Calculations of the Conformations of Iturin A in Relation with NMR Studies

Abstract

Iturin A is an antifungal antibiotic which was isolated from a strain of Bacillus subtilis, and contains a lipophilic β amino acid closing an heptapeptide cycle with polar L and D residues. Iturin A belongs to a lipopeptide family of which the LDDLLDL sequence is kept constant.

NMR spectroscopy and semi-empirical energy calculations are combined to design the conformations of Iturin A in pyridine solution. J coupling constants and nOes (nuclear Overhauser enhancements) are used as guiding line for energy calculations. This preliminary study shows that Iturin A in pyridine appears as rather rigid, especially in the L Pro 5—D Asn 6 region, probably involved in a β turn. The polar side chains can form different networks of intramolecular hydrogen bonds. The Tyr side chain, relatively mobile, could be involved in interactions with an hydrophobic environment as the β amino acid side chain found away from the peptide cycle.     

NMR studies of the conformations of leghemoglobins from soybean and lupin

Phase-sensitive two-dimensional NMR methods have been used to obtain extensive proton resonance assignments for the carbon monoxide complexes of lupin leghemoglobins I and II and soybean leghemoglobin a. The assigned resonances provide information on the solution conformations of the proteins, particularly in the vicinity of the heme. The structure of the CO complex of lupin leghemoglobin II in solution is compared with the X-ray crystal structure of the cyanide complex by comparison of observed and calculated ring current shifts. The structures are generally very similar but significant differences are observed for the ligand contact residues, Phe30, His63 and Val67, and for the proximal His97 ligand. Certain residues are disordered and adopt two interconverting conformations in lupin leghemoglobin II in solution. The proximal heme pocket structure is closely conserved in the lupin leghemoglobins I and II but small differences in conformation in the distal heme pocket are apparent. Larger conformational differences are observed when comparisons are made with the CO complex of soybean leghemoglobin. Altered protein-heme packing is indicated on the proximal side of the heme and some conformational differences are evident in the distal heme pocket. The small conformational differences between the three leghemoglobins probably contribute to the known differences in their O2 and CO association and dissociation kinetics. The heme pocket conformations of the three leghemoglobins are more closely related to each other than to sperm whale myoglobin. The most notable differences between the leghemoglobins and myoglobin are: (a) reduced steric crowding of the ligand binding site in the leghemoglobins, (b) different orientations of the distal histidine, and (c) small but significant differences in proximal histidine coordination geometry. These changes probably contribute to the large differences in ligand binding kinetics between the leghemoglobins and myoglobin.     

NMR studies of differences in the conformations and dynamics of ligand complexes formed with mutant dihydrofolate reductases

Two mutants of Lactobacillus casei dihydrofolate reductase, Trp 21----Leu and Asp 26----Glu, have been prepared by using site-directed mutagenesis methods, and their ligand binding and structural properties have been compared with those of the wild-type enzyme. 1H, 13C, and 31P NMR studies have been carried out to characterize the structural changes in the complexes of the mutant and wild-type enzymes. Replacement of the conserved Trp 21 by a Leu residue causes a decrease in activity of the enzyme and reduces the NADPH binding constant by a factor of 400. The binding of substrates and substrate analogues is only slightly affected. 1H NMR studies of the Trp 21----Leu enzyme complexes have confirmed the original resonance assignments for Trp 21. In complexes formed with methotrexate and the mutant enzyme, the results indicate some small changes in conformation occurring as much as 14 A away from the site of substitution. For the enzyme-NADPH complexes, the chemical shifts of nuclei in the bound coenzyme indicate that the nicotinamide ring binds differently in complexes with the mutant and the wild-type enzyme. There are complexes where the wild-type enzyme has been shown to exist in solution as a mixture of conformations, and studies on the corresponding complexes with the Trp 21----Leu mutant indicate that the delicately poised equilibria can be perturbed. For example, in the case of the ternary complex formed between enzyme, trimethoprim, and NADP+, two almost equally populated conformations (forms I and II) are seen with the wild-type enzyme but only form II (the one in which the nicotinamide ring of the coenzyme is extended away from the enzyme structure and into the solvent) is observed for the mutant enzyme complex. It appears that the Trp 21----Leu substitution has a major effect on the binding of the nicotinamide ring of the coenzyme. For the Asp 26----Glu enzyme there is a change in the bound conformation of the substrate folate. Further indications that some conformational adjustments are required to allow the carboxylate of Glu 26 to bind effectively to the N1 proton of inhibitors such as methotrexate and trimethoprim come from the observation of a change in the dynamics of the bound trimethoprim molecule as seen from the increased rate of the flipping of the 13C-labeled benzyl ring and the increased rate of the N1-H bond breaking.     

1H NMR studies of the solution conformations of an analogue of the C-peptide of ribonuclease A

Two-dimensional NMR experiments have been performed on a peptide, succinyl-AE-TAAAKFLRAHA-NH2, related to the amino-terminal sequence of ribonuclease A. This peptide contains 50-60% helix in 0.1 M NaCl solution, pH 5.2, 3 degrees C, as measured by circular dichroism. NOESY spectra of the peptide in aqueous solution at low temperatures show a number of NOE connectivities that are used to determine the highly populated conformations of the peptide in solution. Short-range dNN(i, i + 1) and d alpha N(i, i + 1) connectivities and medium-range d alpha beta(i, i + 3) and d alpha N(i, i + 3) connectivities are detected. The pattern of NOE connectivities unambiguously establishes the presence of helix in this peptide. The magnitudes of the 3JHN alpha coupling constants and the intensities of the dNN(i, i + 1) and d alpha N(i,i + 1) NOEs allow the evaluation of the position of the helix along the peptide backbone. These data indicate that the amino terminus of the peptide is less helical than the remainder of the peptide. The observation of several long-range NOEs that are atypical of helices indicates the presence of a high population of peptide molecules in which the first three residues are distorted out of the helical conformation. The absence of these NOEs in a related peptide, RN-31, in which Arg 10 has been changed to Ala, suggests that this distortion at the amino-terminal end of the peptide arises from the formation of a salt bridge between Glu 2 and Arg 10.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR studies of multiple conformations in complexes of Lactobacillus casei dihydrofolate reductase with analogues of pyrimethamine

1H and 19F NMR signals from bound ligands have been assigned in one- and two-dimensional NMR spectra of complexes of Lactobacillus casei dihydrofolate reductase with various pyrimethamine analogues (including pyrimethamine [1, 2,4-diamino-5-(4'-chlorophenyl)-6-ethylpyrimidine], fluoropyrimethamine [2, 2,4-diamino-5-(4'-fluorophenyl)-6-ethylpyrimidine], fluoronitropyrimethamine [3, 2,4-diamino-5-(4'-fluoro-3'-nitrophenyl) -6-ethylpyrimidine], and methylbenzoprim [4, 2,4-diamino-5-[4'- (methylbenzylamino)-3'-nitrophenyl]-6-ethylpyrimidine]). The signals were identified mainly by correlating signals from bound and free ligands by using 2D exchange experiments. Analogues (such as 1 and 2) with symmetrically substituted phenyl rings give rise to 1H signals from four nonequivalent aromatic protons, clearly indicating the presence of hindered rotation about the pyrimidine-phenyl bond. Analogues containing asymmetrically substituted aromatic rings (such as 3 and 4) exist as mixtures of two rotational isomers (an enantiomeric pair) because of this hindered rotation and the NMR spectra revealed that both isomers (forms A and B) bind to the enzyme with comparable, though unequal, binding energies. In this case two complete sets of bound proton signals were observed. The phenyl ring protons in each of the two forms experience essentially the same protein environment (same shielding) as that experienced by the corresponding protons in bound pyrimethamine: this confirms that forms A and B correspond to two rotational isomers resulting from approximately 180 degrees rotation about the pyrimidine-phenyl bond, with the 2,4-diaminopyrimidine ring being bound similarly in both forms. The relative orientations of the two forms have been determined from NOE through-space connections between protons on the ligand and protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantum mechanical and NMR spectroscopy studies on the conformations of the hydroxymethyl and methoxymethyl groups in aldohexosides

The potential energy surfaces of the hydroxymethyl and methoxymethyl groups in methyl hexopyranosides have been extensively studied, employing quantum mechanical calculations and high resolution NMR data. The structure and energy of the C-5-C-6 rotamers were calculated at the B3LYP level of the density functional theory (DFT). For all, geometry optimizations were carried out for 264 conformers of 16 methyl D-gluco- and methyl D-galactopyranoside derivatives 1-16 at the B3LYP/6-31G** level. For all calculated minima, single-point calculations were performed at the B3LYP/6-311++G** level. Solvent effects were considered using a self-consistent reaction field method. Values of the vicinal coupling constants 3J(H-5-H-6R), 3J(H-5-H-6S), 3J(C-4-H-6R), and 3J(C-4-H-6S) for methyl D-glucopyranosides, methyl D-galactopyranosides and their 6-O-methyl derivatives 9-16 were measured in two solvents, methanol and water. The calculated gg, gt, and tg rotamer populations of the hydroxymethyl and methoxymethyl groups in 9-16 agreed well with experimental data. The results clearly showed that the population of gg, gt, and tg rotamers is sensitive to solvent effects. It was concluded that the preference of rotamers in 1-16 is due to the hydrogen bonding and solvent effects.     

NMR studies of flexible opiate conformations at monoclonal antibody binding sites. I. Transferred nuclear Overhauser effects show bound conformations

500 MHz H, homonuclear, intra-molecular, transferred Nuclear Overhauser Effect measurements have been performed on the bound forms of a classical opiate antagonist, nalorphine and an agonist, levorphanol at their respective binding sites in two different specific anti-opiate monoclonal antibody fragments. Based upon previous studies of opiate conformations in solution the results clearly show without extensive interpretation that one of these flexible haptens has the opposite (from solution) isomeric conformation in its bound form. For nalorphine the axial isomer of the N-allyl substituent is the bound form whereas in solution the equatorial isomer dominates at a ratio of 5:1. For levorphanol the bound form is that of equatorial N-methyl in accord with the low energy conformation in solution. In this preliminary report we discuss the initial measurements and results and their implications with respect to the conformations of flexible ligands at macromolecular binding sites including opiate receptors.     

Multiple conformations of SAM-II riboswitch detected with SAXS and NMR spectroscopy

Riboswitches are a newly discovered large family of structured functional RNA elements that specifically bind small molecule targets out of a myriad of cellular metabolites to modulate gene expression. Structural studies of ligand-bound riboswitches by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy have provided insights into detailed RNA-ligand recognition and interactions. However, the structures of ligand-free riboswitches remain poorly characterized. In this study, we have used a variety of biochemical, biophysical and computational techniques including small-angle X-ray scattering and NMR spectroscopy to characterize the ligand-free and ligand-bound forms of SAM-II riboswitch. Our data demonstrate that the RNA adopts multiple conformations along its folding pathway and suggest that the RNA undergoes marked conformational changes upon Mg(2+) compaction and S-adenosylmethionine (SAM) metabolite binding. Further studies indicated that Mg(2+) ion is not essential for the ligand binding but can stabilize the complex by facilitating loop/stem interactions. In the presence of millimolar concentration of Mg(2+) ion, the RNA samples a more compact conformation. This conformation is near to, but distinct from, the native fold and competent to bind the metabolite. We conclude that the formation of various secondary and tertiary structural elements, including a pseudoknot, occur to sequester the putative Shine-Dalgarno sequence of the RNA only after metabolite binding.     

NMR studies of the heme pocket conformations of monomeric hemoglobins from Glycera dibranchiata. Implications for ligand binding

Two-dimensional 1H-NMR methods have been used to assign side-chain resonances for the tryptophan residues and for several amino acids located in the heme pockets of the carbon monoxide complexes of the major monomeric hemoglobins from Glycera dibranchiata. The NMR spectra reveal a high degree of conservation of the heme pocket structure in the different hemoglobins. However some conformational differences are evident and residues at positions B10 and G8 on the distal side of the heme pocket are not conserved. From the present NMR studies it appears that the monomeric G. dibranchiata hemoglobin examined by X-ray crystallography [Padlan, E. A. & Love, W. (1974) J. Biol. Chem. 249, 4067-4078] corresponds to HbC. Except that the orientation of the heme in solution is the reverse of that reported in the crystal structure, there is a close correspondence between the heme pocket structure in the crystal and in solution. The proximal histidine coordination geometry is almost identical in the CO complexes of the three monomeric hemoglobins studied. Distal residues are strongly implicated in determining the observed kinetic differences in ligand binding reactions. In particular, steric crowding of the ligand binding site in hemoglobin A is probably a major factor in the slower kinetics of this component.     

NMR studies of chromomycin A3 interaction with DNA

The binding of chromomycin A3 to calf thymus DNA and poly(dG-dC) has been studied by 13C and 1H NMR with emphasis on the mode of binding, the role of Mg2+, and pH effects. The most prominent changes in the DNA base pair 13C NMR resonances upon complexation with chromomycin were observed for G and C bases, consistent with the G-C preference exhibited by this compound. Comparison of the 13C spectrum of DNA-bound chromomycin A3 with that of DNA-bound actinomycin D, a known intercalator, showed many similarities in the base pair resonances. This suggested the possibility that chromomycin A3 binds via an intercalative mechanism. 1H NMR studies in the imino proton, low-field region of the spectrum provided additional evidence in support of this binding mode. In the low-field spectrum of chromomycin A3 bound to calf thymus DNA, a small shoulder was observed on the upfield side of the G-C imino proton peak. Similarly, in the chromomycin A3 complex with poly(dG-dC), a well-resolved peak was found upfield from the G-C imino proton peak. These results are expected for ligands that bind by intercalation. Furthermore, in both the calf thymus and poly(dG-dC) drug complexes (in the presence of Mg2+) a broad peak was also present downfield (approximately 16 ppm from TSP) from the DNA imino protons. This was attributed to the C-9 phenolic hydroxyl proton on the chromomycin chromophore. Visible absorbance spectra at different pH values showed that the role of Mg2+ in the binding of chromomycin A3 to DNA is more than simple neutralization of the drug's anionic change.     

Validation of NMR side-chain conformations by packing calculations

The precision and accuracy of protein structures determined by nuclear magnetic resonance (NMR) spectroscopy depend on the completeness of input experimental data set. Typically, rather than a single structure, an ensemble of up to 20 equally representative conformers is generated and routinely deposited in the Protein Database. There are substantially more experimentally derived restraints available to define the main-chain coordinates than those of the side chains. Consequently, the side-chain conformations among the conformers are more variable and less well defined than those of the backbone. Even when a side chain is determined with high precision and is found to adopt very similar orientations among all the conformers in the ensemble, it is possible that its orientation might still be incorrect. Thus, it would be helpful if there were a method to assess independently the side-chain orientations determined by NMR. Recently, homology modeling by side-chain packing algorithms has been shown to be successful in predicting the side-chain conformations of the buried residues for a protein when the main-chain coordinates and sequence information are given. Since the main-chain coordinates determined by NMR are consistently more reliable than those of the side-chains, we have applied the side-chain packing algorithms to predict side-chain conformations that are compatible with the NMR-derived backbone. Using four test cases where the NMR solution structures and the X-ray crystal structure of the same protein are available, we demonstrate that the side-chain packing method can provide independent validation for the side-chain conformations of NMR structures. Comparison of the side-chain conformations derived by side-chain packing prediction and by NMR spectroscopy demonstrates that when there is agreement between the NMR model and the predicted model, on average 78% of the time the X-ray structure also concurs. While the side-chain packing method can confirm the reliable residue conformations in NMR models, more importantly, it can also identify the questionable residue conformations with an accuracy of 60%. This validation method can serve to increase the confidence level for potential users of structural models determined by NMR.     

Distinguishing multiple chemotaxis Y protein conformations with laser-polarized 129Xe NMR

The chemical shift of the (129)Xe NMR signal has been shown to be extremely sensitive to the local environment around the atom and has been used to follow processes such as ligand binding by bacterial periplasmic binding proteins. Here we show that the (129)Xe shift can sense more subtle changes: magnesium binding, BeF(3)(-) activation, and peptide binding by the Escherichia coli chemotaxis Y protein. (1)H-(15)N correlation spectroscopy and X-ray crystallography were used to identify two xenon-binding cavities in CheY that are primarily responsible for the shift changes. One site is near the active site, and the other is near the peptide binding site.     

Calculations of NMR dipolar coupling strengths in model peptides

Ab initio MP2 and density functional quantum chemistry calculations are used to explore geometries and vibrational properties of N-methylacetamide and of the alanine dipeptide with backbone angles characteristic of helix and sheet regions in proteins. The results are used to explore one-bond direct dipolar couplings for the N–H, C–H, C–N, and C–C bonds, as well as for the two-bond C–H interaction. Vibrational averaging affects these dipolar couplings, and these effects can be expressed as effective bond lengths that are 0.5–3% larger than the true bond lengths; bending and torsion vibrations have a bigger influence on the effective coupling than do stretching vibrations. Because of zero-point motion, these effects are important even at low temperature. Hydrogen bonding interactions at the amide group also increase the N-H effective bond length. Although vibrational contributions to effective bond lengths are small, they can have a significant influence on the extraction of order parameters from relaxation data, and a knowledge of relative bond lengths is needed when several types of dipolar couplings are to be simultaneously used for refinement. The present computational results are compared to both solid- and liquid-state NMR experiments. The analysis suggests that secondary structural elements in many proteins may be more rigid than is commonly thought.     

Theoretical studies on the conformations of higher gangliosides

The possible conformations of higher gangliosides (GD3, GT1a. GT1b, GQ1b) have been determined by computing their potential energy using semi-empirical potential functions. The favoured conformation of the disialic acid fragment in these gangliosides is independent of its position (internal or terminal). The favoured conformations of these gangliosides have also been correlated to their biological activity. The results suggest that tetanus toxin and sendai virus may have a large binding site which can accommodate at least four sugar residues.     

Theoretical studies on the conformations of methyl glycopyranosides

The σ-charges on various atoms of methyl glycosides have been computed by using the MO-LCAO method of Del Re. The potential and free energies of methyl aldohexopyranosides and methyl aldopentopyranosides in their C1(d) and 1C(d) conformations have been calculated. Minimization of the energies of these conformations has been studied by suitably tilting the axial C-C and C-O bonds. Considerable release of strain is achieved when tilts of 4.5 and 3° are given to the axial hydroxymethyl and hydroxyl groups, respectively, that are involved in Hassel-Ottar effect. A tilt of 3° is also found necessary for the axial OMe group involved in the Hassel-Ottar effect. The calculated free-energy values are in accord with experimental ones, after adding a value of 0.8 kcal.mole^?1 for the anomeric effect of -OMe group. These studies predict that all of the methyl aldohexopyranosides, except methyl α-d- and methyl β-d-idopyranosides, favour the C1 conformation. On the other hand, the energy calculations also predict that, of the eight methyl aldopentopyranosides studied, only methyl α-d- and methyl β-d-xylopyranosides and methyl α-d -ribopyranoside favour the C1(d) conformation; for the other pentopyranosides, considerable amounts of both C1(d) and 1C(d) conformations are present in the equilibrium mixture. The calculated values of the percentage of α-anomer present in the equilibrium mixture agree fairly well with those obtained experimentally.     

Theoretical studies on the conformations of aldohexopyranose pentaacetates

The net charges on various atoms of aldohexopyranose pentaacetates were computed by using the MO-LCAO method of Del Re for σ-charges and the Hückel MO method for π-charges. The potential and free energies of sixteen aldohexopyranose pentaacetates in the C1(D) and 1C(D) conformations were estimated. Minimization of the energies of these conformations was studied by suitably tilting the axial CC and CO bonds. As with the free sugars, considerable release of strain is achieved when tilts of 4.5 and 2° are given to the axial CH₂OAc and the axial OAc groups, respectively, involved in the Hassel—Ottar effect in the 1C(D) conformations. In the case of C1(D) conformations, the ideal models have the minimum energy even when the acetate groups are involved in syn-axial interactions, indicating that strain induced by axial acetate groups is less than that of axial hydroxyl groups. The calculated free-energies agree well with the experimental values after adding a value of 0.9 kcal.mole^-1 for the anomeric effect of the acetoxyl group. The free-energy calculations also predict that α-D-idohexopyranose pentaacetate and α-D-altrose pentaacetate favour the C1(D) conformation and β-D-idose pentaacetate a C1?1C equilibrium in solution, in agreement with n.m.r. studies.     

A Practical Implicit Membrane Potential for NMR Structure Calculations of Membrane Proteins

The highly anisotropic environment of the lipid bilayer membrane imposes significant constraints on the structures and functions of membrane proteins. However, NMR structure calculations typically use a simple repulsive potential that neglects the effects of solvation and electrostatics, because explicit atomic representation of the solvent and lipid molecules is computationally expensive and impractical for routine NMR-restrained calculations that start from completely extended polypeptide templates. Here, we describe the extension of a previously described implicit solvation potential, eefxPot, to include a membrane model for NMR-restrained calculations of membrane protein structures in XPLOR-NIH. The key components of eefxPot are an energy term for solvation free energy that works together with other nonbonded energy functions, a dedicated force field for conformational and nonbonded protein interaction parameters, and a membrane function that modulates the solvation free energy and dielectric screening as a function of the atomic distance from the membrane center, relative to the membrane thickness. Initial results obtained for membrane proteins with structures determined experimentally in lipid bilayer membranes show that eefxPot affords significant improvements in structural quality, accuracy, and precision. Calculations with eefxPot are straightforward to implement and can be used to both fold and refine structures, as well as to run unrestrained molecular-dynamics simulations. The potential is entirely compatible with the full range of experimental restraints measured by various techniques. Overall, it provides a useful and practical way to calculate membrane protein structures in a physically realistic environment.