 'A' form have very similar number of bridging sites in both 'A' and 'B' conformations. Those sequences which are found in the 'A' conformation have considerably more bridging sites in this low hydration form than in the 'B' conformation. Sequence related solvent effects for a given conformation have also been analysed.

Solvent bridging sites in A- and B-DNA helices.

Nucleotide hydration is important for the understanding of the stability of and the transitions between the different helical conformations of DNA. We have used energy minimization and geometric criteria in order to look for possible sites for solvent which can bridge more than one polar or charged atomic group on a nucleotide. Such bridging sites between phosphate groups have been seen experimentally and used to explain the A to B transition. We show that these phosphate bridging sites occur at energy minima around A-DNA but do not occur around B-DNA. We also find that there are further low energy bridging sites which depend on sequence and which enable the more economical hydration of the A form.

A fractional charge model for empirical calculations of peptide-water interactions.

The properties of an empirical model of interaction between a water molecule and polar groups of peptides or small peptides are explored. The H2O molecule is represented by a four-point charges distribution. In electron donor groups, a point charge is located on the axis of the lone pairs orbitals in order to introduce some directionality in hydrogen bonds. The effective potential is approximated by the sum of the coulombic interactions between point charges distribution and of a 6--12 atom-atom potential. The coefficients of this last potential are first adjusted by simulating the geometry of the water dimer. Equilibrium configurations of associated polar molecules and H2O predicted by the model are found to be in good agreement with those resulting from more sophisticated ab initio SCF calculations. Interactions between H2O and the side-chains of the cyclic dipeptide C(L-Thr-L-His) are then calculated. It is shown that internal bridging by water is an essential effect of the solvent. The experimental position of the H2O molecule is reproduced, stability of which depends also on intermolecular interactions.

Biomolecular energy calculations using transputer technology.

We report an application of current parallel processing transputer technology which has readily achieved a 25-fold reduction in computational time of peptide-solvent interactions.

Solution structure of the parallel-stranded duplex oligonucleotide alpha-d(TCTAAAC)-beta-d(AGATTTG) via complete relaxation matrix analysis of the NOE effects and molecular mechanics calculations.

The solution structure of the duplex formed by the association of the unnatural oligonucleotide alpha-d(TCTAAAC) with its natural and parallel complementary sequence beta-d(AGATTTG) was investigated by nuclear magnetic resonance spectroscopy and constrained molecular mechanics calculations. The structure was refined on the basis of interproton distances determined by NOE measurements for a series of mixing times. The NOE values were converted to distances by using the complete 134 x 134 relaxation matrix including all proton dipole-dipole interactions and spin diffusion. The computation of the relaxation matrix requires the Cartesian coordinates of the oligonucleotide, which are not known, a priori. To avoid this ambiguity, we used an iterative procedure in which the new distance constraints are obtained by using the complete relaxation matrix calculated from the previous structure. After three iterations, the process converged. The unnatural duplex alpha-d(TCTAAAC)-beta-d(AGATTTG) adopts in solution a right-helical structure with Watson-Crick base pairing, an anti conformation on the glycosyl linkage on the beta-strand, a syn conformation on the alpha-strand, and a 3'-exo conformation of the deoxyriboses for both sugar anomers. The three-dimensional structure obtained allowed us to describe the local heterogeneity of the duplex.

Homology modelling of an antimicrobial protein, Ace-AMP1, from lipid transfer protein structures.

BACKGROUND: Plant nonspecific lipid transfer proteins (ns-LTPs) are small basic proteins that facilitate lipid shuttling between membranes in vitro. The function of ns-LTPs in vivo is still unknown. It has been suggested, in relation to their lipid binding ability, that they may be involved in cutin formation. Alternatively, they may act in the plant defence system against pathogenic agents. Ace-AMP1 is an antimicrobial protein extracted from onion seed that shows sequence homology with ns-LTPs but that is unable to transfer lipids. We have recently determined the three-dimensional structure of wheat and maize ns-LTPs. In order to compare the structural features of Ace-AMP1 and ns-LTPs, we have used the comparative modelling software MODELLER to predict the structure of Ace-AMP1. RESULTS: The global fold of Ace-AMP1 is very similar to those of ns-LTPs, involving four helices and a C-terminal tail without secondary structure elements. The structure of maize and wheat ns-LTP is characterized by the existence of a tunnel-like hydrophobic cavity in which a lipid molecule can be inserted. In the Ace-AMP1 structure, this cavity is blocked by a number of bulky residues. Similarly, the electrostatic potential contours of ns-LTPs show some common features that were not observed in Ace-AMP1. CONCLUSIONS: Although Ace-AMP1 displays a similar global fold to ns-LTPs, it does not present a hydrophobic cavity, which may explain why Ace-AMP1 cannot shuttle lipids between membranes in vitro. The large differences in the electrostatic properties of Ace-AMP1 and ns-LTPs suggest a different mode of interaction with membranes.

The active site of drosomycin, a small insect antifungal protein, delineated by comparison with the modeled structure of Rs-AFP2, a plant antifungal protein.

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 beta-sheet and an alpha-helix, the protein global fold being maintained by four disulfide bridges. Rs-AFP2 is a plant antifungal protein exhibiting 41% sequence similarity with drosomycin. Mutational analysis of Rs-AFP2 showed the importance of some residues in the antifungal activity of the protein against the fungus target. In order to determine the structural features responsible for antifungal activity in both drosomycin and Rs-AFP2, we modeled the three-dimensional structure of Rs-AFP2, and of other antifungal proteins, using the solution structure of drosomycin as a template. Structure analysis of drosomycin and Rs-AFP2, and comparisons with the other modeled antifungal structures, revealed that the two proteins shared a hydrophobic cluster located at the protein surface in which a lysine residue is embedded. Based on these close structural similarities and the experimental data available for Rs-AFP2 mutants, an antifungal active site of the insect protein is proposed.

The wide binding properties of a wheat nonspecific lipid transfer protein. Solution structure of a complex with prostaglandin B2.

The 3D solution structure of wheat nonspecific lipid transfer protein (ns-LTP) complexed with prostaglandin B2, a lipid with both vinyl and hydroxylated groups, has been determined by 1H 2D NMR. The global fold of the protein is close to the previously published structures of wheat, maize, barley and rice ns-LTPs. The ligand is almost completely embedded in the hydrophobic core of the protein. Structure comparisons of free and bound wheat ns-LTP reveal that the binding of prostaglandin B2 hardly affects the global fold of the protein. The structural data on this unusual complex are discussed and compared with other known ns-LTP lipid-complexes.

Peptides corresponding to the N- and C-terminal parts of PEBP are well-structured in solution: new insights into their possible interaction with partners in vivo.

Recently, it has been shown that mammalian PEBPs are implicated in several signalling pathways controlling the cellular cycle. In particular, during brain development, the N-terminal part of mammalian PEBP is specifically cleaved and the resulting 11 amino acid peptide stimulates the growth and activity of acetylcholinergic neurons. The crystallographic structure of bovine and human PEBPs has revealed that their N- and C-terminal parts are accessible and exposed to the solvent suggesting that they may be involved in specific interactions with cellular partners. We have chemically synthetized the two peptides corresponding to these terminal parts and studied their structure in solution by circular dichroism and NMR spectroscopies: both of them are well-structured. The N-terminal peptide is composed of a series of turns, leading to a hook conformation. The C-terminal peptide displays a globally helical conformation similar to that observed in the whole protein; it is characterized by an amphipatic feature with a hydrophobic cluster located on one side. These structural features enlighten previous fluorescence and monolayer experiments and give new insights on the roles of both PEBP termini.

Distortions induced in DNA by cis-platinum interstrand adducts.

A 22 base pair double-stranded oligonucleotide containing a unique interstrand adduct resulting from chelation of the two guanine residues within the central sequence d(TGCT/AGCA) by a cis-platinum residue has been studied by means of gel electrophoresis, chemical probes, and molecular mechanics. The anomalously slow electrophoretic mobility of the multimers of the platinated and ligated oligomers suggests that the platinated oligonucleotide is bent. The two cytosine residues (complementary to the platinated guanines) are hyperreactive to hydroxylamine, indicating a large exposure of the two bases to the solvent. The adduct does not induce a local denaturation within the flanking sequences since the adenine residues are not reactive with diethyl pyrocarbonate. This is confirmed by the nonreactivity of the complementary T residues with osmium tetraoxide. These results and the molecular mechanics modeling suggest that the interstrand adduct bends the double helix by approximately 55 degrees toward the major groove, that the double helix conserves its average twist angle, and that the distortion induced by the adduct is localized at the platinated sequence d(GC/CG).