Solution conformation of endothelin-3 by H NMR and distance geometry calculations

Endothelin-3 dissolved in 10% aqueous acetic acid was studied by nuclear magnetic resonance spectroscopy. A total of 363 distances (143 intra-residue, 108 sequential and 112 long range) was compiled from the nuclear Overhauser effect spectra and used in distance geometry calculations. The molecule assumes a compact conformation stabilized by hydrophobic interactions of the side chains. There is a helix-like structure between the residues 9–15 and an extended strand at the N-terminus. The C-terminus is in close proximity to the bicyclic ring.     

Solution structure of mu-conotoxin GIIIA analysed by 2D-NMR and distance geometry calculations

We have investigated the structure of mu-conotoxin GIIIA by 2D-NMR methods. The assignment of 1H NMR spectra and a quantitative analysis of NOE and J-coupling data are presented. These results were used for the calculation of secondary structure elements of mu-conotoxin GIIIA. Distance geometry calculations were carried out to define the global folding of the peptide.     

Conformation of sarafotoxin-6b in aqueous solution determined by NMR spectroscopy and distance geometry

The solution structure of sarafotoxin-6b in water has been determined using high-resolution NMR spectroscopy. 127 proton-proton distance measurements and three phi dihedral angle constraints derived from NMR spectra were used to calculate the solution structure using a combination of distance geometry and restrained molecular dynamics. The major structural feature of the resulting family of five structures was a right-handed alpha-helix extending from K9 to Q17. In contrast, the C-terminal region of the peptide appears not to adopt a preferred conformation in aqueous solution. The present structure is compared with those previously determined for endothelin peptides in non-aqueous solvents.     

Application of distance geometry to protein tertiary structure calculations

A general approach to the problem of molecular conformation is advanced. We describe a formalism that permits experimental and theoretical information to be incorporated into a set of upper and lower bounds on intramolecular distances. Structures (conformations) meeting these bounds can be readily generated and compared with each other. To illustrate the use of the method, we have employed a simple “firehose” model for protein folding to predict the long-range hydrophobic interactions in a small protein: pancreatic trypsin inhibitor. Models of this type lead to the proper hairpin turns and a reasonable set of long-range contacts for this protein. Application of the distance geometry method then yields backbone conformations with errors of 4–8 Å compared to the native structure. We discuss both the merits and shortcomings of the firehose model and the relation between distance geometry and energy minimization techniques.     

Three-dimensional structure of acyl carrier protein determined by NMR pseudoenergy and distance geometry calculations

Distance constraints from two-dimensional NMR cross-relaxation data are used to derive a three-dimensional structure for acyl carrier protein from Escherichia coli. Several approaches to structure determination are explored. The most successful proves to be an approach that combines the early stages of a distance geometry program with energy minimization in the presence of NMR constraints represented as pseudopotentials. Approximately 450 proton to proton distance constraints including 50 long-range constraints were included in these programs. Starting structures were generated at random by the distance geometry program and energies minimized by a molecular mechanics module to give final structures. Seven of the structures were deemed acceptable on the basis of agreement with experimentally determined distances. Root-mean-square deviations from the mean of these structures for backbone atoms range from 2 to 3 A. All structures show three roughly parallel helices with hydrophobic residues facing inward and hydrophilic residues facing outward. A hydrophobic cleft is recognizable and is identified as a likely site for acyl chain binding.     

Conformation of CD4-derived cyclic hexapeptides by nmr and molecular dynamics

Two cyclic hexapeptides, cyclo[Ala¹-D -Ala²-Ser³-Phe⁴-Gly⁵-Ser⁶] and cyclo[Ala¹-Gly²-Ser³-Phe⁴-Gly⁵-Ser⁶], derived from the loop portion of the C′C″ ridge of CD4, were characterized by high-resolution nmr spectroscopy and simulated annealing studies. In DMSO-d₆ both of these peptides display a single conformer on the nmr time scale with two intramolecular H-bond (1 ← 4) stabilized β-turns at positions 2–3 and 5–6. The nmr derived distance constraints were used in simulated annealing calculations to generate the solution structures. These structures adopt energetically comparable conformational substates that are not resolvable on the nmr time scale. In aqueous solution, the H-bond stabilized β-turn conformation for cyclo [Ala-D -Ala-Ser-Phe-Gly-Ser] is no longer the predominant structural form. Structures generated using molecular dynamics simulations with no experimental constraints were compared with those from nmr analysis. The correlation between these two sets of structures allows the use of molecular simulations as a predictive tool for the conformational analysis of small peptides. © 1994 John Wiley & Sons, Inc.     

Solution conformation of endothelin determined by means of 1H-NMR spectroscopy and distance geometry calculations

The structure of endothelin-1 (ET-1), an endothelial cell-derived peptide with vasoconstricting activity, was determined in an aqueous solution by means of a combination of NMR and distance geometry calculations. The resulting structure is characterized by an alpha-helical conformation in the sequence region, Lys9-Cys15. Furthermore, an extended structure and a turn structure exist in the Cys1-Ser4 and Ser5-Asp8 regions respectively, and no preferred conformation was found for the C-terminal part of the peptide which was not uniquely constrained by the NMR data. These structural elements, the alpha-helical structure in the sequence portion, Cys-X-X-X-Cys, and the extended structure in Cys-X-Cys, are homologous to those found commonly in several neurotoxic peptides.     

Conformation in solution of porcine brain natriuretic peptide determined by combined use of nuclear magnetic resonance and distance geometry

The conformation in solution of porcine brain natriuretic peptide was determined by combined use of NMR spectroscopy and distance geometry. A set of 157 inter-proton-distance constraints was derived from the two-dimensional NOE spectra, and further a set of three hydrogen bond constraints was obtained from analysis of the temperature dependence of labile protons. The five structures with minimal violations were selected after performing distance-geometry calculations starting from 40 random initial conformations. The distance-geometry structures were further refined by the use of restrained energy minimization and restrained molecular dynamics. This structure shows a compact conformation with the carboxy-terminal region, Asn21-Tyr26, folded back to the disulfide-linked loop region, Cys4-Cys20. The characteristics of the conformation determined are as follows: conformations of the three segments interposed by glycine residues, which are Arg7-Ile12, Ser14-Leu18 and Cys20-Arg25, were well defined and the segments Arg7-Ile12 and Cys20-Arg25 are rather close to each other and nearly parallel. The biological significance of these local conformations is discussed on the basis of comparisons with those of atrial natriuretic peptide reported by Kobayashi et al.     

Conformation of parathyroid hormone antagonists by CD,nmr, and molecular dynamics simulations

The conformation of two highly potent parathyroid hormone (PTH) antagonists was investigated in water/2, 2, 2-trifluoroethanol mixtures. The two peptides are derived from the sequence (7-34) of PTH and of PTH-related protein (PTHrP) and have a D -Trp replacing Gly in position 12. In the analogue derived from PTHrP, Lys¹¹ was replaced by Leu to remove the residual agonist activity. The study was conducted by CD and two-dimensional proton magnetic resonance spectroscopy, and the nuclear Overhauser effects found were utilized in restrained distance geometry and molecular dynamics simulations. Both peptides adopt a helical C-terminal conformation, which seems more stable in the case of the PTHrP analogue. A type II′ β-turn centered around D -Trp¹² and Lys¹³ is present inboth structures. © 1995 John Wiley & Sons, Inc.     

Solution conformation of conotoxin GI determined by 1H nuclear magnetic resonance spectroscopy and distance geometry calculations

Conformational analysis of conotoxin GI, one of the neurotoxic peptides produced by a marine snail, genus Conus, was performed by a combination of nuclear magnetic resonance spectroscopy (NMR) and distance geometry calculations. The resulting conformers on minimization of the target function were classified into two groups. The difference in the structures of the conformers is mainly due to the difference in the orientation of the side chain of the tyrosyl residue. The results show that the solution structure of conotoxin GI satisfies the conformational requirements for the biological activity of an antagonist toward nicotinic cholinergic receptors elucidated in a series of studies on alkaloids. The structure is discussed on the basis of the results of comparison of the atomic arrangements of the active sites of snake venom peptides and molecular models based on the results of secondary structure prediction.     

Determining stereo-specific 1H nuclear magnetic resonance assignments from distance geometry calculations

Stereo-specific 1H nuclear magnetic resonance assignments can be obtained following distance geometry structure calculations. The key to this method is to allow stereo-related atoms or methyls to float between pro-R and pro-S configurations, the final configuration being determined by the experimental constraints. Resonances from stereo-related pairs are given initial random assignments (either pro-R or pro-S) for identifying nuclear Overhauser effects (NOEs). A list of distance constraints using these assignments is compiled and a series of structures calculated where the chirality of non-C alpha chiral centers is not constrained; no pseudoatom corrections are required. Calculated structures are both locally and globally well-determined since the assignments rely upon the structure determination rather than the structure quality relying upon stereo-specific assignments. The method represents a global approach to determining stereo-specific assignments versus previously reported methods where only intraresidue NOEs and J-coupling information are used.     

Opioid receptor three-dimensional structures from distance geometry calculations with hydrogen bonding constraints.

Three-dimensional structures of the transmembrane, seven alpha-helical domains and extracellular loops of delta, mu, and kappa opioid receptors, were calculated using the distance geometry algorithm, with hydrogen bonding constraints based on the previously developed general model of the transmembrane alpha-bundle for rhodopsin-like G-protein coupled receptors (Biophys. J. 1997. 70:1963). Each calculated opioid receptor structure has an extensive network of interhelical hydrogen bonds and a ligand-binding crevice that is partially covered by a beta-hairpin formed by the second extracellular loop. The binding cavities consist of an inner "conserved region" composed of 18 residues that are identical in delta, mu, and kappa opioid receptors, and a peripheral "variable region," composed of 19 residues that are different in delta, mu, and kappa subtypes and are responsible for the subtype specificity of various ligands. Sixteen delta-, mu-, or kappa-selective, conformationally constrained peptide and nonpeptide opioid agonists and antagonists and affinity labels were fit into the binding pockets of the opioid receptors. All ligands considered have a similar spatial arrangement in the receptors, with the tyramine moiety of alkaloids or Tyr1 of opioid peptides interacting with conserved residues in the bottom of the pocket and the tyramine N+ and OH groups forming ionic interactions or H-bonds with a conserved aspartate from helix III and a conserved histidine from helix VI, respectively. The central, conformationally constrained fragments of the opioids (the disulfide-bridged cycles of the peptides and various ring structures in the nonpeptide ligands) are oriented approximately perpendicular to the tyramine and directed toward the extracellular surface. The results obtained are qualitatively consistent with ligand affinities, cross-linking studies, and mutagenesis data.     

Solution conformation of salmon calcitonin in sodium dodecyl sulfate micelles as determined by two-dimensional NMR and distance geometry calculations

The 32 amino acid hormone salmon calcitonin was studied at pH 3.7 and 7.4 by two-dimensional NMR in sodium dodecyl sulfate (SDS) micelles at 310 K. The spectrum was fully assigned, and the secondary structure was obtained from nuclear Overhauser enhancement spectroscopy (NOESY), 3JHN alpha coupling constants, and slowly exchanging amide data. Three-dimensional structures consistent with NMR data were generated by using distance geometry calculations. A set of 260 interproton distances, derived from NOESY, and hydrogen-bond constraints, obtained from analysis of the amide exchange, were used. From the initial random conformations, 13 distance geometry structures with minimal violations were selected for further refinement with restrained energy minimization. In SDS, at both pHs, the main conformational feature of the hormone is an alpha-helix from Thr6 through Tyr22, thus including the amphipathic 8-22 segment and two residues of the Cys1-Cys7 N-terminal loop. The C-terminal decapeptide forms a loop folded back toward the helix. The biological significance of this conformation is discussed.     

Recognizing protein folds by cluster distance geometry

Cluster distance geometry is a recent generalization of distance geometry whereby protein structures can be described at even lower levels of detail than one point per residue. With improvements in the clustering technique, protein conformations can be summarized in terms of alternative contact patterns between clusters, where each cluster contains four sequentially adjacent amino acid residues. A very simple potential function involving 210 adjustable parameters can be determined that favors the native contacts of 31 small, monomeric proteins over their respective sets of nonnative contacts. This potential then favors the native contacts for 174 small, monomeric proteins that have low sequence identity with any of the training set. A broader search finds 698 small protein chains from the Protein Data Bank where the native contacts are preferred over all alternatives, even though they have low sequence identity with the training set. This amounts to a highly predictive method for ab initio protein folding at low spatial resolution.     

The transmembrane 7-alpha-bundle of rhodopsin: distance geometry calculations with hydrogen bonding constraints.

A 3D model of the transmembrane 7-alpha-bundle of rhodopsin-like G-protein-coupled receptors (GPCRs) was calculated using an iterative distance geometry refinement with an evolving system of hydrogen bonds, formed by intramembrane polar side chains in various proteins of the family and collectively applied as distance constraints. The alpha-bundle structure thus obtained provides H bonding of nearly all buried polar side chains simultaneously in the 410 GPCRs considered. Forty evolutionarily conserved GPCR residues form a single continuous domain, with an aliphatic "core" surrounded by six clusters of polar and aromatic side chains. The 7-alpha-bundle of a specific GPCR can be calculated using its own set of H bonds as distance constraints and the common "average" model to restrain positions of the helices. The bovine rhodopsin model thus determined is closely packed, but has a few small polar cavities, presumably filled by water, and has a binding pocket that is complementary to 11-cis (6-s-cis, 12-s-trans, C = N anti)-retinal or to all-trans-retinal, depending on conformations of the Lys296 and Trp265 side chains. A suggested mechanism of rhodopsin photoactivation, triggered by the cis-trans isomerization of retinal, involves rotations of Glu134, Tyr223, Trp265, Lys296, and Tyr306 side chains and rearrangement of their H bonds. The model is in agreement with published electron cryomicroscopy, mutagenesis, chemical modification, cross-linking, Fourier transform infrared spectroscopy, Raman spectroscopy, electron paramagnetic resonance spectroscopy, NMR, and optical spectroscopy data. The rhodopsin model and the published structure of bacteriorhodopsin have very similar retinal-binding pockets.     

Studies of the solution structure of the bleomycin-A2-zinc complex by means of two-dimensional NMR spectroscopy and distance geometry calculations

Application of various two-dimensional NMR techniques (SECSY, COSY and NOESY) enabled the complete assignment of the 1H-NMR spectrum of the bleomycin-A2-zinc complex in H2O and D2O at pH 6.7. The spectra were interpreted at 277 K as well as at 300 K. Identification of the resonances permitted a vicinal coupling constant analysis which revealed that the conformation around the C alpha and C beta bond of the beta-aminoalanine and beta-hydroxyhistidine residues is fixed. From this finding it was concluded that both amino functions of the beta-aminoalanine fragment and the amide and imidazole groups of the beta-hydroxyhistidine moiety are involved in zinc coordination. Also, for the mannose carbamoyl group and the pyrimidine ring active participation in zinc coordination could be established. NOE data together with the six coordination sites proposed above were used as interpoint distance constraints in distance geometry calculations for the bleomycin-A2-zinc complex in H2O. Sets of ten structures, randomly chosen within the distance constraints, were calculated (with and without the zinc ion). The calculated structures were very similar but in case that the zinc ion was omitted some flexibility was observed, within the distance constraints, in the pyrimidine-aminoalanine region. Because of the great overall similarity between the structures, a reliable representation of the solution conformation of the bleomycin-zinc complex was reached. Surprisingly, no regular symmetry around the zinc ion was found to be present in the generated structures.     

Statistical mechanics of protein folding by cluster distance geometry

This is our second type of model for protein folding where the configurational parameters and the effective potential energy function are chosen in such a way that all conformations are described and the canonical partition function can be evaluated analytically. Structure is described in terms of distances between pairs of sequentially contiguous blocks of eight residues, and all possible conformations are grouped into 71 subsets in terms of bounds on these distances. The energy is taken to be a sum of pairwise interactions between such blocks. The 210 energy parameters were adjusted so that the native folds of 32 small proteins are favored in free energy over the denatured state. We then found 146 proteins having negligible sequence similarity to any of the training proteins, yet the free energy of the respective correct native states were favored over the denatured state.     

The theory and practice of distance geometry

The mathematics of distance geometry constitutes the basis of a group of algorithms for revealing the structural consequences of diverse forms of information about a macromolecule's conformation. These algorithms are of proven utility in the analysis of experimental conformational data. This paper presents the basic theorems of distance geometry in Euclidean space and gives formal proofs of the correctness and, where possible, of the complexity of these algorithms. The implications of distance geometry for the energy minimization of macromolecules are also discussed.