Brownian dynamics simulation of the association of Bacillus amyloliquefaciens ribonuclease (barnase) and Bacillus intermedius ribonuclease (binase) with barstar

A comparative study of the association of two ribonucleases, barnase and binase, with the polypeptide inhibitor barstar has been performed by the Brownian dynamics simulation method. It was shown that the method adequately reproduced the dependence of the association rate on pH and ionic strength of solution and the influence of mutations of some ribonuclease amino acids. Two types of energetically favorable complexes of binase-barstar encounter were determined. In the type I complex, the amino acids of binase active center take part in the complex formation. In the second complex, the active center is free. It was supposed that the temporary binding of barstar into complex of type II is competitive relative to the inhibition reaction. This can partially explain the decrease in the rate of binase inhibition as compared with the corresponding reaction of barnase.     

A comparative study of the interaction of Bacillus amyloliquefaciens ribonuclease (barnase) and Bacillus intermedius ribonuclease (binase) with barstar by brownian dynamics simulation

A comparative study of the interaction of two RNases (binase and barnase) with the polypeptide inhibitor barstar was performed by Brownian dynamics simulation. It was demonstrated that this method adequately reproduced the dependence of the association rate on the pH of solution as well as the effect of mutations at individual amino acid residues on the inhibition of barnase by barstar. Two types of energy-favorable binase-barstar encounter complexes were found. In type I complex, the amino acid residues of the binase active center are involved in formation of the complex; in type II complex, the active center remains free. It is suggested that temporary binding of free barstar into type II complex competes with the inhibition reaction. Presumably, this explains the decrease in the rate of binase inhibition by barstar as compared with the analogous reaction of barnase.     

X-ray diffraction and biochemical studies of W34F mutant ribonuclease binase

The structures of two crystal modifications of the W34F mutant ribonuclease from the bacterium Bacillus intermedius (binase) were solved and refined at 1.7 and 1.1 Å resolution. The kinetic parameters of the hydrolysis of substrates of different length (GpU, GpUp, and poly(I)) by binase and its W34F mutant were investigated and compared. The catalytic activity of the enzymes was shown to increase with increasing length of the substrate. The substitution of tryptophan for phenylalanine does not lead to a change in the activity of the enzyme but results in a decrease of the binding constants for substrates containing more than one phosphate groups. A comparison of the structure of the mutant enzyme with the previously established structures of binase and its complexes with sulfate ions and guanosine monophosphate showed that the difference in their kinetic parameters is related to the fact that the mutant ribonuclease cannot bind the second phosphate group. Both crystal modifications of the mutant binase contain dimers, like in the crystal structure of binase studied previously. In these dimers, only one enzyme molecule can bind the substrate molecule. Since the dimers were found in the crystals grown under four different conditions, it can be suggested that the enzyme can exist as dimers in solution as well. Mutants of binase, which could exclude the formation of dimers, are suggested.     

Induction of apoptosis of tumor cells by binase

An induction of apoptosis by RNase from Bacillus intermedius (binase) and its mutants characterized with low catalytic activity (Lys26Ala and His101Glu) in human myelogenic erythroleukemia K562 cells, human lung carcinoma A549 cells and human peripheral blood mononuclear cells was studied. For the first time selective apoptogenic effects of binase toward leukemic blood cells was determined. Neither antiproliferative nor apoptotic effects of binase were detected in normal human peripheral blood mononuclear cells. Formation of low molecular weight oligonucleosomal DNA fragments (less than 50 Kb) which are an early marks of apoptosis was registered in solid tumor cells treated by binase. Using mutant RNases it was shown that decrease of catalytic activity to 2.5% of wild type enzyme activity leads to the loss of apoptogenic properties of enzyme. Selective apoptogenicity of binase found towards malignant cells confirmed that antitumor agents based on bacterial RNases could be considered as an alternative to standard chemotherapeutic drugs.     

Backbone dynamics of the ribonuclease binase active site area using multinuclear ((15)N and (13)CO) NMR relaxation and computational molecular dynamics

The nano-pico second backbone dynamics of the ribonuclease binase, homologous to barnase, is investigated with (15)N, (13)C NMR relaxation at 11.74 and 18.78 T and with a 1.1 ns molecular dynamics simulation. The data are compared with the temperature factors reported for the X-ray structure of this enzyme. The molecular dynamics and X-ray data correspond well and predict motions in the loops 56-61 and 99-104 that contain residues that specifically recognize substrate and are catalytic (His101), respectively. In contrast, the (15)N relaxation data indicate that these loops are mostly ordered at the nano-pico second time scale. Nano-pico second motions in the recognition loop 56-61 are evident from (13)CO-(13)C cross relaxation data, but the mobility of the catalytic loop 99-104 is not detected by (13)CO cross relaxation either. From the results of this and previous work [Wang, L., Pang, Y., Holder, T., Brender, J. R., Kurochkin, A., and Zuiderweg, E. R. P. (2001) Proc. Natl. Acad. Sci. U.S.A., 98, 7684-7689], the following dynamical characterization of the active site area of binase emerges: a beta sheet, rigid at all probed time scales, supports the catalytic residue Glu 72. Both substrate-encapsulating loops are mobile on both fast and slow time scales, but the fast motions of the loop which contains the other catalytic residue, His 101, as predicted by B-factors and computational molecular dynamics is not detected by NMR relaxation. This work strongly argues for the use of several measures in the study of protein dynamics.     

Ribonuclease binase inhibits primary tumor growth and metastases via apoptosis induction in tumor cells

Exogenous ribonucleases are known to inhibit tumor growth via apoptosis induction in tumor cells, allowing to consider them as promising anticancer drugs for clinical application. In this work the antitumor potential of binase was evaluated in vivo and the mechanism of cytotoxic effect of binase on tumor cells was comprehensively studied in vitro. We investigated tumoricidal activity of binase using three murine tumor models of Lewis lung carcinoma (LLC), lymphosarcoma RLS₄₀ and melanoma B-16. We show for the first time that intraperitoneal injection of binase at a dose range 0.1–5 mg/kg results in retardation of primary tumor growth up to 45% in LLC and RLS₄₀ and inhibits metastasis up to 50% in LLC and RLS₄₀ and up to 70% in B-16 melanoma. Binase does not exhibit overall toxic effect and displays a general systemic and immunomodulatory effects. Treatment of RLS₄₀-bearing animals with binase together with polychemotherapy revealed that binase decreases the hepatotoxicity of polychemotherapy while maintaining its antitumor effect. It was demonstrated that the cytotoxic effect of binase is realized via the induction of the intrinsic and extrinsic apoptotic pathways. Activation of intrinsic apoptotic pathway is manifested by a drop of mitochondrial potential, increase in calcium concentration and inhibition of respiratory activity. Subsequent synthesis of TNF-α in the cells under the action of binase triggers extrinsic apoptotic pathway through the binding of TNF with cell-death receptors and activation of caspase 8. Thus binase is a potential anticancer therapeutics inducing apoptosis in cancer cells.     

The mechanisms of the manifestation of the genotoxic effects of binase]

The high concentrations of commercial sample of Bacillus intermedius 7P ribonuclease (binase) showed a weak mutagenic effect in Ames test and Ara-test. It has been established that binase induces the SOS-functions of microbial cell and prophage induction. The way of exogenic enzyme action through activation of RecA protein is proposed.     

The combined action of binase and bleomycin on human lung adenocarcinoma cells

Some microbial ribonucleases (RNases) demonstrate selective cytotoxic effect against a wide range of tumor cells. In this context combined use of cytotoxic RNases in complex therapy with other chemotherapeutic agents appears to be especially promising. In this study we have investigated the apoptosisinduced effect of Bacillus pumilus RNase (binase) in combination with known antitumor antibiotic bleomycin on human lung adenocarcinoma A549 cells. The combined effect of high concentrations of these agents did not have any mutual increase in their apoptosisinduced action, while a combination of nonapoptotic concentrations resulted in the increase of proportion of apoptotic cells up to 22% as compared with individual effect of bleomycin (6%) and binase (12%) used separately. These results indicate that binase and bleomycin are effective in combination of their low concentrations and ineffective in combination of their high concentrations.     

Barnase and binase: twins with distinct fates

RNases are enzymes that cleave RNAs, resulting in remarkably diverse biological consequences. Many RNases are cytotoxic. In some cases, they attack selectively malignant cells triggering an apoptotic response. A number of eukaryotic and bacterial RNase-based strategies are being developed for use in anticancer and antiviral therapy. However, the physiological functions of these RNases are often poorly understood. This review focuses on the properties of the extracellular RNases from Bacillus amyloliquefaciens (barnase) and Bacillus intermedius (binase), the characteristics of their biosynthesis regulation and their physiological role, with an emphasis on the similarities and differences. Barnase and binase can be regarded as molecular twins according to their highly similar structure, physical-chemical and catalytic properties. Nevertheless, the 'life paths' of these enzymes are not the same, as their expression in bacteria is controlled by diverse signals. Binase is predominantly synthesized under phosphate starvation, whereas barnase production is strictly dependent on the multifunctional Spo0A regulator controlling sporulation, biofilm formation and cannibalism. Barnase and binase also have some distinctions in practical applications. Barnase was initially suggested to be useful in research and biotechnology as a tool for studying protein-protein interactions, for RNA elimination from biological samples, for affinity purification of RNase fusion proteins, for the development of cloning vectors and for sterility acquisition by transgenic plants. Binase, as later barnase, was tested for antiviral, antitumour and immunogenic effects. Both RNases have found their own niche in cancer research as a result of success in targeted delivery and selectivity towards tumour cells.     

Exogenous <Emphasis Type="Italic">Bacillus pumilus</Emphasis> RNase (binase) suppresses the reproduction of reovirus serotype 1

The experimental study identified the antiviral activity of Bacillus pumilus RNase (binase) against the reovirus of serotype 1/strain Lang. For the first time, it has been found that 50 μg/mL of binase effectively reduced the hemagglutinin and cytocidal activity of reovirus in Vero cell line. The preincubation of the enzyme with reovirus before infection of the cells inhibited the viral replication. To determine the stagedependent effect of reovirus reproduction upon binase inhibition, the infected cells were treated with binase or RNase A at different phases of the infectious cycle. The treatment of virus-infected cells has revealed that both enzymes have a maximal antiviral effect on the reovirus propagation during early phases of the reovirus reproduction cycle, with binase being more effective than RNase A. It has been hypothesized that the combined action of the oncolytic reovirus and binase is promising for the elimination of tumor cells carrying mutated RAS gene.     

Genotoxicity of the preparation "binase" in tests on Salmonella: Ames test and ARA-test]

The present work deals with mutagenicity determination of enzyme sample "binase" (Bacillus intermedius ribonuclease) in microbial test-systems: Ames test and Ara-test. The weak mutagenic effect of "binase" high concentration was established in both tests by induction of forward Ara-mutations and Histidine-reverse mutations. A metabolic activation is seen to remove this effect.     

Expression of the FLT3-ITD oncogene sensitizes murine progenitor B-cell line BAF3 to cytotoxic action of binase

Acute myeloid leukemia makes up about 30% of all leukemia cases in adults. Mutations in the genes of the receptor tyrosine kinases KIT and FLT3, along with chromosomal translocations, are frequently found in leukemic cells. In the current work, we show that the transgenic B-cells BAF3/FLT3-ITD are significantly more sensitive to cytotoxic action of the ribonuclease binase than original BAF3 cells. BAF3/FLT3-ITD cells differ from BAF3 in expression of the FLT3-ITD oncogene, which results in the alteration of normal signaling pathways. We observed a similar effect previously when studying binase cytotoxic action in cells Kasumi-1 and FDC-P1-N822K, in which the activated oncogene KIT-N822K was expressed. An elevated cytotoxicity of binase to the cells that express the FLT3-ITD oncogene indicates that, as in case of the FDC-P1 cells transduced by the KIT oncogene, the expression of an activated oncogene determines the cell’s sensitivity to the binase action.     

Ribonuclease binase apoptotic signature in leukemic Kasumi-1 cells

Cytotoxic exogenous RNases triggering apoptotic response in malignant cells have potential as anticancer drugs; surprisingly, detailed characterization of the RNase-induced apoptosis has not been conducted so far. Here we show that a cytotoxic RNase from Bacillus intermedius (binase) induces extrinsic and intrinsic apoptotic pathways in leukemic Kasumi-1 cells. The experiments were performed using TaqMan Array Human Apoptosis 96-well Plate for gene expression analysis, and flow cytometry. Cytometric studies demonstrated dissipation of the mitochondrial membrane potential, opening of mitochondrial permeability transition pores, activation of caspases, increase of intracellular Ca²⁺ and decrease of reactive oxygen species levels. We found that expression of 62 apoptotic genes is up-regulated, including 16 genes that are highly up-regulated, and only one gene was found to be down-regulated. The highest, 16 fold increase of the expression level was observed for TNF gene. Highly up-regulated genes also include the non-canonical NF-κB signaling pathway and inflammatory caspases 1,4. The obtained results suggest that binase induces evolutionary acquired cellular response to a microbial agent and triggers unusual apoptosis pathway.     

Simulation of the diffusion-controlled reaction between superoxide and superoxide dismutase. I. Simple models

A Brownian dynamics simulation method is used to study the diffusion-influenced bimolecular reaction between superoxide and superoxide dismutase (SOD). Using simple models, the details of which are based on the crystallographic structure of SOD, it is found that the electrostatic charge distribution of SOD serves to guide superoxide into the active site and enhance the diffusion-controlled rate constant by about 40%.     

Thermodynamics of denaturation of complexes of barnase and binase with barstar

Differential scanning calorimetry was used to study the thermodynamics of denaturation of protein complexes for which the free energy stabilizing the complexes varied between -8 and -16 kcal/mol. The proteins studied were the ribonucleases barnase and binase, their inhibitor barstar and mutants thereof, and complexes between the two. The results are in good agreement with the model developed by Brandts and Lin for studying the thermodynamics of denaturation for tight complexes between two proteins which undergo two-state thermal unfolding transitions.     

Bimolecular reaction simulation using Weighted Ensemble Brownian dynamics and the University of Houston Brownian Dynamics program

We discuss here the implementation of the Weighted Ensemble Brownian (WEB) dynamics algorithm of Huber and Kim in the University of Houston Brownian Dynamics (UHBD) suite of programs and its application to bimolecular association problems. WEB dynamics is a biased Brownian dynamics (BD) algorithm that is more efficient than the standard Northrup-Allison-McCammon (NAM) method in cases where reaction events are infrequent because of intervening free energy barriers. Test cases reported here include the Smoluchowski rate for association of spheres, the association of the enzyme copper-zinc superoxide dismutase with superoxide anion, and the binding of the superpotent sweetener N-(p-cyanophenyl)-N'-(diphenylmethyl)-guanidinium acetic acid to a monoclonal antibody fragment, NC6.8. Our results show that the WEB dynamics algorithm is a superior simulation method for enzyme-substrate reaction encounters with large free energy barriers.     

Diffusional channeling in the sulfate-activating complex: combined continuum modeling and coarse-grained brownian dynamics studies

Enzymes required for sulfur metabolism have been suggested to gain efficiency by restricted diffusion (i.e., channeling) of an intermediate APS^2- between active sites. This article describes modeling of the whole channeling process by numerical solution of the Smoluchowski diffusion equation, as well as by coarse-grained Brownian dynamics. The results suggest that electrostatics plays an essential role in the APS^2- channeling. Furthermore, with coarse-grained Brownian dynamics, the substrate channeling process has been studied with reactions in multiple active sites. Our simulations provide a bridge for numerical modeling with Brownian dynamics to simulate the complicated reaction and diffusion and raise important questions relating to the electrostatically mediated substrate channeling in vitro, in situ, and in vivo.     

New Insight into Secreted Ribonuclease Structure: Binase Is a Natural Dimer

The biological effects of ribonucleases (RNases), such as the control of the blood vessels growth, the toxicity towards tumour cells and antiviral activity, require a detailed explanation. One of the most intriguing properties of RNases which can contribute to their biological effects is the ability to form dimers, which facilitates efficient RNA hydrolysis and the evasion of ribonuclease inhibitor. Dimeric forms of microbial RNase binase secreted by Bacillus pumilus (former B. intermedius) have only been found in crystals to date. Our study is the first report directly confirming the existence of binase dimers in solution and under natural conditions of enzyme biosynthesis and secretion by bacilli. Using different variants of gel electrophoresis, immunoblotting, size-exclusion chromatography and mass-spectrometry, we revealed that binase is a stable natural dimer with high catalytic activity.