Solution structures of the rabbit neutrophil defensin NP-5

Solution structures of the rabbit neutrophil defensin NP-5 have been determined by 1H nuclear magnetic resonance (n.m.r.) spectroscopy and distance geometry techniques. This 33 amino acid peptide is part of the oxygen-independent mammalian defense system against microbial infection. The structures were generated from 107 n.m.r. derived inter-residue proton-proton distance constraints. A distance geometry algorithm was then used to determine the range of structures consistent with these distance constraints. These distance geometry calculations employed an improved algorithm that allowed the chirality constraints to be relaxed on prochiral centers when it was not possible to make stereo-specific assignments of protons on these centers. This procedure gave superior results compared with standard distance geometry methods and also produced structures that were more consistent with the original n.m.r. data. Analysis of the NP-5 structures shows that the overall folding of the peptide backbone is well defined by the n.m.r. distance information but that the side-chain group conformations are generally less well defined.     

Analysis of side-chain conformational distributions in neutrophil peptide-5 NMR structures

The side-chain conformations have been analyzed in the antimicrobial peptide, Neutrophil Peptide-5 (NP-5), whose structure was independently generated from nmr-derived distance constraints using a distance geometry algorithm. The side-chain and peptide dihedral angle distributions in the nmr structures were compared with those constructed from a data base of high-resolution protein crystal structures. The side-chain conformational preferences for NP-5 in solution are significantly different from those observed in the crystal structure data base. These results indicate that the side-chain conformations are quite disordered for many of the residues of NP-5. The absence of a correlation between the width of the conformational distribution and surface accessibility suggests that the disorder may be due to limitations in the structural information extracted from the nmr data rather than to molecular motion. However, it is also observed that the degree of conformational disorder is only weakly correlated with the number of nuclear Overhauser enhancements to a given side chain. Possible reasons for this are discussed. Molecular mechanics refinement of these structures did not significantly change the side-chain populations. Anomolously wide distributions are observed for rotations about the peptide bonds and the disulfide bonds in the NP-5 distance geometry structures, which are improved by the refinement. The very high degree of order observed for the central dihedral angle of the disulfide bond in the high-resolution crystal data base suggests that the rotation about this bond in proteins is determined by the local potential.     

Side-chain modeling with an optimized scoring function

Modeling side-chain conformations on a fixed protein backbone has a wide application in structure prediction and molecular design. Each effort in this field requires decisions about a rotamer set, scoring function, and search strategy. We have developed a new and simple scoring function, which operates on side-chain rotamers and consists of the following energy terms: contact surface, volume overlap, backbone dependency, electrostatic interactions, and desolvation energy. The weights of these energy terms were optimized to achieve the minimal average root mean square (rms) deviation between the lowest energy rotamer and real side-chain conformation on a training set of high-resolution protein structures. In the course of optimization, for every residue, its side chain was replaced by varying rotamers, whereas conformations for all other residues were kept as they appeared in the crystal structure. We obtained prediction accuracy of 90.4% for chi(1), 78.3% for chi(1 + 2), and 1.18 A overall rms deviation. Furthermore, the derived scoring function combined with a Monte Carlo search algorithm was used to place all side chains onto a protein backbone simultaneously. The average prediction accuracy was 87.9% for chi(1), 73.2% for chi(1 + 2), and 1.34 A rms deviation for 30 protein structures. Our approach was compared with available side-chain construction methods and showed improvement over the best among them: 4.4% for chi(1), 4.7% for chi(1 + 2), and 0.21 A for rms deviation. We hypothesize that the scoring function instead of the search strategy is the main obstacle in side-chain modeling. Additionally, we show that a more detailed rotamer library is expected to increase chi(1 + 2) prediction accuracy but may have little effect on chi(1) prediction accuracy.     

A method for predicting protein structure from sequence

BACKGROUND: The ability to predict the native conformation of a globular protein from its amino-acid sequence is an important unsolved problem of molecular biology. We have previously reported a method in which reduced representations of proteins are folded on a lattice by Monte Carlo simulation, using statistically-derived potentials. When applied to sequences designed to fold into four-helix bundles, this method generated predicted conformations closely resembling the real ones. RESULTS: We now report a hierarchical approach to protein-structure prediction, in which two cycles of the above-mentioned lattice method (the second on a finer lattice) are followed by a full-atom molecular dynamics simulation. The end product of the simulations is thus a full-atom representation of the predicted structure. The application of this procedure to the 60 residue, B domain of staphylococcal protein A predicts a three-helix bundle with a backbone root mean square (rms) deviation of 2.25-3 A from the experimentally determined structure. Further application to a designed, 120 residue monomeric protein, mROP, based on the dimeric ROP protein of Escherichia coli, predicts a left turning, four-helix bundle native state. Although the ultimate assessment of the quality of this prediction awaits the experimental determination of the mROP structure, a comparison of this structure with the set of equivalent residues in the ROP dime- crystal structure indicates that they have a rms deviation of approximately 3.6-4.2 A. CONCLUSION: Thus, for a set of helical proteins that have simple native topologies, the native folds of the proteins can be predicted with reasonable accuracy from their sequences alone. Our approach suggest a direction for future work addressing the protein-folding problem.     

Solution structure of apamin determined by nuclear magnetic resonance and distance geometry

The solution structure of the bee venom neurotoxin apamin has been determined with a distance geometry program using distance constraints derived from NMR. Twenty embedded structures were generated and refined by using the program DSPACE. After error minimization using both conjugate gradient and dynamics algorithms, six structures had very low residual error. Comparisons of these show that the backbone of the peptide is quite well-defined with the largest rms difference between backbone atoms in these structures of 1.34 A. The side chains have far fewer constraints and show greater variability in their positions. The structure derived here is generally consistent with the qualitative model previously described, with most differences occurring in the loop between the beta-turn (residues 2-5) and the C-terminal alpha-helix (residues 9-17). Comparisons are made with previously derived models from NMR data and other methods.     

NMR studies of defensin antimicrobial peptides. 1. Resonance assignment and secondary structure determination of rabbit NP-2 and human HNP-1.

Two-dimensional nuclear magnetic resonance spectroscopy has been used to make resonance assignments of the proton spectra of two defensin antimicrobial peptides, human neutrophil peptide HNP-1 and rabbit neutrophil peptide NP-2. The secondary structures of these peptides were determined from analysis of the proton-proton NOEs and from the positions of slowly exchanging amide protons. Both peptides contain a long stretch of a double-stranded antiparallel beta-sheet in a hairpin conformation that contains a beta-bulge, a short region of triple-stranded beta-sheet, and several tight turns. The NMR results clearly show that HNP-1 forms a dimer or higher order aggregate in solution and that Pro8 exists as a cis peptide bond. The NMR data on these peptides are compared with NMR data for a homologous peptide NP-5 [Bach, A. C., Selsted, M. E., & Pardi, A. (1987) Biochemistry 26, 4389-4397]. Analysis of the conformation-dependent proton chemical shifts shows that it is not possible to confidently judge the structural similarity of the three defensins from chemical shift data alone. However, comparison of the 3JHN alpha coupling constants in NP-2 and NP-5 indicates that the backbone conformations for these peptides are very similar. A more detailed comparison of the solution conformations of the defensins peptides is made in the following paper in this issue where the NMR data are used as input for distance geometry and molecular dynamics calculations to determine the three-dimensional structures of HNP-1 and NP-2.     

The reconstruction of side-chain conformations from protein Cα coordinates

A model of nine proteins including side-chain atoms have been built from the known C^α coordinates and amino acid sequences using a Monte Carlo Protein Building Annealing method. The Cartesian coordinates for the side-chain atoms were established with bond lengths and angles selected randomly from within previously determined ranges. A simulated annealing technique is used to generate some 300 structures with differing side-chain conformations. The atomic coordinates of the backbone atoms are fixed during the simulated annealing process. The coordinates of the side-chain atoms of 300 low energy conformations are averaged to obtain a mean structure that is minimized with the C^α atoms constrained to their position in the x-ray structure using the OPLS/AMBER force field with the GB/SA water model. The rms deviation of the main-chain atoms (without C^β) compared with the corresponding crystal structures is in the range 0.20–0.64 Å. The rms deviation of the side-chain atoms is between 1.72 and 2.71 Å and for all atoms is between 1.19 and 1.99 Å. The method is insensitive to random errors in the C^α positions and the computational requirement is modest. © 1997 John Wiley & Sons, Inc.     

Solution structures of alpha-conotoxin G1 determined by two-dimensional NMR spectroscopy

Two-dimensional NMR data have been used to generate solution structures of alpha-conotoxin G1, a potent peptide antagonist of the acetylcholine receptor. Structural information was obtained in the form of proton-proton internuclear distance constraints, and initial structures were produced with a distance geometry algorithm. Energetically more favorable structures were generated by using the distance geometry structures as input for a constrained energy minimization program. The results of both of these calculations indicate that the overall backbone conformation of the molecule is well-defined by the NMR data whereas the side-chain conformations are generally less well-defined. The main structural features derived from the NMR data were the presence of tight turns centered on residues Pro5 and Arg9. The solution structures are compared with previous proposed models of conotoxin G1, and the NMR data are interpreted in conjunction with chemical modification studies and structural properties of other antagonists of the acetylcholine receptor to gain insight into structure-activity relationships in these peptide toxins.     

Three-dimensional structure of potato carboxypeptidase inhibitor in solution. A study using nuclear magnetic resonance, distance geometry, and restrained molecular dynamics

The solution conformation of potato carboxypeptidase inhibitor (CPI) has been investigated by 1H NMR spectroscopy. The spectrum is assigned in a sequential manner by using two-dimensional NMR techniques to identify through-bond and through-space (less than 5 A) connectivities. A set of 309 approximate interproton distance restraints is derived from the two-dimensional nuclear Overhauser enhancement spectra and used as the basis of a three-dimensional structure determination by a combination of metric matrix distance geometry and restrained molecular dynamics calculations. A total of 11 converged distance geometry structures were computed and refined by using restrained molecular dynamics. The average atomic root mean square (rms) difference between the final 11 structures and the mean structure obtained by averaging their coordinates is 1.4 +/- 0.3 A for residues 2-39 and 0.9 +/- 0.2 A for residues 5-37. The corresponding values for all atoms are 1.9 +/- 0.3 and 1.4 +/- 0.2 A, respectively. The larger values for residues 2-38 relative to those for residues 5-37 arise from the fact that the positions of the N- (residues 1-4) and C- (residues 38-39) terminal tails are rather poorly determined, whereas those of the core of the protein (residues 5-37) are well determined by the experimental interproton distance data. The computed structures are very close to the X-ray structure of CPI in its complex with carboxypeptidase, and the backbone atomic rms difference between the mean of the computed structures and the X-ray structure is only 1.2 A. Nevertheless, there are some real differences present which are evidenced by significant deviations between the experimental upper interproton distance limits and the corresponding interproton distances derived from the X-ray structure. These principally occur in two regions, residues 18-20 and residues 28-30, the latter comprising part of the region of secondary contacts between CPI and carboxypeptidase in the X-ray structure.     

The solution structure of a leucine-zipper motif peptide

We report the complete structure determination of a 34 residue synthetic peptide with the amino acid sequence of the dimerization domain (leucine zipper) of GCN4. A high resolution structure in solution was obtained by 1H-NMR studies and distance geometry calculations followed by restrained energy minimization. A set of 20 final structures was obtained with an average root mean square deviation of 1.3 A for the backbone atoms (excluding the first and the last two residues). The structure contains an uninterrupted helix. A comparison with a structure previously determined for a larger peptide containing both the DNA-binding region (basic region) and the leucine-zipper motif shows the structural independence of the leucine-zipper domain from the contiguous DNA binding region.     

The determination of the three-dimensional structure of barley serine proteinase inhibitor 2 by nuclear magnetic resonance, distance geometry and restrained molecular dynamics

The solution structure of the 64 residue structured domain (residues 20-83) of barley serine proteinase inhibitor 2 (BSPI-2) is determined on the basis of 403 interproton distance, 34 phi backbone torsion angle and 26 hydrogen bonding restraints derived from n.m.r. measurements. A total of 11 converged structures were computed using a metric matrix distance geometry algorithm and refined by restrained molecular dynamics. The average rms difference between the final 11 structures and the mean structure obtained by averaging their coordinates is 1.4 +/- 0.2 A for the backbone atoms and 2.1 +/- 0.1 A for all atoms. The overall structure, which is almost identical to that found by X-ray crystallography, is disc shaped and consists of a central four component mixed parallel and antiparallel beta-sheet flanked by a 13 residue alpha-helix on one side and the reactive site loop on the other.     

Modeling of protein loops by simulated annealing.

A method is presented to model loops of protein to be used in homology modeling of proteins. This method employs the ESAP program of Higo et al. (Higo, J., Collura, V., & Garnier, J., 1992, Biopolymers 32, 33-43) and is based on a fast Monte Carlo simulation and a simulated annealing algorithm. The method is tested on different loops or peptide segments from immunoglobulin, bovine pancreatic trypsin inhibitor, and bovine trypsin. The predicted structure is obtained from the ensemble average of the coordinates of the Monte Carlo simulation at 300 K, which exhibits the lowest internal energy. The starting conformation of the loop prior to modeling is chosen to be completely extended, and a closing harmonic potential is applied to N, CA, C, and O atoms of the terminal residues. A rigid geometry potential of Robson and Platt (1986, J. Mol. Biol. 188, 259-281) with a united atom representation is used. This we demonstrate to yield a loop structure with good hydrogen bonding and torsion angles in the allowed regions of the Ramachandran map. The average accuracy of the modeling evaluated on the eight modeled loops is 1 A root mean square deviation (rmsd) for the backbone atoms and 2.3 A rmsd for all heavy atoms.     

Use of residual dipolar couplings as restraints in ab initio protein structure prediction

NMR residual dipolar couplings (RDCs), in the form of the projection angles between the respective internuclear bond vectors, are used as structural restraints in the ab initio structure prediction of a test set of six proteins. The restraints are applied using a recently developed SICHO (SIde-CHain-Only) lattice protein model that employs a replica exchange Monte Carlo (MC) algorithm to search conformational space. Using a small number of RDC restraints, the quality of the predicted structures is improved as reflected by lower RMSD/dRMSD (root mean square deviation/distance root mean square deviation) values from the corresponding native structures and by the higher correlation of the most cooperative mode of motion of each predicted structure with that of the native structure. The latter, in particular, has possible implications for the structure-based functional analysis of predicted structures.     

Prediction of the native conformation of a polypeptide by a statistical-mechanical procedure. I. Backbone structure of enkephalin

A new methodology for theoretically predicting the native, three-dimensional structure of a polypeptide is presented. Based on equilibrium statistical mechanics, an algorithm has been designed to determine the probable conformation of a polypeptide by calculating conditional free-energy maps for each residue of the macromolecule. The conditional free-energy map of each residue is computed from a set of probability integrals, obtained by summing over the interaction energies of all pairs of nonbonded atoms of the whole molecule. By locating the region(s) of lowest free energy for each map, the probable conformation for each residue can be identified. The native structure of the polypeptide is assumed to be the combination of the probable conformations of the individual residues. All multidimensional probability integrals are evaluated by an adaptive Monte Carlo algorithm (SMAPPS —Statistical-Mechanical Algorithm for Predicting Protein Structure). The Monte Carlo algorithm searches the entire conformational space, adjusting itself automatically to concentrate its sampling in regions where the magnitude of the integrand is largest (“importance sampling”). No assumptions are made about the native conformation. The only prior knowledge necessary for the prediction of the native conformation is the amino acid sequence of the polypeptide. To test the effectiveness of the algorithm, SMAPPS was applied to the prediction of the native conformation of the backbone of Met-enkephalin, a pentapeptide. In the calculations, only the backbone dihedral angles (? and ψ) were allowed to vary; all side-chain (χ) and peptide-bond (ω) dihedral angles were kept fixed at the values corresponding to the alleged global minimum energy previously determined by direct energy minimization. For each conformation generated randomly by the Monte Carlo algorithm, the total conformational energy of the polypeptide was obtained from established empirical potential energy functions. Solvent effects were not included in the computations. With this initial application of SMAPPS , three distinct low-free-energy β-bend structures of Met-enkephalin were found. In particular, one of the structures has a conformation remarkably similar to the one associated with the previously alleged global minimum energy. The two additional structures of the pentapeptide have conformational energies lower than the previously computed low-energy structure. However, the Monte Carlo results are in agreement with an improved energy-minimization procedure. These initial results on the backbone structure of Met-enkephalin indicate that an equilibrium statistical-mechanical procedure, coupled with an adaptive Monte Carlo algorithm, can overcome many of the problems associated with the standard methods of direct energy minimization.     

Solution structure of tertiapin determined using nuclear magnetic resonance and distance geometry

The solution structure of tertiapin, a 21-residue bee venom peptide, has been characterized by circular dichroism (CD), two-dimensional nuclear magnetic resonance (NMR) spectroscopy, and distance geometry. A total of 21 lowest error structures were obtained from distance geometry calculations. Superimposition of these structures shows that the backbone of tertiapin is very well defined. One type-I reverse turn from residue 4 to 7 and an α-helix from residue 12 to 19 exist in the structure of tertiapin. The α-helical region is best defined from both conformational analysis and structural superimposition. The overall three-dimensional structure of tertiapin is highly compact resulting from side chain interactions. The structural information obtained from CD and NMR are compared for both tertiapin and apamin (ref. 3), another bee venom peptide. Tertiapin and apamin have some similar secondary structure, but display different tertiary structures. © 1993 Wiley-Liss, Inc.     

A comparison of the restrained molecular dynamics and distance geometry methods for determining three-dimensional structures of proteins on the basis of interproton distances

A direct comparison of the metric matrix distance geometry and restrained molecular dynamics methods for determining three-dimensional structures of proteins on the basis of interproton distances is presented using crambin as a model system. It is shown that both methods reproduce the overall features of the secondary and tertiary structure (shape and polypeptide fold). The region of conformational space sampled by the converged structures generated by the two methods is similar in size, and in both cases the converged structures are distributed about mean structures which are closer to the X-ray structure than any of the individual structures. The restrained molecular dynamics structures are superior to those obtained from distance geometry as regards local backbone conformation, side chain positions and non-bonding energies.     

The influence of stereospecific assignments on the determination of three-dimensional structures of proteins by nuclear magnetic resonance spectroscopy. Application to the sea anemone protein BDS-I

The influence of the stereospecific assignments of beta-methylene protons and the classification of chi 1 torsion angles on the definition of the three-dimensional structures of proteins determined from NMR data is investigated using the sea anemone protein BDS-I (43 residues) as a model system. Two sets of structures are computed. The first set comprises 42 converged structures (denoted STEREO structures) calculated on the basis of the complete list of restraints derived from the NMR data, consisting of 489 interproton and 24 hydrogen bonding distance restraints, supplemented by 23 phi backbone and 21 chi 1 side chain torsion angle restraints. The second set comprises 31 converged structures (denoted NOSTEREO structures) calculated from a reduced data set in which those restraints arising from stereospecific assignments, and the corresponding chi 1 torsion angle restraints, are explicitly omitted. The results show that the inclusion of the stereospecific restraints leads to a significant improvement in the definition of the structure of BDS-I, both with respect to the backbone and the detailed arrangement of the side chains. Average atomic rms differences between the individual structures and the mean structures for the backbone atoms are 0.67 +/- 0.12 A and 0.93 +/- 0.16 A for the STEREO and NOSTEREO structures, respectively; the corresponding values for all atoms are 0.90 +/- 0.17 A and 1.17 +/- 0.17 A, respectively. In addition, while the overall fold remains unchanged, there is a small but significant atomic displacement between the two sets of structures.     

Database algorithm for generating protein backbone and side-chain co-ordinates from a C alpha trace application to model building and detection of co-ordinate errors

The problem of constructing all-atom model co-ordinates of a protein from an outline of the polypeptide chain is encountered in protein structure determination by crystallography or nuclear magnetic resonance spectroscopy, in model building by homology and in protein design. Here, we present an automatic procedure for generating full protein co-ordinates (backbone and, optionally, side-chains) given the C alpha trace and amino acid sequence. To construct backbones, a protein structure database is first scanned for fragments that locally fit the chain trace according to distance criteria. A best path algorithm then sifts through these segments and selects an optimal path with minimal mismatch at fragment joints. In blind tests, using fully known protein structures, backbones (C alpha, C, N, O) can be reconstructed with a reliability of 0.4 to 0.6 A root-mean-square position deviation and not more than 0 to 5% peptide flips. This accuracy is sufficient to identify possible errors in protein co-ordinate sets. To construct full co-ordinates, side-chains are added from a library of frequently occurring rotamers using a simple and fast Monte Carlo procedure with simulated annealing. In tests on X-ray structures determined at better than 2.5 A resolution, the positions of side-chain atoms in the protein core (less than 20% relative accessibility) have an accuracy of 1.6 A (r.m.s. deviation) and 70% of chi 1 angles are within 30 degrees of the X-ray structure. The computer program MaxSprout is available on request.     

Modeling structurally variable regions in homologous proteins with rosetta

A major limitation of current comparative modeling methods is the accuracy with which regions that are structurally divergent from homologues of known structure can be modeled. Because structural differences between homologous proteins are responsible for variations in protein function and specificity, the ability to model these differences has important functional consequences. Although existing methods can provide reasonably accurate models of short loop regions, modeling longer structurally divergent regions is an unsolved problem. Here we describe a method based on the de novo structure prediction algorithm, Rosetta, for predicting conformations of structurally divergent regions in comparative models. Initial conformations for short segments are selected from the protein structure database, whereas longer segments are built up by using three- and nine-residue fragments drawn from the database and combined by using the Rosetta algorithm. A gap closure term in the potential in combination with modified Newton's method for gradient descent minimization is used to ensure continuity of the peptide backbone. Conformations of variable regions are refined in the context of a fixed template structure using Monte Carlo minimization together with rapid repacking of side-chains to iteratively optimize backbone torsion angles and side-chain rotamers. For short loops, mean accuracies of 0.69, 1.45, and 3.62 A are obtained for 4, 8, and 12 residue loops, respectively. In addition, the method can provide reasonable models of conformations of longer protein segments: predicted conformations of 3A root-mean-square deviation or better were obtained for 5 of 10 examples of segments ranging from 13 to 34 residues. In combination with a sequence alignment algorithm, this method generates complete, ungapped models of protein structures, including regions both similar to and divergent from a homologous structure. This combined method was used to make predictions for 28 protein domains in the Critical Assessment of Protein Structure 4 (CASP 4) and 59 domains in CASP 5, where the method ranked highly among comparative modeling and fold recognition methods. Model accuracy in these blind predictions is dominated by alignment quality, but in the context of accurate alignments, long protein segments can be accurately modeled. Notably, the method correctly predicted the local structure of a 39-residue insertion into a TIM barrel in CASP 5 target T0186.     

Three-dimensional structure of ectatomin from Ectatomma tuberculatum ant venom

Summary Two-dimensional ¹H NMR techniques were used to determine the spatial structure of ectatomin, a toxin from the venom of the ant Ectatomma tuberculatum. Nearly complete proton resonance assignments for two chains of ectatomin (37 and 34 amino acid residues, respectively) were obtained using 2D TOCSY, DQF-COSY and NOESY experiments. The cross-peak volumes in NOESY spectra were used to define the local structure of the protein and generate accurate proton-proton distance constraints employing the MARDIGRAS program. Disulfide bonds were located by analyzing the global fold of ectatomin, calculated with the distance geometry program DIANA. These data, combined with data on the rate of exchange of amide protons with deuterium, were used to obtain a final set of 20 structures by DIANA. These structures were refined by unrestrained energy minimization using the CHARMm program. The resulting rms deviations over 20 structures (excluding the mobile N- and C-termini of each chain) are 0.75 ? for backbone heavy atoms, and 1.25 ? for all heavy atoms. The conformations of the two chains are similar. Each chain consists of two α-helices and a hinge region of four residues; this forms a hairpin structure which is stabilized by disulfide bridges. The hinge regions of the two chains are connected together by a third disulfide bridge. Thus, ectatomin forms a four-α-helical bundle structure.