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.     

Solution structure of the octamer motif in immunoglobulin genes via restrained molecular dynamics calculations

The solution structure of the DNA decamer d(CATTTGCATC)-d(GATGCAAATG), comprising the octamer motif of immunoglobulin genes, is determined by restrained molecular dynamics (rMD) simulations. The restraint data set includes interproton distances and torsion angles for the deoxyribose sugar ring which were previously obtained by a complete relaxation matrix analysis of the two-dimensional nuclear Overhauser enhancement (2D NOE) intensities and by the quantitative simulation of cross-peaks in double-quantum-filtered correlated (2QF-COSY) spectra. The influence of torsion angles and the number of experimental distance restraints on the structural refinement has been systematically examined. Omitting part of the experimental NOE-derived distances results in reduced restraint violations and lower R factors but impairs structural convergence in the rMD refinement. Eight separate restrained molecular dynamics simulations were carried out for 20 ps each, starting from either energy-minimized A- or B-DNA. Mutual atomic root-mean-square (rms) differences among the refined structures are well below 1 A and comparable to the rms fluctuations of the atoms about their average position, indicating convergence to essentially identical structures. The average refined structure was subjected to an additional 100 ps of rMD simulations and analyzed in terms of average torsion angles and helical parameters. The B-type duplex exhibits clear sequence-dependent variations in its geometry with a narrow minor groove at the T3.A3 tract and a large positive roll at the subsequent TG.CA step. This is accompanied by a noticeable bend of the global helix axis into the major groove. There is also evidence of significant flexibility of the sugar-phosphate backbone with rapid interconversion among different conformers.     

Automated 2D NOESY assignment and structure calculation of Crambin(S22/I25) with the self-correcting distance geometry based NOAH/DIAMOD programs

The NOAH/DIAMOD program suite was used to automatically assign an experimental 2D NOESY spectrum of the 46 residue protein crambin(S22/I25), using feedback filtering and self-correcting distance geometry (SECODG). Automatically picked NOESY cross peaks were combined with 157 manually assigned peaks to start NOAH/DIAMOD calculations. At each cycle, DIAMOD was used to calculate an ensemble of 40 structures from these NOE distance constraints and random starting structures. The 10 structures with smallest target function values were analyzed by the structure-based filter, NOAH, and a new set of possible assignments was automatically generated based on chemical shifts and distance constraints violations. After 60 iterations and final energy minimization, the 10 structures with smallest target functions converged to 1.48 A for backbone atoms. Despite several missing chemical shifts, 426 of 613 NOE peaks were unambiguously assigned; 59 peaks were ambiguously assigned. The remaining 128 peaks picked automatically by FELIX are probably primarily noise peaks, with a few real peaks that were not assigned by NOAH due to the incomplete proton chemical shifts list.     

On the mobility of riboflavin 5'-phosphate in Megasphaera elsdenii flavodoxin as studied by 13C-nuclear-magnetic-resonance relaxation

The mobility of the isoalloxazine ring of the prosthetic group of Megasphaera elsdenii flavodoxin was investigated by a 13C relaxation study of the non-protonated ring atoms 2, 4, 4a and 10a. In this study a selectively enriched (greater than 90% 13C) prosthetic group was bound to the apoprotein. T1 and T2 values were determined at two magnetic field strengths, i.e. 8.46 T (90.5 MHz) and 5.88 T (62.8 MHz). Values of nuclear Overhauser effects (NOE) were determined at 5.88 T. It is shown that both the dipole-dipole interaction and the chemical shift anisotropy are important relaxation sources for all the carbon atoms investigated. The results are in agreement with a spectral density function of the isoalloxazine ring in which only the overall reorientational motion of the protein is accounted for. From this it is concluded that the isoalloxazine ring is tightly associated with the apoprotein. The protein-bound isoalloxazine ring does not exhibit large fluctuations on the nanosecond time scale, although small amplitude fluctuations cannot be excluded. This information was obtained by a combination of field-dependent T1 and NOE measurements. T2 values are in agreement with these results. On the basis of the dipolar part of the overall T1 values, the distance between the carbon investigated and the nearest proton was calculated and found to be in fair agreement with the crystallographic results of the related flavodoxin from Clostridium MP. In addition, it is shown that, based on the chemical shift anisotropy as a relaxation source, information on the internal mobility is difficult to obtain. The main reason for this is the low precision in the determination of the chemical shift anisotropy tensor.     

Light and electron microscopic distribution of the AMPA receptor subunit, GluR2, in the spinal cord of control and G86R mutant superoxide dismutase transgenic mice

The NMR structure of the 98 residue beta-elicitin, cryptogein, which induces a defence response in tobacco, was determined using 15N and 13C/15N labelled protein samples. In aqueous solution conditions in the millimolar range, the protein forms a discrete homodimer where the N-terminal helices of each monomer form an interface. The structure was calculated with 1047 intrasubunit and 40 intersubunit NOE derived distance constraints and 236 dihedral angle constraints for each subunit using the molecular dynamics program DYANA. The twenty best conformers were energy-minimized in OPAL to give a root-mean-square deviation to the mean structure of 0.82 A for the backbone atoms and 1.03 A for all heavy atoms. The monomeric structure is nearly identical to the recently derived X-ray crystal structure (backbone rmsd 0.86 A for residues 2 to 97) and shows five helices, a two stranded antiparallel beta-sheet and an omega-loop. Using 1H,15N HSQC spectroscopy the pKa of the N- and C-termini, Tyr12, Asp21, Asp30, Asp72, and Tyr85 were determined and support the proposal of several stabilizing ionic interactions including a salt bridge between Asp21 and Lys62. The hydroxyl hydrogens of Tyr33 and Ser78 are clearly observed indicating that these residues are buried and hydrogen bonded. Two other tyrosines, Tyr47 and Tyr87, show pKa's > 12, however, there is no indication that their hydroxyls are hydrogen bonded. Calculations of theoretical pKa's show general agreement with the experimentally determined values and are similar for both the crystal and solution structures.     

Solution structure of the DNA-binding domain of a human papillomavirus E2 protein: evidence for flexible DNA-binding regions

The three-dimensional structure of the DNA-binding domain of the E2 protein from human papillomavirus-31 was determined by using multidimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy. A total of 1429 NMR-derived distance and dihedral angle restraints were obtained for each of the 83-residue subunits of this symmetric dimer. The average root mean square deviations of 20 structures calculated using a distance geometry-simulated annealing protocol are 0.59 and 0.90 angstroms for the backbone and all heavy atoms, respectively, for residues 2-83. The structure of the human virus protein free in solution consists of an eight-stranded beta-barrel and two pairs of alpha-helices. Although the overall fold of the protein is similar to the crystal structure of the bovine papillomavirus-1 E2 protein when complexed to DNA, several small but interesting differences were observed between these two structures at the subunit interface. In addition, a beta-hairpin that contacts DNA in the crystal structure of the protein-DNA complex is disordered in the NMR structures, and steady-state 1H-15N heteronuclear NOE measurements indicate that this region is highly mobile in the absence of DNA. The recognition helix also appears to be flexible, as evidenced by fast amide exchange rates. This phenomenon has also been observed for a number of other DNA-binding proteins and may constitute a common theme in protein/DNA recognition.     

Solution structure of nodularin. An inhibitor of serine/threonine-specific protein phosphatases

The three-dimensional solution structure of nodularin was studied by NMR and molecular dynamics simulations. The conformation in water was determined from the distance and dihedral data by distance geometry and refined by iterative relaxation matrix analysis. The cyclic backbone adopts a well defined conformation but the remote parts of the side chains of arginine as well as the amino acid derivative Adda have a large spatial dispersion. For the unusual amino acids the partial charges were calculated and nodularin was subjected to molecular dynamic simulations in water. A good agreement was found between experimental and computational data with hydrogen bonds, solvent accessibility, molecular motion, and conformational exchange. The three-dimensional structure resembles very closely that of microcystin-LR in the chemically equivalent segment. Therefore, it is expected that the binding of both microcystins and nodularins to serine/threonine-specific protein phosphatases is similar on an atomic level.     

Solution conformation of the Pseudomonas syringae MSU 16H phytotoxic lipodepsipeptide Pseudomycin A determined by computer simulations using distance geometry and molecular dynamics from NMR data

Pseudomycin A is a cyclic lipodepsinonapeptide phytotoxin produced by a strain of the plant pathogenic bacterium Pseudomonas syringae. Like other members of this family of bacterial metabolites, it is characterised by a fatty acylated cyclic peptide with mixed chirality and lactonic closure. Several biological activities of Pseudomycin A are lower than those found for some of its congeners, a difference which might depend on the diverse number and distribution of charged residues in the peptide moiety. Hence, it was of interest to investigate its conformation in solution. After the complete interpretation of the two-dimensional NMR spectra, NOE data were obtained and the structure was determined by computer simulations, applying distance geometry and molecular dynamics procedures. The conformation of the large ring of Pseudomycin A in solution includes three rigid structural regions interrupted by three short flexible regions that act as hinges. The overall three-dimensional structure of the cyclic moiety is similar to that of previously studied bioactive lipodepsinonapeptides produced by other pseudomonads.     

Insights into the dynamic nature of DNA duplex structure via analysis of nuclear Overhauser effect intensities

Sequence-dependent structures of DNA duplexes in solution can be reliably determined using NMR data if care is taken to determine restraint bounds accurately. This entails use of complete relaxation matrix methods to analyze nuclear Overhauser effect (NOE) spectroscopic cross-peak intensities, yielding accurate distance restraints. NMR studies of various DNA duplexes have suggested that there may be some limited internal motions. First, it is typically not possible to reconcile all vicinal proton coupling constants in deoxyribose rings with a single conformer. In addition, with the increased accuracy of interproton distance measurements afforded by the complete relaxation matrix algorithm MARDIGRAS, we find inconsistencies in certain distances which can most readily be ascribed to limited conformational flexibility, since conformational averaging is nonlinear. As base-sugar interproton distances depend on both sugar pucker and glycosidic torsion angle chi, motion involving these structural variables should be reflected by experimental data. Possible motional models have been considered to account for all of the data for three DNA duplexes. Analysis of intraresidue base-sugar interproton NOE bounds patterns suggests a motional model with individual sugars in equilibrium between S (2'-endo) and N (3'-endo) conformations, with S being preferred. As sugar repuckering is correlated with changes in glycosidic torsion angle chi, different sugar conformers imply different values for chi, but this is insufficient to account for all data. A two-state jump between anti and syn glycosidic conformers was considered, but it was incapable of accounting for all data. However, a model with restricted diffusion (rocking) about the glycosidic bond in addition to sugar repuckering was capable of accommodating all experimental data. This motional model is in qualitative agreement with experimental 13C relaxation-derived order parameter values in a DNA duplex.     

Observation of the A-DNA to B-DNA transition during unrestrained molecular dynamics in aqueous solution

A large challenge in molecular dynamics (MD) simulations of proteins and nucleic acids is to find the correct "experimental" geometry when a simulation is started a significant distance away from it. In this study, we have carried out four unrestrained approximately 1 ns length MD trajectories in aqueous solution on the DNA duplex d(CCAACGTTGG)2, two beginning in a canonical A-DNA structure and two beginning in a canonical B-DNA structure. As judged by root-mean-squared coordinate deviations, average structures computed from all four of the trajectories converge to within approximately 0.8 to 1.6 angstroms (all atoms) of each other, which is 1.3 to 1.7 angstroms (all atoms of the central six residues from each strand) and 3.1 to 3.6 angstroms (all atoms) away from the B-DNA-like X-ray structure reported for this sequence. To our knowledge, this is the first example of multiple nanosecond molecular dynamics trajectories with full representation of DNA charges, solvent and long range electrostatics that demonstrate both internal consistency (two different starting structures and four different trajectories lead to a consistent average structure) and considerable agreement with the X-ray crystal structure of this sequence and NMR data on duplex DNA in aqueous solution. This internal consistency of structure for a given sequence suggests that one can now begin to realistically examine sequence-dependent structural effects in DNA duplexes using molecular dynamics.     

Solution structure and backbone dynamics of component IV Glycera dibranchiata monomeric hemoglobin-CO

The solution structure and backbone dynamics of the recombinant, ferrous CO-ligated form of component IV monomeric hemoglobin from Glycera dibranchiata (GMH4CO) have been characterized by NMR spectroscopy. Distance geometry and simulated annealing calculations utilizing a total of 2550 distance and torsion angle constraints yielded an ensemble of 29 structures with an overall average backbone rmsd of 0.48 A from the average structure. Differences between the solution structure and a related crystal structure are confined to regions of lower precision in either the NMR or X-ray structure, or in regions where the amino acid sequences differ. 15N relaxation measurements at 76.0 and 60.8 MHz were analyzed with an extended model-free approach, and revealed low-frequency motions in the vicinity of the heme, concentrated in the F helix. Amide proton protection factors were obtained from H-D amide exchange measurements on 15N-labeled protein. Patterns in the backbone dynamics and protection factors were shown to correlate with regions of heterogeneity and disorder in the ensemble of NMR structures and with large crystallographic B-factors in the X-ray structures. Surprisingly, while the backbone atoms of the F helix have higher rmsds and larger measures of dynamics on the microsecond to millisecond time scale than the other helices, amide protection factors for residues in the F helix were observed to be similar to those of the other helices. This contrasts with H-D amide exchange measurements on sperm whale myoglobin which indicated low protection for the F helix (S. N. Loh and B. F. Volkman, unpublished results). These results for GMH4 suggest a model in which the F helix undergoes collective motions as a relatively rigid hydrogen-bonded unit, possibly pivoting about a central position near residue Val87.     

Structure of a DNA duplex containing a single 2'-O-methyl-beta-D-araT: combined use of NMR, restrained molecular dynamics, and full relaxation matrix refinement

Two-dimensional 1H NMR spectroscopy was used to determine the solution structure of the double-stranded DNA oligonucleotide d(5'-CGCATATAGCC-3'): d(5'-GGCTAXATGCG-3'), where X is 1-(2-O-methyl-beta-D-arabinofuranosyl)thymine. The structure determination was based on a total relaxation matrix analysis of NOESY cross-peak intensities using the MARDIGRAS program. The improved RANDMARDI procedure was used during the calculations to include the experimental "noise" in the NOESY spectra. The NOE-derived distance restraints were applied in restrained molecular dynamics calculations. Twenty final structures each were generated for the modified DNA duplex from both A-form and B-form DNA starting structures. The root-mean-square deviation of the coordinates for the 40 structures was 0.82 A. The duplex adopts a normal B-DNA-type helix, and the spectra as well as the structure show that the modified nucleotide X adopts a C2'-endo (S) sugar conformation. There are no significant changes in the helix originating from the modified nucleotide. The CH3O group on X is directed toward the major groove, and there seems to be free space for further modifications at this position.     

Solution structure of the SH3 domain from Bruton's tyrosine kinase

X-linked agammaglobulinemia (XLA) is a heritable immunodeficiency caused by mutations in the gene coding for Bruton's tyrosine kinase (Btk). Btk belongs to the Tec family of tyrosine kinases. Each member of the family contains five regions and mutations causing XLA have been isolated in all five regions. We have determined the solution structure of the Src homology 3 (SH3) domain of Btk using two- and three-dimensional nuclear magnetic resonance (NMR) spectroscopy on natural abundance and 15N-labeled protein material. The structure determination is complemented by investigation of backbone dynamics based on 15N NMR relaxation. The Btk SH3 forms a well-defined structure and shows the typical SH3 topology of two short antiparallel beta-sheets packed almost perpendicular to each other in a sandwich-like fold. The N- and C-termini are more flexible as are peptide fragments in the RT and n-Src loops. The studied Btk SH3 fragment adopts two slowly interconverting conformations with a relative concentration ratio of 7:1. The overall fold of the minor form is similar to that of the major form, as judged on the basis of observed NOE connectivities and small chemical shift differences. A tryptophan (W251) ring flip is the favored mechanism for interconversion, although other possibilities cannot be excluded. The side chain of Y223, which becomes autophosphorylated upon activation of Btk, is exposed within the potential SH3 ligand binding site. Finally, we compare the present Btk SH3 structure with other SH3 structures.     

Solution structure of Lqh-8/6, a toxin-like peptide from a scorpion venom--structural heterogeneity induced by proline cis/trans isomerization

Lqh-8/6 is a minor fraction isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus. Here we describe the purification, amino acid sequencing and solution structure determination by NMR and molecular modeling of this peptide. Lqh-8/6 is a small polypeptide (38 residues) which contains 8 half-cystines and is highly similar to another venom component, chlorotoxin. Standard homonuclear methods were used to sequentially assign the proton NMR spectra and to collect spatial restraints for structure determination. Two populations, identified early in the assignment step, are in slow interconversion on the NMR timescale. The two conformers were shown to originate from a cis/trans peptidyl-prolyl isomerization. Using a distance geometry program and simulated annealing protocol under the NMR restraints we obtained 10 final structures for the major conformation (trans isomer). None of the structures showed NOE violations larger than 0.05 nm, and the rmsd value relative to the mean structure (considering the main chain atoms in well-defined secondary structure) is 0.07 nm. The three-dimensional structure contains a short alpha-helix strapped on a small antiparallel beta-strand and an N-terminal extended fragment. The sequence/structure and structure/function relationships of the new scorpion toxin-like peptide are discussed in the context of the present structure determination. This toxin shows a stable, highly populated cis conformer of a peptidyl-prolyl peptide bond.     

Genetic influences on cellular reactions to CNS injury: the reactive response of astrocytes in denervated neuropil regions in mice carrying a mutation (Wld(S)) that causes delayed Wallerian degeneration

Complete relaxation matrix analysis of the off-resonance ROESY (O-ROESY) spectra is presented and demonstrated for a synthetic DNA duplex with two G-A mismatches, d(GCTGTC-GAAAGC)2, in solution. The internuclear distance and the rotational correlation time of the internuclear vector could be garnered simultaneously using complete relaxation matrix analysis of O-ROESY, by which spin diffusion effects could be accommodated. Correlation times in the terminal and the mismatched regions were significantly reduced compared to those in other regions, indicating the conformational flexibility of the mismatched pair. The average structure obtained by restrained molecular dynamics simulation with inclusion of variations of the rotational correlation times also indicated a general tendency of the mismatched and contiguous bases to flip to the outside of the double strand. Off-resonance ROESY combined with the complete relaxation matrix analysis method may offer an alternative way to investigate the structures and dynamics of biological macromolecules.     

NMR assignment and conformational analysis of the antigenic capsular polysaccharide from Streptococcus pneumoniae type 9N in aqueous solution

Complete 1H and 13C NMR assignments, determined by one- and two-dimensional homo- and hetero-nuclear experiments, are reported for the antigenic capsular polysaccharide (CPS) from Streptococcus pneumoniae serotype 9N (S9 in American nomenclature). Distance constraints derived from 1D NOE difference experiments were combined with energy minimisation (simulated annealing) and molecular dynamics (MD) calculations to determine the most favoured conformation of S9 in aqueous solution at 70 degrees C. NOE values simulated for several static conformational models using the NOEMOL program did not correlate well with experimental values, whereas time averaged interproton distances calculated from 500 ps of restrained MD (using the Tropp formalism to account for rapid internal mobility) were in close agreement with experimentally derived distance estimates.     

Nuclear magnetic resonance solution structure of the plasminogen-activator protein staphylokinase

Staphylokinase, a 15.5 kDa protein from Staphylococcus aureus, is a plasminogen activator which is currently undergoing clinical trials for the therapy of myocardial infarction and peripheral thrombosis. The three-dimensional (3D) NMR solution structure has been determined by multidimensional heteronuclear NMR spectroscopy on uniformly 15N- and 15N,13C-labeled samples of staphylokinase. Structural constraints were obtained from 82 3JHNH alpha as well as 22 3JNH beta scalar coupling constants and 2345 NOE cross-peaks, derived from 15N-edited and 13C-edited 3D NOE spectra. NOE cross-peak assignments were confirmed by analysis of ?15N,13C?-edited and ?13C,13C?-edited 4D NOE spectra. The structure is presented as a family of 20 conformers which show an average rmsd of 1.02 +/- 0.15 A from the mean structure for the backbone atoms. The tertiary structure of staphylokinase shows a well-defined global structure consisting of a central 13-residue alpha-helix flanked by a two-stranded beta-sheet, both of which are located above a five-stranded beta-sheet. Two of the connecting loops exhibit a higher conformational heterogeneity. Overall, staphylokinase shows a strong asymmetry of hydrophilic and hydrophobic surfaces. The N-terminal sequence, including Lys10 which is the site of the initial proteolytic cleavage during activation of plasminogen, folds back onto the protein core, thereby shielding amino acids with functional importance in the plasminogen activation process. From a comparison of the structure with mutational studies, a binding region for plasminogen is proposed.     

Molecular dynamics simulation of protein folding with supersecondary structure constraints

We integrate molecular dynamics simulation methods with a newly developed supersecondary structure prediction method and compute the structure of a protein molecule, crambin. The computed structure is similar to the crystal structure with an rms error of 3.94 A.     

Localized solution structure refinement of an F45W variant of ubiquitin using stochastic boundary molecular dynamics and NMR distance restraints

The local structure within an 8-A radius around residue 45 of a recombinant F45W variant of human ubiquitin has been determined using 67 interproton distance restraints measured by two-dimensional proton NMR. Proton chemical shift evidence indicates that structural perturbations due to the F45W mutation are minimal and limited to the immediate vicinity of the site of mutation. Simulated annealing implemented with stochastic boundary molecular dynamics was applied to refine the structure of Trp 45 and 10 neighboring residues. The stochastic boundary method allowed the entire protein to be reassembled from the refined coordinates and the outlying unrefined coordinates with little distortion at the boundary. Refinement began with four low-energy indole ring orientations of F45W-substituted wild-type (WT) ubiquitin crystal coordinates. Distance restraints were derived from mostly long-range NOE cross peaks with 51 restraints involving the Trp 45 indole ring. Tandem refinements of 64 structures were done using either (1) upper and lower bounds derived from qualitative inspection of NOE crosspeak intensities or (2) quantitative analysis of cross-peak heights using the program MARDIGRAS. Though similar to those based on qualitative restraint, structures obtained using quantitative NOE analysis were superior in terms of precision and accuracy as measured by back-calculated sixth-root R factors. The six-membered portion of the indole ring is nearly coincident with the phenyl ring of the WT and the indole NH is exposed to solvent. Accommodation of the larger ring is accompanied by small perturbations in the backbone and a 120 degrees rotation of the chi 2 dihedral angle of Leu 50.     

Epidemiology of clonorchiasis in Ninh Binh Province, Vietnam

We present the spatial structure of binase, a small extracellular ribonuclease, derived from 1H-NMR* data in aqueous solution. The total of 20 structures were obtained via torsion angle dynamics using DYANA program with experimental NOE and hydrogen bond distance constraints and phi and chi1 dihedral angle constraints. The final structures were energy minimised with ECEPP/2 potential in FANTOM program. Binase consists of three alpha-helices in N-terminal part (residues 6-16, 26-32 and 41-44), five-stranded antiparallel beta-sheet in C-terminal part (residues 51-55, 70-75, 86-90, 94-99 and 104-108) and two-stranded parallel beta-sheet (residues 22-24 and 49-51). Three loops (residues 36-39, 56-67 and 76-83), which play significant role in biological functioning of binase, are flexible in solution. The differences between binase and barnase spatial structures in solution explain the differences in thermostability of binase, barnase and their hybrids.