NMR solution structure of the pathogenesis-related protein P14a

The nuclear magnetic resonance (NMR) structure of the 15 kDa pathogenesis-related protein P14a, which displays antifungicidal activity and is induced in tomato leaves as a response to pathogen infection, was determined using 15N/13C doubly labeled and unlabeled protein samples. In all, 2030 conformational constraints were collected as input for the distance geometry program DIANA. After energy-minimization with the program OPAL the 20 best conformers had an average root-mean-square deviation value relative to the mean coordinates of 0.88 A for the backbone atoms N, C(alpha) and C', and 1.30 A for all heavy atoms. P14a contains four alpha-helices (I to IV) comprising residues 4 to 17, 27 to 40, 64 to 72 and 93 to 98, a short 3(10)-helix of residues 73 to 75 directly following helix III, and a mixed, four-stranded beta-sheet with topology +3x, -2x, +1, containing the residues 24-25, 53 to 58, 104 to 111 and 117 to 124. These regular secondary structure elements form a novel, complex alpha + beta topology in which the alpha-helices I, III and IV and the 3(10)-helix are located above the plane defined by the beta-sheet, and the alpha-helix II lies below this plane. The alpha-helices and beta-strands are thus arranged in three stacked layers, which are stabilized by two distinct hydrophobic cores associated with the two layer interfaces, giving rise to an "alpha-beta-alpha sandwich". The three-dimensional structure of P14a provides initial leads for identification of the so far unknown active sites and the mode of action of the protein, which is of direct interest for the generation of transgenic plants with improved host defense properties.     

Structures of the reduced and mercury-bound forms of MerP, the periplasmic protein from the bacterial mercury detoxification system

Bacteria carrying plasmids with the mer operon, which encodes the proteins responsible for the bacterial mercury detoxification system, have the ability to transport Hg(II) across the cell membrane into the cytoplasm where it is reduced to Hg(0). This is significant because metallic mercury is relatively nontoxic and volatile and thus can be passively eliminated. The structures of the reduced and mercury-bound forms of merP, the periplasmic protein, which binds Hg(II) and transfers it to the membrane transport protein merT, have been determined in aqueous solution by multidimensional NMR spectroscopy. The 72-residue merP protein has a betaalpha betabeta alphabeta fold with the two alpha helices overlaying a four-strand antiparallel beta sheet. Structural differences between the reduced and mercury-bound forms of merP are localized to the metal binding loop containing the consensus sequence GMTCXXC. The structure of the mercury-bound form of merP shows that Hg(II) is bicoordinate with the Cys side chain ligands, and this is confirmed by the chemical shift frequency of the 199Hg resonance.     

NMR assignments, secondary structure and hydration of oxidized Escherichia coli flavodoxin

Recombinant flavodoxin from Escherichia coli was uniformly enriched with 15N and 13C isotopes and its oxidized form in aqueous solution investigated by three-dimensional NMR spectroscopy. Nearly complete 1H, 15N and 13C resonance assignments were obtained. The secondary structure was determined from chemical shift, NOE and 3J(HNH alpha) coupling constant data. Like homologous long-chain flavodoxins, E. coli flavodoxin contains a five-stranded parallel beta-sheet and five helices. The beta-strands were found to comprise the residues 3-8, 29-34, 48-56, 80-89, 114-116 and 141-145. The helices comprise residues 12-25, 40-45, 62-73, 98-108 and 152-166. The FMN-binding site was determined by intermolecular NOEs and low-field shifted amide proton resonances induced by the phosphoester group of the cofactor. The data are in good agreement with a previously predicted model of E. coli flavodoxin [Havel, T. F. (1993) Mol. Sim. 10, 175-210]. The analysis of of water-flavodoxin NOEs revealed the presence of two, possibly three, buried hydration water molecules which are located at sites, where homologous flavodoxins from Anacystis nidulans and Anabena 7120 contain conserved hydration water molecules. One of these water molecules mediates hydrogen bonds between the protein backbone and the ribityl chain of the FMN cofactor.     

NMR solution structure of the receptor binding domain of Pseudomonas aeruginosa pilin strain P1. Identification of a beta-turn

The solution structure of the peptide antigen from the receptor binding domain of Pseudomonas aeruginosa strain P1 has been determined using two-dimensional 1H NMR techniques. Ensembles of solution conformations for the trans form of this 23-residue disulfide bridged peptide have been generated using a simulated annealing procedure in conjunction with distance and torsion angle restraints derived from NMR data. Comparison of the NMR-derived solution structures of the P1 peptide with those previously determined for the 17-residue PAK, PAO and KB7 strain peptides [McInnes, C., et al. (1993) Biochemistry 32, 13432-13440; Campbell, A.P., et al. (1995) Biochemistry 34, 16255-16268] reveals the common structural motif of a beta-turn, which may be the necessary structural requirement for recognition of a common cell surface receptor and a common cross-reactive antibody to which all four strains bind. The importance of this conserved beta-turn in the PAK, PAO, KB7 and P1 peptides is discussed with regard to the design of a synthetic peptide vaccine effective against multiple strains of Pseudomonas aeruginosa infections.     

Use of proline mutants to help solve the NMR solution structure of type III antifreeze protein

To help understand the structure/function relationships in antifreeze proteins (AFP), and to define the motifs required for ice binding, a Type III AFP suitable for two-dimensional (2D) NMR studies was produced in Escherichia coli. A synthetic gene for one of the Type III AFP isoforms was assembled in a T7 polymerase-directed expression vector. The 67-amino acid-long gene product differed from the natural AFP by inclusion of an N-terminal methionine but was indistinguishable in activity. The NMR spectra of this AFP were complicated by cis-trans proline isomerization from the C-terminal sequence YPPA. Substitution of this sequence by YAA eliminated isomer signals without altering the activity or structure of the mutant AFP. This variant (rQAE m1.1) was selected for sequential assignment and the secondary structure determination using 2D 1H NMR spectroscopy. Nine beta-strands are paired to form two triple-stranded antiparallel sheets and one double-stranded antiparallel sheet. Two further proline replacements, P29A and P33A, were made to delineate the role of conserved prolines in Type III AFP. These mutants were valuable in clarifying ambiguous NMR spectral assignments amongst the remaining six prolines of rQAE m1.1. In contrast to the replacement of the C-terminal prolyl residues, the exchange of P29 and P33 caused some structural changes and significantly decreased protein solubility and antifreeze activity.     

The three-dimensional NMR solution structure of alpha-cobratoxin at pH 7.5 and conformational differences with the NMR solution structure at pH 3.2

The 3D solution structure of alpha-cobratoxin, a neurotoxin purified from the Naja naja siamensis snake venom, has been determined by Nuclear Magnetic Resonance spectroscopy, in conjunction with distance geometry and restrained molecular dynamics, at pH 7.5. A total of 490 distance restraints were obtained from NOE intensities and 25 phi dihedral angle restraints deduced from J-coupling data. The generated structures are well defined with root mean square deviations from a geometrical mean structure of 0.107 +/- 0.036 nm for the backbone atoms and 0.128 +/- 0.073 nm for the side-chain atoms (considering residues 1 to 66 minus 26 to 35). A comparison between the generated structures at pH 7.5 and the mean NMR solution structure at pH 3.2 revealed that the 3D structure of alpha-cobratoxin is more compact at neutral pH. This major difference is mainly due to the pH-dependent conformational variations of three residues His18, Thr44 and Thr59.     

Determination of the three-dimensional solution structure of Raphanus sativus antifungal protein 1 by 1H NMR

Raphanus sativus Antifungal Protein 1 (Rs-AFP1) is a 51 amino acid residue plant defensin isolated from radish (Raphanus sativus L.) seeds. The three-dimensional structure in aqueous solution has been determined from two-dimensional 1H NMR data recorded at 500 MHz using the DIANA/REDAC calculation protocols. Experimental constraints consisted of 787 interproton distances extracted from NOE cross-peaks, 89 torsional constraints from 106 vicinal interproton coupling constants and 32 stereospecific assignments of prochiral protons. Further refinement by simulated annealing resulted in a set of 20 structures having pairwise root-mean-square differences of 1.35(+/- 0.35) A over the backbone heavy atoms and 2.11(+/- 0.46) A over all heavy atoms. The molecule adopts a compact globular fold comprising an alpha-helix from Asn18 till Leu28 and a triple-stranded beta-sheet (beta 1 = Lys2-Arg6, beta 2 = His33-Tyr38 and beta 3 = His43-Pro50). The central strand of this beta-sheet is connected by two disulfide bridges (Cys21-Cys45 and Cys25-Cys47) to the alpha-helix. The connection between beta-strand 2 and 3 is formed by a type VIa beta-turn. Even the loop (Pro7 to Asn17) between beta-strand 1 and the alpha-helix is relatively well defined. The structure of Raphanus sativus Antifungal Protein 1 features all the characteristics of the "cysteine stabilized alpha beta motif". A comparison of the complete structure and of the regions important for interaction with the fungal receptor according to a mutational study, is made with the structure of gamma-thionin, a plant defensin that has no antifungal activity. It is concluded that this interaction is both electrostatic and specific, and some possible scenarios for the mode of action are given.     

The baculovirus single-stranded DNA binding protein, LEF-3, forms a homotrimer in solution

LEF-3 is one of six proteins from Autographa californica multinucleocapsid polyhedrosis virus required for transient DNA replication and has the properties of a single-stranded DNA binding protein. In this report we demonstrate that LEF-3 interacts with itself in both yeast two-hybrid assays and glutathione S-transferase fusion affinity assays. LEF-3 deletion clones which were unable to interact with full-length LEF-3 also failed to support transient DNA replication, suggesting that this interaction is required for the proper function of LEF-3. LEF-3 was purified to homogeneity and characterized by analytical ultracentrifugation and native polyacrylamide gel electrophoresis. These studies revealed that LEF-3 was present as a 132-kDa complex, indicating that its native conformation is that of a homotrimer. This result was confirmed by cross-linking with glutaraldehyde followed by matrix-assisted laser desorption/ionization mass spectrometry.     

Overexpression of MerT, the mercuric ion transport protein of transposon Tn501, and genetic selection of mercury hypersensitivity mutations

The small (116 amino acids) inner membrane protein MerT encoded by the transposon Tn501 has been overexpressed under the control of the bacteriophage T7 expression system. Random mutants of MerT were made and screened for loss of mercuric ion hypersensitivity. Several mutant merT genes were selected and sequenced: Cys24Arg and Cys25Tyr mutations abolish mercury resistance, as do charge-substitution mutations in the first predicted transmembrane helix (Gly14Arg, Gly15Arg, Gly27Arg, Ala18Asp), and the termination mutations Trp66Ter and Cys82Ter.     

High-resolution solution NMR structure of the minimal active domain of the human immunodeficiency virus type-2 nucleocapsid protein

The retroviral nucleocapsid (NC) protein is a multifunctional protein essential for RNA genome packaging and viral infectivity. The NC protein, NCp8, of the human immunodeficiency virus type-II (HIV-2) is a 49 amino acid peptide containing two zinc fingers, of the type C-X2-C-X4-H-X4-C, connected by seven amino acid residues, called the "basic amino acid cluster." It has been shown that the N-terminal zinc finger flanked by the basic amino acid cluster is the minimal active domain for the specific binding to viral RNA and other functions. However, the structure-activity relationships of NCp8 have not been investigated in detail. In the present study, the three-dimensional structure of a 29 amino acid peptide, including the minimal active domain (NCp8-fl), was determined by two-dimensional 1H NMR spectroscopy with simulated annealing calculations. A total of 15 converged structures of NCp8-fl were obtained on the basis of 355 experimental constraints, including 343 distance constraints obtained from nuclear Overhauser effect connectivities, 12 torsion angle (phi, chi1) constraints, and four constraints for zinc binding. The root-mean-square deviation of the 15 converged structures was 0.29 +/- 0.04 A for the backbone atoms (N, C(alpha), C) and 1.27 +/- 0.13 A for all heavy atoms. Interestingly, the basic amino acid cluster itself was defined well, with a loop-like conformation in which three arginine residues in the cluster and one arginine residue in the zinc finger are located approximately in the same plane of the molecule and are exposed to the solvent. The structure-activity relationships are discussed on the basis of the comparison of this well-defined structure with those of other NC proteins.     

Solution structure of the recombinant oxidized rabbit uteroglobin using homonuclear and heteronuclear multidimensional NMR

Rabbit uteroglobin (rab-UG) is a 16-kDa homodimeric secretory protein with potent anti-inflammatory/immunomodulatory properties. Its physiological role is still unclear, although it was observed that several small hydrophobic molecules bind to the oxidized and the reduced uteroglobin. It is suggested that the formation and/or disruption of the two disulphide bridges not only regulates this binding itself, but also the affinity to the ligand. The determination of the solution structure has been started with the assignment of 1H, 15N and 13C resonances of the oxidized rabbit uteroglobin, based on several two-dimensional and three-dimensional homonuclear and heteronuclear double and triple resonance experiments. The assignment was possible with the overproduction of the wild-type as well as of uniformly 15N-labeled and 15N/13C-labeled samples of the recombinant protein. A complete assignment of 1H, 15N and 13C resonances, the secondary-structure elements and the tertiary structure in solution is presented. The tertiary solution structure was found to be in good agreement with the previously determined crystal structure of rab-UG and with the solution structure of human uteroglobin (h-UG). h-UG and rab-UG are extremely stable proteins within a wide range of pH and temperatures. Some of the binding characteristics of ligands of rab-UG and a mutant with all cysteine residues exchanged to serine residues are discussed.     

Partitioning of rhodanese onto GroEL. Chaperonin binds a reversibly oxidized form derived from the native protein

The mammalian mitochondrial enzyme, rhodanese, can form stable complexes with the Escherichia coli chaperonin GroEL if it is either refolded from 8 M urea in the presence of chaperonin or is simply added to the chaperonin as the folded conformer at 37 degreesC. In the presence of GroEL, the kinetic profile of the inactivation of native rhodanese followed a single exponential decay. Initially, the inactivation rates showed a dependence on the chaperonin concentration but reached a constant maximum value as the GroEL concentration increased. Over the same time period, in the absence of GroEL, native rhodanese showed only a small decline in activity. The addition of a non-denaturing concentration of urea accelerated the inactivation and partitioning of rhodanese onto GroEL. These results suggest that the GroEL chaperonin may facilitate protein unfolding indirectly by interacting with intermediates that exist in equilibrium with native rhodanese. The activity of GroEL-bound rhodanese can be completely recovered upon addition of GroES and ATP. The reactivation kinetics and commitment rates for GroEL-rhodanese complexes prepared from either unfolded or native rhodanese were identical. However, when rhodanese was allowed to inactivate spontaneously in the absence of GroEL, no recovery of activity was observed upon addition of GroEL, GroES, and ATP. Interestingly, the partitioning of rhodanese and its subsequent inactivation did not occur when native rhodanese and GroEL were incubated under anaerobic conditions. Thus, our results strongly suggest that the inactive intermediate that partitions onto GroEL is the reversibly oxidized form of rhodanese.     

19F NMR in the measurement of binding affinities of chloroeremomycin to model bacterial cell-wall surfaces that mimic VanA and VanB resistance

The reviews of research, summarizing a great amount of studies in a manageable format, are invaluable tools for physicians, inundated with enormous amount of biomedical information. However, narrative reviews are often misleading because, mixing together opinions of authors and results of research, the relation between clinical recommendation and evidence is partial and based on a biased citation of primary studies. In contrast to narrative reviews, the systematic reviews assemble, critically appraise, and synthesize the results of primary studies addressing a specific topic. Additionally their authors use strategies for minimizing bias and random error. The science of systematic reviews is now supported by the Cochrane Collaboration, an international network established for "preparing, maintaining and disseminating systematic reviews of the effects of health care". The authors provide tools for searching, critically appraising and using in practice the systematic reviews, which use can help physicians to improve the transfer of research in clinical practice, a task obliged by limitation of financial resources to physicians of any health service.     

Lysophosphatidylcholine is involved in the antigenicity of oxidized LDL

Lysophosphatidylcholine (LPC) is formed by hydrolysis of PC in low density lipoprotein (LDL) and cell membranes by phospholipase A2 or by oxidation. Oxidized (ox) LDL activates endothelial cells, an effect mimicked by LPC. oxLDL also has the capacity to activate T and B cells, and antibody titers to oxLDL are related to the degree of atherosclerosis. The antigen in oxLDL responsible for its immune-stimulatory capacity is not well characterized, and we hypothesized that LPC was involved. We demonstrate herein the presence of antibodies against LPC, both of the IgG and IgM isotype, in 210 healthy individuals. This antibody reactivity was not specifically related to oxidation of the fatty acid moiety in LPC, since LPC containing only palmitic acid showed antibody titers equivalent to those of LPC containing unsaturated fatty acids. Antibody titers to PC were low compared with LPC, and hydrolysis of PC at the sn-2 position is thus essential for immune reactivity. There was a close correlation between anti-oxLDL and anti-LPC antibodies. Furthermore, LPC competitively inhibited anti-oxLDL reactivity, which indicates that LPC may explain a significant part of the immune-stimulatory properties of oxLDL. LPC, being a lipid, is not likely to be an antigen itself. Instead, LPC could form immunogenic complexes with peptides, which may induce and potentiate immune reactions in the vessel wall. This study adds to the evidence that LPC is an important component of oxLDL and emphasizes the potential role of phospholipase A2 in atherosclerosis.     

The NMR solution structure of intestinal fatty acid-binding protein complexed with palmitate: application of a novel distance geometry algorithm

The three-dimensional solution structure of rat intestinal fatty acid-binding protein (I-FABP) complexed with palmitate has been determined using multidimensional triple-resonance NMR methods. The structure is based on 3889 conformational restraints derived mostly from 3-D 13C- and 15N-resolved nuclear Overhauser (NOESY) experiments. The 3-D NOESY data for this 15.4 kDa complex contained an average of nine possible interpretations per cross-peak. To circumvent this ambiguity, an eight-stage iterative procedure was employed to gradually interpret and introduce unambiguous distance restraints during subsequent rounds of structure calculations. The first stage of this procedure relied critically upon an initial structural model based on the consensus 1H/13C chemical shift-derived secondary structure and a set of symmetry-checked restraints derived from the 3-D 13C-resolved NOESY spectrum. The structures were calculated using DISTGEOM, a program that implements a novel distance geometry algorithm with pairwise Gaussian metrization. A central feature of this algorithm is the use of an iteratively optimized Gaussian distribution for the selection of trial distances, which overcomes the tendency of metrization to produce crushed structures. In addition, this algorithm randomly selects pairwise elements of the distance matrix, which results in an improved sampling of conformational space for a given computational effort. The final family of 20 distance geometry/simulated annealing structures exhibited an average pairwise C(alpha) root-mean-square deviation of 0.98 A, and their stereochemical quality, as assessed by PROCHECK, was comparable to that of 2.5 A X-ray crystal structures. The NMR structure was compared with the X-ray crystal structure of the same ligand/protein complex and was found to be essentially identical within the precision of the results. The NMR structure was also compared with that of the palmitate complex with bovine heart FABP, which shares 30% sequence identity with rat I-FABP. The overall folds were the same, but differences were noted with respect to the presence or absence of apparent conformational heterogeneity and the location and conformation of the bound fatty acid.     

Preliminary crystallographic analysis of the oxidized form of a two mono-nuclear iron centres protein from Desulfovibrio desulfuricans ATCC 27774

Crystals of the fully oxidized form of desulfoferrodoxin were obtained by vapor diffusion from a solution containing 20% PEG 4000, 0.1 M HEPES buffer, pH 7.5, and 0.2 M CaCl2. Trigonal and/or rectangular prisms could be obtained, depending on the temperature used for the crystal growth. Trigonal prisms belong to the rhombohedral space group R32, with a = 112.5 A and c = 63.2 A; rectangular prisms belong to the monoclinic space group C2, with a = 77.7 A, b = 80.9 A, c = 53.9 A, and beta = 98.1 degrees. The crystallographic asymmetric unit of the rhombohedral crystal form contains one molecule. There are two molecules in the asymmetric unit of the monoclinic form, in agreement with the self-rotation function.     

The 30-kD domain of protein 4.1 mediates its binding to the carboxyl terminus of pICln, a protein involved in cellular volume regulation

Erythrocyte protein 4.1 (P4.1) is an 80-kD cytoskeletal protein that is important for the maintenance of the structural integrity and flexibility of the red blood cell membrane. Limited chymotryptic digestion of erythroid P4.1 yields 4 structural domains corresponding to the 30-, 16-, 10-, and 22/24-kD domains. Using a yeast two-hybrid system, we isolated cDNA clones encoding pICln that specifically interacts with the 30-kD domain of P4.1. In this report, we show that the carboxyl-terminus (amino acid residues 103-237) of pICln binds to the 30-kD domain of P4.1 in a yeast two-hybrid system. The direct association between the 30-kD domain of P4.1 and pICln was further confirmed by the following findings: (1) the S35-methione-labeled pICln specifically bound to both GST/P4.1-80 (80 kD) and GST/P4.1-30 (30 kD) fusion proteins, but not to the proteins that lack the 30-kD domain; (2) coimmunoprecipitation analysis of the cell extracts from transfected SiHa cells showed that pICln and P4.1 associate in transfected cells. It was reported that pICln can form a complex with actin and may play a role involved in cellular volume regulation. The direct association between P4.1 and pICln suggests that pICln may link P4.1-bound cytoskeletal elements to an unidentified volume-sensitive chloride channel.     

NMR solution structure of the lead-dependent ribozyme: evidence for dynamics in RNA catalysis

The function of social calls emitted by foraging bats has received little study. Here we use observations of free-ranging greater spear-nosed bats, Phyllostomus hastatus, and field playbacks to determine whether audible, broad-band 'screech' calls attract mates, warn conspecifics or influence access to food. Five lines of evidence suggest that screech calls enable adult females from the same roosting group to fly together from the day roost to feeding sites. (1) Seasonal differences in diet influenced the rate of screech calling recorded outside the cave roost, as well as how often bats departed together. Bats called more often and flew in larger groups when feeding on a concentrated resource, balsa, Ochroma lagopus, flowers, in winter than on more dispersed Cecropia peltata fruit in spring. (2) Observations of bats flying outside the cave, in flyways and at feeding sites indicated that screech calls occurred more often when bats flew in groups than alone. (3) Females from the same roosting group were netted at the same feeding site, sometimes simultaneously, several kilometres from the cave. (4) Calling colour-marked adult females outside the cave were joined by a female group member, both on initial departures and on second foraging trips, more often than non-calling bats. (5) Playbacks attracted conspecifics at roost and feeding sites. Screech calls appear to function as contact calls that recruit and coordinate foraging among group members. We postulate that females benefit from foraging with unrelated roost-mates because they can defend feeding sites more effectively. Copyright 1998 The Association for the Study of Animal Behaviour.     

NMR study suggests a major role for Arg111 in maintaining the structure and dynamical properties of type II human cellular retinoic acid binding protein

The solution structure of a site-directed mutant of type-II human cellular retinoic acid binding protein (CRABPII) with Arg111 replaced by methionine (R111M) has been determined by NMR spectroscopy. The sequential assignments of the 1H and 15N resonances of apo-R111M were established by multinuclear multidimensional NMR. The solution structure was calculated from 2302 distance restraints and 77 phi dihedral restraints derived from the NMR data. The root-mean-square deviation of the ensemble of 28 refined conformers that represent the structure from the mean coordinate set derived from them was 0.54 +/- 0.26 and 0.98 +/- 0.23 A for the backbone atoms and all heavy atoms, respectively. The solution structure of apo-R111M is similar to that of wild-type apo-CRABPII. However, there are significant conformational differences between the two proteins, localized mainly to three segments (Leu19-Ala36, Glu73-Cys81, and Leu99-Pro105) clustered around the ligand entrance more than 17 A away from the point mutation. In apo-R111M, all the three segments move toward the center of the ligand entrance so that the opening of the ligand-binding pocket in apo-R111M is much smaller than that in wild-type apo-CRABPII. Furthermore, the ligand-binding pocket of apo-R111M, especially the ligand entrance, is much less flexible than that of apo-CRABPII. Surprisingly, apo-R111M is more similar to holo-CRABPII than to apo-CRABPII in both structure and dynamical properties. The conformational and dynamical changes caused by the mutation are similar to those induced by binding of RA, although the magnitudes of the changes caused by the mutation are smaller than those induced by binding of RA. The results suggest that Arg111 plays a critical role in determining the structure and dynamical properties of CRABPII.