Structure of the Bacillus subtilis phage SPO1-encoded type II DNA-binding protein TF1 in solution.

The solution structure of a type II DNA-binding protein, the bacteriophage SPO1-encoded transcription factor 1 (TF1), was determined using NMR spectroscopy. Selective 2H-labeling, 13C-labeling and isotopic heterodimers were used to distinguish contacts between and within monomers of the dimeric protein. A total of 1914 distance and dihedral angle constraints derived from NMR experiments were used in structure calculations using restrained molecular dynamics and simulated annealing protocols. The ensemble of 30 calculated structures has a root-mean-square deviation (r.m.s.d.) of 0.9 A, about the average structure for the backbone atoms, and 1.2 A for all heavy-atoms of the dimeric core (helices 1 and 2) and the beta-sheets. A severe helix distortion at residues 92-93 in the middle of helix 3 is associated with r.m.s.d. of approximately 1.5 A for the helix 3 backbone. Deviations of approximately 5 A or larger are noted for the very flexible beta-ribbon arms that constitute part of a proposed DNA-binding region. A structural model of TF1 has been calculated based on the previously reported crystal structure of the homologous HU protein and this model was used as the starting structure for calculations. A comparison between the calculated average solution structure of TF1 and a solution structure of HU indicates a similarity in the dimeric core (excluding the nine amino acid residue tail) with pairwise deviations of 2 to 3 A. The largest deviations between the average structure and the HU solution structure were found in the beta-ribbon arms, as expected. A 4 A deviation is found at residue 15 of TF1 which is in a loop connecting two helical segments; it has been reported that substitution of Glu15 by Gly increases the thermostability of TF1. The homology between TF1 and other proteins of this family leads us to anticipate similar tertiary structures.

Structural elements pertinent to the interaction of cyclosporin A with its specific receptor protein, cyclophilin.

Cyclophilin (163 amino acids; 17,737 daltons) is a ubiquitous cytosolic protein that specifically binds the potent immunosuppressive drug cyclosporin A (CsA). To characterize the structural details of this interaction, extensive use has been made of two-dimensional (2D) NMR methods. For studies on CsA, these methods are being used to assign the conformational space accessible to CsA by analysis of the spectra from the multiple CsA conformers present in slow exchange in mixed solvent systems. These same 2D NMR methods also have been used for extensive studies of the major bovine thymus cyclophilin (CyP) isoform and its complex with stoichiometric amounts of CsA. In the former case, these studies have revealed 81% of the 156 expected HN-H alpha crosspeaks. The complete spin-coupled spin systems for one-third of these amide resonances have been assigned according to amino acid type. After exhaustive D2O exchange, there remain 44 amide protons which exhibit 2D NMR features indicative of a hydrophobic domain with beta-sheet secondary structure. The CsA-complexed form of CyP exhibits a discrete structure and set of resonances in slow exchange with the drug-free CyP. The amino acids that have been specifically identified to be affected by the interaction are limited in number and include three Phe residues, the unique Trp at position 120, and two Ala residues.

Multidimensional NMR spectroscopy of DNA-binding proteins: structure and function of a transcription factor.

The solution structure of a type II DNA-binding protein (DBPII), transcription factor 1 (TF1), has been determined using NMR spectroscopy. A multidimensional, heteronuclear strategy was employed to overcome assignment ambiguities due to resonance overlap and broadened crosspeaks. This approach involved the use of selectively deuteriated, 13C- and 15N-labeled samples and 'isotopic heterodimers' to distinguish between intra- and intermonomeric NOEs. A comparison with the crystal structure and NMR analysis of the E. coli HU protein suggests that other homologous proteins in this family will possess similar tertiary structures. This NMR strategy is applicable to the study of other proteins and their biomolecular complexes.

Characterization of sinapyl derivatives in pineapple (Ananas comosus [L.] Merill) juice.

Three previously unidentified phenolic compounds were found in pineapple (Ananas comosus [L.] Merill) juice in substantial concentrations and were isolated by semipreparative reverse phase HPLC. The structures were elucidated from UV spectra, acid hydrolysis, and subsequent amino acid analysis, mass spectrometry, and two-dimensional NMR spectroscopy. The compounds are identified as S-sinapyl-L-cysteine, N-L-gamma-glutamyl-S-sinapyl-L-cysteine, and S-sinapylglutathione.

The rotating frame nuclear Overhauser effect spectroscopy experiment as an aid to determining solution structures of DNA oligomers.

Important intrinsic characteristics of the rotating frame nuclear Overhauser effect spectroscopy (ROESY) experiment were found to be advantageous in DNA solution structure determination. In a ROESY experiment, the different mechanisms of relaxation result in different signs of cross peaks, enabling a clear distinction between H2' resonances and H2" resonances of the DNA sugar backbone. This method is of particular importance in crowded spectra, for purine resonances whose H2', H2" protons typically resonate closely, as well as in conditions where line broadening makes coupling constants in a correlated spectroscopy experiment impossible to determine. By observing the signs of cross peaks in the base proton to H2', H2" sugar proton region, the ROESY spectrum can be used to distinguish A-form, B-form, and Z-form DNA.

Solution structure of cyclosporin A and a nonimmunosuppressive analog bound to fully deuterated cyclophilin.

A simple strategy involving 1H nuclear magnetic resonance (NMR) spectroscopy and complete protein deuteration was used to determine the structures of two receptor-bound drugs. A potent immunosuppressive, cyclosporin A (CsA) binds tightly to the ubiquitous and highly conserved 17.7-kDa immunophilin, cyclophilin (CyP). Fully deuterated CyP was produced by overexpressing the human CyP gene in Escherichia coli grown on deuterated algal hydrolysate in 98% D2O. As only the CsA molecule is protonated in the CsA-CyP complex, we were able to make a complete sequential assignment of the bound drug using standard two-dimensional proton NMR experiments. The structure determination was accomplished using dynamical simulated annealing calculations with a total of 124 NMR-derived distance and torsion angle restraints. Aside from binding CsA, CyP also acts as a peptidyl-prolyl cis-trans isomerase. Thus, much importance had been ascribed to the cis peptide bond present in the structures reported for free CsA in organic solvents and in crystal studies. Interestingly, CyP-bound CsA exists in an all-trans conformation with no detectable elements of regular secondary structure and no intramolecular hydrogen bonds. A nonactive CsA analog, MeAla6-CsA, was studied using the same CyP deuteration strategy. In addition to structural elucidation of the two bound drugs, we were able to differentiate between the bound and surface-exposed residues of the drugs and also validate our previous hypothesis that the single CyP tryptophan is located in the CsA-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermodynamics of cyclophilin catalyzed peptidyl-prolyl isomerization by NMR spectroscopy.

One-dimensional nmr exchange spectroscopy was carried out to determine thermodynamic parameters of cyclophilin-induced cis-trans isomerization of succinyl-Ala-Phe-Pro-Phe-p-nitroanilide. Rate measurements were possible at physiological temperatures. The kc/Km of rat cyclophilin was found to be 12.8 (+/- 0.5) s-1 microM-1 at 37 degrees C, intermediate to previously reported values that used a coupled enzyme assay extrapolated to this temperature. Activation energies (delta G not equal to) for the uncatalyzed and catalyzed reaction at 37 degrees C were found to be 19.7 and 17.1 kcal/mol, respectively, and were primarily due to an enthalpic barrier.

Formation of multiple complexes between actinomycin D and a DNA hairpin: structural characterization by multinuclear NMR.

The solution conformations of a DNA oligomer and its complexes with the anticancer drug actinomycin D (ActD) were characterized using homo- and heteronuclear NMR techniques. Previous high-resolution NMR investigations of ActD-DNA complexes employed symmetric double-stranded DNA oligomers, yielding two identical symmetry-related complexes. In order to understand the important effects that neighboring base pairs and/or unusual nucleic acid structures may have on ActD binding specificity and orientation, we chose to study the oligonucleotide d(TCGCGTTTTCGCGA), which adopts a hairpin structure in solution. NOE cross-peak intensities were used to generate distance constraints for molecular dynamics simulations and structure determinations of the free oligonucleotide and for both complexes. A total of 86 intermolecular NOEs were identified for each complex, 27 of which involve exchangeable protons. These intermolecular NOEs along with changes in the phosphorus chemical shifts were used to determine the drug binding site on the DNA. As expected, ActD intercalated exclusively at the single d(GC) step in the DNA hairpin. Interestingly, although the two complexes, which differ by the orientation with which the asymmetric drug chromophore intercalates the DNA, were not formed in equal concentrations, their conformations are very similar. The RMS difference of the DNA hairpin in the two complexes is only 1.10 A. The structures of the minor groove binding pentapeptide rings are not affected by any of the changes in the normal double-helical structure imposed by the hairpin loop. The total pairwise RMS difference over all atoms for the four peptides (two per complex) in the calculated structures is 0.72 A. Conversely, the structure of the hairpin loop is not appreciably changed upon binding--the RMS difference between the free DNA loop region and the loop region in the two complexes is 1.68 A and only 0.43 A between the two complexes. Our data also support a possible conformation of the d(T)4 loop that does not possess a thymine-thymine "wobble" base pair.

Proton and nitrogen NMR sequence-specific assignments and secondary structure determination of the Bacillus subtilis SPO1-encoded transcription factor 1.

Sequence-specific 1H and 15N NMR1 assignments are reported for the transcription factor 1 (TF1), a 22-kDa type II DNA-binding protein (DBPII) that consists of two 99-residue monomers. An assignment strategy is employed that uses six complementary selectively deuterium-labeled TF1 variants and an uniformly 15N-labeled TF1 variant. Two-dimensional and three-dimensional homonuclear and heteronuclear NMR correlated spectra are analyzed and yield nearly complete assignments for the 1H and 15N resonances. Discrete protein secondary structure domains are also defined; in each monomer, three alpha-helices, an antiparallel beta-sheet, and an antiparallel beta-ribbon are identified. Analyses of two dimers formed from two distinct selectively deuteriated monomers serve to identify a number of interproton contacts as either intermonomeric or intramonomeric. An analysis of amide proton exchange reveals that the carboxy-terminal alpha-helix is less stable than the other two alpha-helices in each monomer. A previously proposed working structural model of the TF1 dimer [Geiduschek et al. (1990) J. Struct. Biol. 104, 84-90], based on the crystal structure of a highly homologous DBPII, the Bacillus stearothermophilus-encoded HU protein, is generally supported by our results. Several departures from this model, however, are noted. Most notably, the carboxy-terminal tail of TF1 adopts an alpha-helical conformation with a backbone distortion at Lys93.

Maprouneacin, a new daphnane diterpenoid with potent antihyperglycemic activity from Maprounea africana.

Bioassay-guided fractionation of the EtOH extract of M. africana, using the in vivo noninsulin-dependent diabetes mellitus db/db mouse model, resulted in the isolation of the new daphnane-type diterpenoid maprouneacin (2). Compound 2 showed potent glucose-lowering properties when given by the oral route.

A 1H-NMR study of the transcription factor 1 from Bacillus subtilis phage SPO1 by selective 2H-labeling. Complete assignment and structural analysis of the aromatic resonances for a 22-kDa homodimer.

1H-NMR experiments have been performed on transcription factor 1 (TF1) encoded by Bacillus subtilis phage SPO1. To study this 22-kDa homodimeric DNA-binding protein, a selective 2H-labeling strategy has been employed. Complete sequence-specific assignments of all the resonances from the five aromatic residues were determined by a modified standard sequential-assignment procedure. The reduced contribution of spin diffusion upon the long-mixing-time nuclear-Overhauser-enhancement spectroscopy for the selectively 2H-labeled variants, as opposed to the fully 1H-containing protein, has allowed for the identification of the spin systems and of the long-range dipolar contacts between Phe28 and Phe47 protons in the protein core and between Phe61 and Phe97 protons. The latter suggests an interaction between the proposed beta-ribbon DNA-binding arm and the carboxy terminus of the paired monomer. A previously proposed TF1 structural model [Geiduschek, E. P., Schneider, G. J. & Sayre, M. H. (1990) J. Struct. Biol. 104, 84-90)] has been modified using constrained-energy-minimization calculations incorporating the experimentally determined set of aromatic-to-aromatic contacts. This new model has been analyzed with regard to the relative mobility and the relative solvent accessibility of the aromatic residues which have been measured by the nonselective T1 relaxation times of the aromatic resonances for the fully 1H-containing protein and the relaxation time enhancements upon selective 2H-labeling, respectively.