Crystal structure of human factor VIIa/tissue factor in complex with peptide mimetic inhibitor

The 3D structure of human factor VIIa/soluble tissue factor in complex with a peptide mimetic inhibitor, propylsulfonamide-D-Thr-Met-p-aminobenzamidine, is determined by X-ray crystallography. As compared with the interactions between thrombin and thrombin inhibitors, the interactions at S2 and S3 sites characteristic of factor VIIa and factor VIIa inhibitors are revealed. The S2 site has a small pocket, which is filled by the hydrophobic methionine side chain in P2. The small S3 site fits the small size residue, D-threonine in P3. The structural data and SAR data of the peptide mimetic inhibitor show that these interactions in the S2 and S3 sites play an important role for the improvement of selectivity versus thrombin. The results will provide valuable information for the structure-based drug design of specific inhibitors for FVIIa/TF.     

Structural Basis for Conformational Dynamics of GTP-bound Ras Protein

Ras family small GTPases assume two interconverting conformations, “inactive” state 1 and “active” state 2, in their GTP-bound forms. Here, to clarify the mechanism of state transition, we have carried out x-ray crystal structure analyses of a series of mutant H-Ras and M-Ras in complex with guanosine 5′-(β,γ-imido)triphosphate (GppNHp), representing various intermediate states of the transition. Crystallization of H-RasT35S-GppNHp enables us to solve the first complete tertiary structure of H-Ras state 1 possessing two surface pockets unseen in the state 2 or H-Ras-GDP structure. Moreover, determination of the two distinct crystal structures of H-RasT35S-GppNHp, showing prominent polysterism in the switch I and switch II regions, reveals a pivotal role of the guanine nucleotide-mediated interaction between the two switch regions and its rearrangement by a nucleotide positional change in the state 2 to state 1 transition. Furthermore, the ³¹P NMR spectra and crystal structures of the GppNHp-bound forms of M-Ras mutants, carrying various H-Ras-type amino acid substitutions, also reveal the existence of a surface pocket in state 1 and support a similar mechanism based on the nucleotide-mediated interaction and its rearrangement in the state 1 to state 2 transition. Intriguingly, the conformational changes accompanying the state transition mimic those that occurred upon GDP/GTP exchange, indicating a common mechanistic basis inherent in the high flexibility of the switch regions. Collectively, these results clarify the structural features distinguishing the two states and provide new insights into the molecular basis for the state transition of Ras protein.     

Genetic diversity of soybean-nodulating rhizobia in Nepal in relation to climate and soil properties

Backgroud and aims

This study was conducted to reveal the genetic diversity of soybean-nodulating rhizobia in Nepal in relation to climate and soil properties.

Method

A total of 102 bradyrhizobial strains were isolated from the root nodules of soybeans cultivated in 12 locations in Nepal varying in climate and soil properties, and their genetic diversity was examined based on 16S rDNA, ITS regions of 16S–23S rDNA, nodC and nifH. In vitro growth properties of some representative strains were examined to elucidate their characteristic distribution in Nepal.

Results

Four species of the genus Bradyrhizobium were isolated, and B. japonicum dominated at temperate locations, while in subtropical locations, B. elkanii, B. yuanmingense, and B. liaoningense dominated at acidic, moderately acidic, and slightly alkaline soils, respectively. The relative nodule occupancies could not be fully explained by their in vitro growth properties. Similar nodC and nifH genes among the strains suggested co-evolution of these genes also in Nepal, probably through horizontal gene transfer.

Conclusions

The influence of climate and soil pH on diversity at the sub-species level was revealed. It is concluded that the highly diverse climate and soils in Nepal might be conducive for the existence of diverse soybean rhizobial strains.     

Effect of penicillin on GABA-gated chloride ion influx

To investigate the mechanism of penicillin-induced convulsions, we have studied the effects of penicillin G (PC-G) on GABA-gated chloride ion influx in brain microsac preparations of mice. In the presence of 10^–4 M GABA, PC-G inhibited GABA-gated chloride ion influx in a dose-dependent manner. The dose-response curve for GABA in the presence of 10^–3 M PC-G was shifted rightward and there was a decrease in maximum response. The inhibitory effects of PC-G were not reversed by RO 15-1788, an antagonist of benzodiazepine (BZ) receptors, but were reversed by washing the microsac membranes. Therefore, PC-G probably exerts its proconvulsant effect by inhibiting GABA-gated chloride ion influx. However, it appears not to act through the BZ receptor of the GABA/BZ receptor complex.     

Functional morphology of Gené's organ inHaemaphysalis longicornis (Acari: Ixodidae)

The structure and function of Gené's organ, which secretes the egg wax, were investigated inHaemaphysalis longicornis. The hatching rates of eggs deposited before and after the prevention of the eversion of Gené's organ were compared at the conditions of 30°C, 100% RH and soaking in liquid paraffin. The movement of Gené's organ was observed, and the structures of this organ in unfed and ovipositing females were examined with both light and scanning electron microscopes.The hatching rates of eggs without contact with Gené's organ were markedly lower (4.9%) than that of normal eggs (94.5%) even at the suitable condition for eggs (30°C, 100% RH). In contrast, the hatching rates of eggs soaked in liquid paraffin without contact of Gené's organ were considerably higher (71.8%) than that of eggs without both Gené's organ contact and soaking in liquid paraffin.When oviposition commenced, the camerostome of the female was widened by a downswing of the capitulum, the paired horns of Gené's organ emerged through it, and the oviposited egg from the genital aperture was coated with egg wax. At the same time of retraction of the horns which grip an egg, the capitulum was swung up and the egg was loaded on the scutum.Gené's organ included the outer and inner cuticles, and the outer one is highly folded like bellows when the horns of this organ were retracted into the haemocoel. The undeveloped glands lying in the peripheral region of Gené's organ in the unfed stage commenced their enlargement at the time of the onset of feeding, and they became large tubular glands in the 1-day ovipositing female. The columnar glandular cells of the tubular glands extended their domed apical region into the lumen and contained numerous secretory granules during oviposition.These results suggest that the tubular glands secrete the egg wax which is indispensable for egg survival by serving as a waterproofing agent.     

A posttranslationally regulated protease, VheA, is involved in the liberation of juveniles from parental spheroids in Volvox carteri

The lineage of volvocine algae includes unicellular Chlamydomonas and multicellular Volvox in addition to their colonial relatives intermediate in size and cell number. In an asexual life cycle, daughter cells of Chlamydomonas hatch from parental cell walls soon after cell division, while Volvox juveniles are released from parental spheroids after the completion of various developmental events required for the survival of multicellular juveniles. Thus, heterochronic change in the timing of hatching is considered to have played an important role in the evolution of multicellularity in volvocine algae. To study the hatching process in Volvox carteri, we purified a 125-kD Volvox hatching enzyme (VheA) from a culture medium with enzymatic activity to degrade the parental spheroids. The coding region of vheA contains a prodomain with a transmembrane segment, a subtilisin-like Ser protease domain, and a functionally unknown domain, although purified 125-kD VheA does not contain a prodomain. While 143-kD VheA with a prodomain is synthesized long before the hatching stage, 125-kD VheA is released into the culture medium during hatching due to cleavage processing at the site between the prodomain and the subtilisin-like Ser protease domain, indicating that posttranslational regulation is involved in the determination of the timing of hatching.     

Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase.

Spinach leaf NADH:nitrate reductase (NR) responds to light/dark signals and photosynthetic activity in part as a result of rapid regulation by reversible protein phosphorylation. We have identified the major regulatory phosphorylation site as Ser-543, which is located in the hinge 1 region connecting the cytochrome b domain with the molybdenum-pterin cofactor binding domain of NR, using recombinant NR fragments containing or lacking the phosphorylation site sequence. Studies with NR partial reactions indicated that the block in electron flow caused by phosphorylation also could be localized to the hinge 1 region. A synthetic peptide (NR6) based on the phosphorylation site sequence was phosphorylated readily by NR kinase (NRk) in vitro. NR6 kinase activity tracked the ATP-dependent inactivation of NR during several chromatographic steps and completely inhibited inactivation/phosphorylation of native NR in vitro. Two forms of NRk were resolved by using anion exchange chromatography. Studies with synthetic peptide analogs indicated that both forms of NRk had similar specificity determinants, requiring a basic residue at P-3 (i.e., three amino acids N-terminal to the phosphorylated serine) and a hydrophobic residue at P-5. Both forms are strictly calcium dependent but belong to distinct families of protein kinases because they are distinct immunochemically.