Improved NMR spectra of a protein–DNA complex through rational mutagenesis and the application of a sensitivity optimized isotope-filtered NOESY experiment

The NMR spectra of the complex between the DNA-binding domain of the Dead ringer protein (DRI-DBD, Gly262-Gly398) and its DNA binding site (DRI-DBD:DNA, 26 kDa) have been optimized by biochemical and spectroscopic means. First, we demonstrate the utility of a modified 2D [F1,F2] ¹³C-filtered NOESY experiment that employs a ¹J_HC versus chemical shift optimized adiabatic ¹³C inversion pulse [Zwahlen, C. et al. (1997) J. Am. Chem. Soc., 119, 6711–6721]. The new sequence is shown to be more sensitive than previously published pulse schemes (up to 40% in favorable cases) and its utility is demonstrated using two protein–DNA complexes. Second, we demonstrate that the targeted replacement of an interfacial aromatic residue in the DRI-DBD:DNA complex substantially reduces line broadening within its NMR spectra. The spectral changes are dramatic, salvaging a protein–DNA complex that was originally ill suited for structural analysis by NMR. This biochemical approach is not a general method, but may prove useful in the spectral optimization of other protein complexes that suffer from interfacial line broadening caused by dynamic changes in proximal aromatic rings.     

Monitoring of Bacillus thermodenitrificans OHT-1 in compost by whole cell hybridization

Thermophilic aerobic composting is a widely practiced method for the disposal of exhaust materials. We isolated a thermophilic bacteria strain from a compost sample under aerobic conditions at 60 degrees C. On the basis of its 16S rRNA sequence and physiological characteristics, this strain was identified as Bacillus thermodenitrificans OHT-1. An 18-subunit oligonucleotide probe for 16S rRNA, labeled with fluorescein isothiocyanate, was developed for the detection of B. thermodenitrificans. Spores and vegetative cells of B. thermodenitrificans OHT-1 were detected in liquid culture and laboratory compost by whole cell hybridization using this oligonucleotide probe. The results obtained by whole cell hybridization were evaluated in growth experiments of B. thermodenitrificans OHT-1 in laboratory compost and were used to enumerate spores and vegetative cells.     

A comparative study on the hydroperoxide and thiol specificity of the glutathione peroxidase family and selenoprotein P

Glutathione peroxidase catalyzes the reduction of hydrogen peroxide and organic hydroperoxide by glutathione and functions in the protection of cells against oxidative damage. Glutathione peroxidase exists in several forms that differ in their primary structure and localization. We have also shown that selenoprotein P exhibits a glutathione peroxidase-like activity (Saito, Y., Hayashi, T., Tanaka, A., Watanabe, Y., Suzuki, M., Saito, E., and Takahashi, K. (1999) J. Biol. Chem. 274, 2866-2871). To understand the physiological significance of the diversity among these enzymes, a comparative study on the peroxide substrate specificity of three types of ubiquitous glutathione peroxidase (cellular glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, and extracellular glutathione peroxidase) and of selenoprotein P purified from human origins was done. The specific activities and kinetic parameters against two hydroperoxides (hydrogen peroxide and phosphatidylcholine hydroperoxide) were determined. We next examined the thiol specificity and found that thioredoxin is the preferred electron donor for selenoprotein P. These four enzymes exhibit different peroxide and thiol specificities and collaborate to protect biological molecules from oxidative stress both inside and outside the cells.     

Two-state conformational changes in inositol 1,4,5-trisphosphate receptor regulated by calcium

Inositol 1,4,5-trisphosphate receptor (IP3R) is a highly controlled calcium (Ca2+) channel gated by inositol 1,4,5-trisphosphate (IP3). Multiple regulators modulate IP3-triggered pore opening by binding to discrete allosteric sites within IP3R. Accordingly we have postulated that these regulators structurally control ligand gating behavior; however, no structural evidence has been available. Here we show that Ca2+, the most pivotal regulator, induced marked structural changes in the tetrameric IP3R purified from mouse cerebella. Electron microscopy of the IP3R particles revealed two distinct structures with 4-fold symmetry: a windmill structure and a square structure. Ca2+ reversibly promoted a transition from the square to the windmill with relocations of four peripheral IP3-binding domains, assigned by binding to heparin-gold. Ca2+-dependent susceptibilities to limited digestion strongly support the notion that these alterations exist. Thus, Ca2+ appeared to regulate IP3 gating activity through the rearrangement of functional domains.     

Disruption of adiponectin causes insulin resistance and neointimal formation

The adipocyte-derived hormone adiponectin has been proposed to play important roles in the regulation of energy homeostasis and insulin sensitivity, and it has been reported to exhibit putative antiatherogenic properties in vitro. In this study we generated adiponectin-deficient mice to directly investigate whether adiponectin has a physiological protective role against diabetes and atherosclerosis in vivo. Heterozygous adiponectin-deficient (adipo(+/-)) mice showed mild insulin resistance, while homozygous adiponectin-deficient (adipo(-/-)) mice showed moderate insulin resistance with glucose intolerance despite body weight gain similar to that of wild-type mice. Moreover, adipo(-/-) mice showed 2-fold more neointimal formation in response to external vascular cuff injury than wild-type mice (p = 0.01). This study provides the first direct evidence that adiponectin plays a protective role against insulin resistance and atherosclerosis in vivo.     

barS1, a gene for biosynthesis of a gamma-butyrolactone autoregulator,a microbial signaling molecule eliciting antibiotic production in Streptomyces species

From Streptomyces virginiae, in which production of streptogramin antibiotic virginiamycin M(1) and S is tightly regulated by a low-molecular-weight Streptomyces hormone called virginiae butanolide (VB), which is a member of the gamma-butyrolactone autoregulators, the hormone biosynthetic gene (barS1) was cloned and characterized by heterologous expression in Escherichia coli and by gene disruption in S. virginiae. The barS1 gene (a 774-bp open reading frame encoding a 257-amino-acid protein [M(r), 27,095]) is situated in the 10-kb regulator island surrounding the VB-specific receptor gene, barA. The deduced BarS1 protein is weakly homologous to beta-ketoacyl-acyl carrier protein/coenzyme A reductase and belongs to the superfamily of short-chain alcohol dehydrogenase. The function of the BarS1 protein in VB biosynthesis was confirmed by BarS1-dependent in vitro conversion of 6-dehydro-VB-A to VB-A, the last catalytic step in VB biosynthesis. Of the four possible enantiomeric products from racemic 6-dehydro-VB-A as a substrate, only the natural enantiomer of (2R,3R,6S)-VB-A was produced by the purified recombinant BarS1 (rBarS1), indicating that rBarS1 is the stereospecific reductase recognizing (3R)-isomer as a substrate and reducing it stereospecifically to the (6S) product. In the DeltabarS1 mutant created by homologous recombination, the production of VB as well as the production of virginiamycin was lost. The production of virginiamycin by the DeltabarS1 mutant was fully recovered by the external addition of VB to the culture, which indicates that the barS1 gene is essential in the biosynthesis of the autoregulator VBs in S. virginiae and that the failure of virginiamycin production was a result of the loss of VB production.     

The slender rice mutant,with constitutively activated gibberellin signal transduction,has enhanced capacity for abscisic acid level

The slender rice (slr1-1) mutant, carrying a lethal and recessive single mutation, has a constitutive gibberellin (GA)-response phenotype and behaves as if it were saturated with GAs [Ikeda et al. (2001) Plant Cell 13, 999]. The SLR1 gene, with sequence homology to members of the plant-specific GRAS gene family, is a mediator of the GA signal transduction process. In the slender rice, GA-inducible alpha-amylase was produced from the aleurone layer without applying GA. GA-independent alpha-amylase production in the mutant was inhibited by applying abscisic acid (ABA). Shoot elongation in the mutant was also suppressed by ABA, indicating that the slender rice responds normally to ABA. Interestingly, shoot ABA content was 10-fold higher in the mutant than in the wild type, while there was no difference in root ABA content. Expression of the Rab16A gene, which is known to be ABA inducible, was about 10-fold higher in shoots of the mutant than in those of the wild type. These results indicate that constitutive activation of the GA signal transduction pathway by the slr1-1 mutation promotes the endogenous ABA level.     

Solution structure determination of the two DNA-binding domains in the Schizosaccharomyces pombe Abp1 protein by a combination of dipolar coupling and diffusion anisotropy restraints

We have solved the solution structure of the N-terminal region of the fission yeast centromere protein, Abp1, bound to a 21-base pair DNA fragment bearing its recognition site (Mw = 30 kDa). Although the two DNA-binding domains in the Abp1 protein were defined well by a conventional NOE-based NMR methodology, the overall structure of the Abp1 protein was poorly defined, due to the lack of interdomain distance restraints. Therefore, we additionally used residual dipolar couplings measured in a weakly aligned state, and rotational diffusion anisotropies. Neither the NH residual dipolar couplings nor the backbone ¹⁵N T ₁/T ₂ data were sufficient to determine the overall structure of the Abp1 protein, due to spectral overlap. We used a combination of these two orientational restraints (residual dipolar coupling and rotational diffusion anisotropy), which significantly improved the convergence of the overall structures. The range of the observed T ₁/T ₂ ratios was wider (20–50 for the secondary structure regions of Abp1) than the previously reported data for several globular proteins, indicating that the overall shape of the Abp1DNA complex is ellipsoid. This extended form would facilitate the recognition of the two separate sites in the relatively long DNA sequence by the DNA-binding domains of Apb1.