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Klebsiella pneumoniae is one of the most critical opportunistic pathogens. TA systems are promising drug targets because they are related to the survival of bacterial pathogens. However, structural information on TA systems in K. pneumoniae remains lacking; therefore, it is necessary to explore this information for the development of antibacterial agents. Here, we present the first crystal structure of the VapBC complex from K. pneumoniae at a resolution of 2.00 A. We determined the toxin inhibitory mechanism of the VapB antitoxin through an Mg2+ switch, in which Mg2+ is displaced by R79 of VapB. This inhibitory mechanism of the active site is a novel finding and the first to be identified in a bacterial TA system. Furthermore, inhibitors, including peptides and small molecules, that activate the VapC toxin were discovered and investigated. These inhibitors can act as antimicrobial agents by disrupting the VapBC complex and activating VapC. Our comprehensive investigation of the K. pneumoniae VapBC system will help elucidate an unsolved conundrum in VapBC systems and develop potential antimicrobial agents.

The serine protease inhibitor Rv3364c of

Although protein-protein interactions (PPIs) have emerged as an attractive therapeutic target space, the identification of chemicals that effectively inhibit PPIs remains challenging. Here, we identified through library screening a chemical probe (compound 1) that can inhibit the tumor-promoting interaction between the oncogenic factor exon 2–depleted splice variant of aminoacyl–transfer RNA synthetase-interacting multifunctional protein 2 (AIMP2-DX2) and heat shock protein 70 (HSP70). We found that compound 1 binds to the N-terminal subdomain of glutathione S-transferase (GST-N) of AIMP2-DX2, causing a direct steric clash with HSP70 and an intramolecular interaction between the N-terminal flexible region and the GST-N of AIMP2-DX2, which induces masking of the HSP70 binding region during molecular dynamics and mutation studies. Compound 1 thus interferes with the AIMP2-DX2 and HSP70 interaction and suppresses the growth of cancer cells that express high levels of AIMP2-DX2 in vitro and in preliminary in vivo experiment. This work provides an example showing that allosteric conformational changes induced by chemicals can be a way to control pathologic PPIs. SIGNIFICANCE STATEMENT Compound 1 is a promising protein-protein interaction inhibitor between AIMP2-DX2 and HSP70 for cancer therapy by the mechanism with allosteric modulation as well as competitive binding. It seems to induce allosteric conformational change of AIMP2-DX2 proteins and direct binding clash between AIMP2-DX2 and HSP70. The compound reduced the level of AIMP2-DX2 in ubiquitin-dependent manner via suppression of binding between AIMP2-DX2 and HSP70 and suppressed the growth of cancer cells highly expressing AIMP2-DX2 in vitro and in preliminary in vivo experiment.

Recent development of the chemical inhibitors specific to oncogenic KRAS (Kirsten Rat Sarcoma 2 Viral Oncogene Homolog) mutants revives much interest to control KRAS-driven cancers. Here, we report that AIMP2-DX2, a variant of the tumor suppressor AIMP2 (aminoacyl-tRNA synthetase-interacting multi-functional protein 2), acts as a cancer-specific regulator of KRAS stability, augmenting KRAS-driven tumorigenesis. AIMP2-DX2 specifically binds to the hypervariable region and G-domain of KRAS in the cytosol prior to farnesylation. Then, AIMP2-DX2 competitively blocks the access of Smurf2 (SMAD Ubiquitination Regulatory Factor 2) to KRAS, thus preventing ubiquitin-mediated degradation. Moreover, AIMP2-DX2 levels are positively correlated with KRAS levels in colon and lung cancer cell lines and tissues. We also identified a small molecule that specifically bound to the KRAS-binding region of AIMP2-DX2 and inhibited the interaction between these two factors. Treatment with this compound reduces the cellular levels of KRAS, leading to the suppression of KRAS-dependent cancer cell growth in vitro and in vivo. These results suggest the interface of AIMP2-DX2 and KRAS as a route to control KRAS-driven cancers.

Modulation of γ-aminobutyric acid (GABA) levels has been required in various disorders. GABA itself cannot be directly introduced into central nervous system (CNS) because of the blood brain barrier; inhibition of GABA aminotransferase (GABA-AT), which degrades GABA in CNS, has been the target for the modulation of GABA levels in CNS. Given that root extract of valerian (Valeriana officinalis) has been used for millennia as anti-anxiolytic and sedative, in silico approach was carried out to investigate valerian compounds exhibiting GABA-AT inhibiting activity. The 3D structure of human GABA-AT was created from pig crystal structure via homology modeling. Inhibition of GABA-AT by 18 valerian compounds was analyzed using molecular docking and molecular dynamics simulations and compared with known GABA-AT inhibitors such as vigabatrin and valproic acid. Isovaleric acid and didrovaltrate exhibited GABA-AT inhibiting activity in computational analysis, albeit less potent compared with vigabatrin. However, multiple compounds with low activity may have additive effects when the total extract of valeriana root was used in traditional usage. In addition, isovaleric acid shares similar backbone structure to GABA, suggesting that isovaleric acid might be a valuable starting structure for the development of more efficient GABA-AT inhibitors for disorders related with low level of GABA in the CNS.

LK-3, an amphipathic dimeric peptide linked by two disulfide bonds, and related isomeric bundles were synthesized, and their cell penetrating abilities were investigated. The measurements using size exclusion chromatography and dynamic light scattering show that LK-3 and its isomers form cell penetrating oligomers. Calculations, performed for various types of peptide isomers, elucidate a strong correlation between the amphipathic character of dimers and cell penetration ability. The results suggest that the amphipathicities of LK-3 and related bundle dimers are responsible for their oligomerization propensities which in turn determine their cell penetrating abilities. The observations made in this study provide detailed information about the mechanism of cell uptake of LK-3 and suggest a plausible insight of the early stage of nanoparticle formation of the cell penetrating amphipathic peptides., LK-3, an amphipathic dimeric peptide, shows good cell penetrating ability. Calculations show that the amphipathicity of LK-3 is responsible for its oligomerization and the cell penetrating abilities.

Peroxiredoxins (Prx) have received considerable attention during recent years. This study demonstrates that two typical Pseudomonas-derived 2-Cys Prx proteins, PpPrx and PaPrx can alternatively function as a peroxidase and chaperone. The amino acid sequences of these two Prx proteins exhibit 93% homology, but PpPrx possesses an additional cysteine residue, Cys112, instead of the alanine found in PaPrx. PpPrx predominates with a high molecular weight (HMW) complex and chaperone activity, whereas PaPrx has mainly low molecular weight (LMW) structures and peroxidase activity. Mass spectrometry and structural analyses showed the involvement of Cys112 in the formation of an inter-disulfide bond, the instability of LMW structures, the formation of HMW complexes, and increased hydrophobicity leading to functional switching of Prx proteins between peroxidase and chaperone.

Guanosine triphosphatases (GTPases) function as molecular switches in signal transduction pathways that enable cells to respond to extracellular stimuli. Saccharomyces cerevisiae yeast protein two 1 protein (Ypt1p) is a monomeric small GTPase that is essential for endoplasmic reticulum-to-Golgi trafficking. By size-exclusion chromatography, SDS-PAGE, and native PAGE, followed by immunoblot analysis with an anti-Ypt1p antibody, we found that Ypt1p structurally changed from low-molecular-weight (LMW) forms to high-molecular-weight (HMW) complexes after heat shock. Based on our results, Ypt1p exhibited dual functions both as a GTPase and a molecular chaperone, and furthermore, heat shock induced a functional switch from that of a GTPase to a molecular chaperone driven by the structural change from LMW to HMW forms. Subsequently, we found, by using a galactose-inducible expression system, that conditional overexpression of YPT1 in yeast cells enhanced the thermotolerance of cells by increasing the survival rate at 55°C by ∼60%, compared with the control cells expressing YPT1 in the wild-type level. Altogether, our results suggest that Ypt1p is involved in the cellular protection process under heat stress conditions. Also, these findings provide new insight into the in vivo roles of small GTP-binding proteins and have an impact on research and the investigation of human diseases.

Background and aims The 2-Cys peroxiredoxin (Prx) A protein of Arabidopsis thaliana performs the dual functions of a peroxidase and a molecular chaperone depending on its conformation and the metabolic conditions. However, the precise mechanism responsible for the functional switching of 2-Cys Prx A is poorly known. This study examines various serine-to-cysteine substitutions on α-helix regions of 2-Cys Prx A in Arabidopsis mutants and the effects they have on the dual function of the protein. Methods Various mutants of 2-Cys Prx A were generated by replacing serine (Ser) with cysteine (Cys) at different locations by site-directed mutagenesis. The mutants were then over-expressed in Escherichia coli. The purified protein was further analysed by size exclusion chromatography, polyacrylamide gel electrophoresis, circular dichroism spectroscopy and transmission electron microscopy (TEM) and image analysis. Peroxidase activity, molecular chaperone activity and hydrophobicity of the proteins were also determined. Molecular modelling analysis was performed in order to demonstrate the relationship between mutation positions and switching of 2-Cys Prx A activity. Key results Replacement of Ser(150) with Cys(150) led to a marked increase in holdase chaperone and peroxidase activities of 2-Cys Prx A, which was associated with a change in the structure of an important domain of the protein. Molecular modelling demonstrated the relationship between mutation positions and the switching of 2-Cys Prx A activity. Examination of the α2 helix, dimer-dimer interface and C-term loop indicated that the peroxidase function is associated with a fully folded α2 helix and easy formation of a stable reduced decamer, while a more flexible C-term loop makes the chaperone function less likely. Conclusions Substitution of Cys for Ser at amino acid location 150 of the α-helix of 2-Cys Prx A regulates/enhances the dual enzymatic functions of the 2-Cys Prx A protein. If confirmed in planta, this leads to the potential for it to be used to maximize the functional utility of 2-Cys Prx A protein for improved metabolic functions and stress resistance in plants.

Recent evidence suggests that aldo-keto reductase family 1 B10 (AKR1B10) may be a potential diagnostic or prognostic marker of human tumors, and that AKR1B10 inhibitors offer a promising choice for treatment of many types of human cancers. The aim of this study was to identify novel chemical scaffolds of AKR1B10 inhibitors using in silico approaches. The 3D QSAR pharmacophore models were generated using HypoGen. A validated pharmacophore model was selected for virtual screening of 4 chemical databases. The best mapped compounds were assessed for their drug-like properties. The binding orientations of the resulting compounds were predicted by molecular docking. Density functional theory calculations were carried out using B3LYP. The stability of the protein-ligand complexes and the final binding modes of the hit compounds were analyzed using 10 ns molecular dynamics (MD) simulations. The best pharmacophore model (Hypo 1) showed the highest correlation coefficient (0.979), lowest total cost (102.89) and least RMSD value (0.59). Hypo 1 consisted of one hydrogen-bond acceptor, one hydrogen-bond donor, one ring aromatic and one hydrophobic feature. This model was validated by Fischer's randomization and 40 test set compounds. Virtual screening of chemical databases and the docking studies resulted in 30 representative compounds. Frontier orbital analysis confirmed that only 3 compounds had sufficiently low energy band gaps. MD simulations revealed the binding modes of the 3 hit compounds: all of them showed a large number of hydrogen bonds and hydrophobic interactions with the active site and specificity pocket residues of AKR1B10. Three compounds with new structural scaffolds have been identified, which have stronger binding affinities for AKR1B10 than known inhibitors.

In contrast to a number of studies on the humanization of non-human antibodies, the reshaping of a non-human antibody into a chicken antibody has never been attempted. Therefore, nothing is known about the animal species-dependent compatibility of the framework regions (FRs) that sustain the appropriate conformation of the complementarity-determining regions (CDRs). In this study, we attempted the reshaping of the variable domains of the mouse catalytic anti-nucleic acid antibody 3D8 (m3D8) into the FRs of a chicken antibody (“chickenization”) by CDR grafting, which is a common method for the humanization of antibodies. CDRs of the acceptor chicken antibody that showed a high homology to the FRs of m3D8 were replaced with those of m3D8, resulting in the chickenized antibody (ck3D8). ck3D8 retained the biochemical properties (DNA binding, DNA hydrolysis, and cellular internalizing activities) and three-dimensional structure of m3D8 and showed reduced immunogenicity in chickens. Our study demonstrates that CDR grafting can be applied to the chickenization of a mouse antibody, probably due to the interspecies compatibility of the FRs.

Metal ions play a vital role in many biological processes. An important factor in these processes is the dynamics of exchange between ion bound-water molecules and the bulk. Although structural and dynamical properties of labile waters bound to metal ions, such as Na+ and Ca2+, can be elucidated using molecular dynamics simulations, direct evaluation of rates of exchange of waters rigidly bound to high charge density Mg2+, has been elusive. Here, we report a universal relationship, allowing us to determine the water exchange time on metal ions as a function of valence and hydration radius. The proposed relationship, which covers times spanning 14 orders of magnitude, highlights the ultrasensitivity of water lifetime to the ion size, as exemplified by divalent ions, Ca2+ (∼100 ps) and Mg2+ (∼1.5 μs). We show that even when structures, characterized by radial distributions are similar, a small difference in hydration radius leads to a qualitatively different (associative or dissociative) mechanism of water ...

The modular auxiliary activity (AA) family of proteins is believed to cause amorphogenesis in addition to oxidative cleavage of crystalline cellulose although the supporting evidence is limited. HcAA10-2 is a modular AA10 family protein (58 kDa) composed of a AA10 module and a family two carbohydrate binding module (CBM2), joined by a long stretch of 222 amino acids of unknown function. The protein was expressed in Escherichia coli and purified to homogeneity. Scanning electron microscopy and X-ray diffraction analysis of Avicel treated with HcAA10-2 provided evidence for the disruption of the cellulose microfibrils ("amorphogenesis") and reduction of the crystallinity index, resulting in a twofold increase of cellulase adsorption on the polysaccharide surface. HcAA10-2 exhibited weak endoglucanase-like activity toward soluble cellulose and cello-oligosaccharides with an optimum at pH 6.5 and 45 °C. HcAA10-2 catalyzed oxidative cleavage of crystalline cellulose released native and oxidized cello-oligosaccharides in the presence of copper and an electron donor such as ascorbic acid. Multiple sequence alignment indicated that His1, His109, and Phe197 in the AA10 module formed the conserved copper-binding site. The reducing sugar released from Avicel by the endoglucanase Cel5 and Celluclast accompanying HcAA10-2 was increased by four- and sixfold, respectively. Moreover, HcAA10-2 and Celluclast acted synergistically on pretreated wheat straw biomass resulting in a threefold increase in reducing sugar than Celluclast alone. Taken together, these results suggest that HcAA10-2 is a novel multifunctional modular AA10 protein possessing amorphogenesis, weak endoglucanase, and oxidative cleavage activities useful for efficient degradation of crystalline cellulose.

Microtubules play an important role in intracellular transport, mobility, and particularly mitosis. Paclitaxel (Taxol) and paclitaxel-like compounds have been shown to be anti-tumor agents useful for various human tumors. Paclitaxel-like compounds operate by stabilizing microtubules through interface binding at the interface between two -tubulin monomers in adjacent protofilaments. In this paper we present the elucidation of the structural features of paclitaxel and paclitaxel-like compounds (e.g., epothilones) with microtubule stabilizing activities, and relate their activities to spatial and chemical features of the molecules. CATALYST program was used to generate three-dimensional quantitative structure activity relationships (3D-QSARs) resulting in 3D pharmacophore models of epothilone- and paclitaxel-derivatives. Pharmacophore models were generated from diverse conformers of these compounds resulting in a high correlation between experimental and predicted biological activities (r = 0.83 and 0.91 for epothilone and paclitaxel derivatives, respectively). On the basis of biological activities of the training sets, five- and four-feature pharmacophore hypotheses were generated in the epothilone and paclitaxel series. The validation of generated hypotheses was achieved by using twelve epothilones and ten paclitaxels, respectively, which are not in the training sets. The clustering (grouping) and merging techniques were used in order to supplement spatial restrictions of each of hypothesis and to develop more comprehensive models. This approach may be of use in developing novel inhibitor candidates as well as contributing a better understanding of structural characters of many compounds useful as anticancer agents targeting microtubules.

Despite much effort to probe the properties of dimethyl sulfoxide (DMSO) solution, effects of DMSO on water, especially near plasma membrane surfaces still remain elusive. By performing molecular dynamics (MD) simulations at varying DMSO concentrations ($X_{\text{DMSO}}$), we study how DMSO affects structural and dynamical properties of water in the vicinity of phospholipid bilayers. As proposed by a number of experiments, our simulations confirm that DMSO induces dehydration from bilayer surfaces and disrupts the H-bond structure of water. However, DMSO enhanced water diffusivity at solvent-bilayer interfaces, an intriguing discovery reported by a spin-label measurement, is not confirmed in our simulations. In order to resolve this discrepancy, we examine the location of the spin-label (Tempo), relative to the solvent-bilayer interface. In accord with the evidence in the literature, our simulations, which explicitly model Tempo-PC, find that the Tempo moiety is equilibrated at $\sim 8-10$ \AA\ \emph{below} the bilayer surface. Furthermore, the DMSO-enhanced surface water diffusion is confirmed only when water diffusion is analyzed around the Tempo moiety that is immersed below the bilayer surface, which implies that the experimentally detected signal of water using Tempo stems from the interior of bilayers, not from the interface. Our analysis finds that the increase of water diffusion below the bilayer surface is coupled to the increase of area per lipid with an increasing $X_{\text{DMSO}}$ $(\lesssim 10\text{ mol\%})$. Underscoring the hydrophobic nature of Tempo moiety, our study calls for careful re-evaluation of the use of Tempo in the measurement on lipid bilayer surfaces., Comment: 18 pages, 16 figures

Using all atom molecular dynamics simulations at various DMSO concentrations, we study the structural and dynamical properties of water and DMSO in the vicinity of phospholipid bilayers. In accord with the recent experimental studies, we confirmed the DMSO-induced dehydration and the enhancement of water diffusivity unique to lipid-DMSO-H2O system, and further revealed the microscopic origin of these observations. At higher DMSO concentrations the extent of DMSO depletion from the phospholipid bilayer surface further increases, resulting in a development of the hydration layer in which the composition of water is greater than in the bulk. Modeling TEMPO-PC explicitly in the simulations, our study also reveals that due to the hydrophobic moiety, the spin label tends to be buried ∼ 7 A interior from the head groups.

Grb2 is an adapter protein involved in the signal transduction and cell communication. The Grb2 is responsible for initiation of kinase signaling by Ras activation which leads to the modification in transcription. Ligand based pharmacophore approach was applied to built the suitable pharmacophore model for Grb2. The best pharmacophore model was selected based on the statistical values and then validated by Fischer's randomization method and test set. Hypo1 was selected as a best pharmacophore model based on its statistical values like high cost difference (182.22), lowest RMSD (1.273), and total cost (80.68). It contains four chemical features, one hydrogen bond acceptor (HBA), two hydrophobic (HY), and one ring aromatic (RA). Fischer's randomization results also shows that Hypo1 have a 95% significant level. The correlation coefficient of test set was 0.97 which was close to the training set value (0.94). Thus Hypo1 was used for virtual screening to find the potent inhibitors from various chemical databases. The screened compounds were filtered by Lipinski's rule of five, ADMET and subjected to molecular docking studies. Totally, 11 compounds were selected as a best potent leads from docking studies based on the consensus scoring function and critical interactions with the amino acids in Grb2 active site.

As previously reported, the activity of the large-conductance calcium (Ca(2+))-activated potassium (K(+)) (BK(Ca)) channel is strongly potentiated from the extracellular side of the cell membrane by certain benzofuroindole derivatives. Here, the mechanism of action of one of the most potent activators, 4-chloro-7-(trifluoromethyl)-10H-benzofuro[3,2-b]indole-1-carboxylic acid (CTBIC), is characterized. This compound, Compound 22 in the previous report (Chembiochem 6:1745-1748, 2005), potentiated the activity of the channel by shifting its conductance-voltage relationship toward the more negative direction. Cotreatment with CTBIC reduced the affinity of charybdotoxin, a peptide pore-blocker, whereas that of tetraethylammonium, a small pore-blocking quaternary ammonium, was not significantly altered. Guided by these results, scanning mutagenesis of the outer vestibule of the BK(Ca) channel was launched to uncover the molecular determinants that affect CTBIC binding. Alanine substitution of several amino acid residues in the turret region and the S6 helix of the channel decreased potentiation by CTBIC. Homology modeling and molecular dynamics simulation showed that some of these residues formed a CTBIC binding pocket between two adjacent α-subunits in the outer vestibule of the channel. Thus, it can be envisioned that benzofuroindole derivatives stabilize the open conformation of the channel by binding to the residues clustered across the extracellular part of the subunit interface. The present results indicate that the interface between different α-subunits of the BK(Ca) channel may play a critical role in the modulation of channel activity. Therefore, this interface represents a potential therapeutic target site for the regulation of K(+) channels.

Hsp33, a prokaryotic molecular chaperone, exerts holdase activity in response to oxidative stress. In this study, the stepwise conformational change of Hsp33 upon oxidation was monitored by NMR. In order to overcome its high molecular weight (33 kDa as a monomer and 66 kDa as a dimer), spectra were simplified using a selectively ( 15 N)His-labeled protein. All of the eight histidines were observed in the TROSY spectrum of the reduced Hsp33. Among them, three peaks showed dramatic resonance shifts dependent on the stepwise oxidation, indicating a remarkable conformational change. The results suggest that unfolding of the linker domain is associated with dimerization, but not entire region of the linker domain is unfolded.

Histone deacetylase 8 (HDAC8) is an enzyme involved in deacetylating the amino groups of terminal lysine residues, thereby repressing the transcription of various genes including tumor suppressor gene. The over expression of HDAC8 was observed in many cancers and thus inhibition of this enzyme has emerged as an efficient cancer therapeutic strategy. In an effort to facilitate the future discovery of HDAC8 inhibitors, we developed two pharmacophore models containing six and five pharmacophoric features, respectively, using the representative structures from two molecular dynamic (MD) simulations performed in Gromacs 4.0.5 package. Various analyses of trajectories obtained from MD simulations have displayed the changes upon inhibitor binding. Thus utilization of the dynamically-responded protein structures in pharmacophore development has the added advantage of considering the conformational flexibility of protein. The MD trajectories were clustered based on single-linkage method and representative structures were taken to be used in the pharmacophore model development. Active site complimenting structure-based pharmacophore models were developed using Discovery Studio 2.5 program and validated using a dataset of known HDAC8 inhibitors. Virtual screening of chemical database coupled with drug-like filter has identified drug-like hit compounds that match the pharmacophore models. Molecular docking of these hits reduced the false positives and identified two potential compounds to be used in future HDAC8 inhibitor design.

The acetylcholinesterase (AChE) is important to terminate acetylcholine-mediated neurotransmission at cholinergic synapses. The pivotal role of AChE in apoptosome formation through the interactions with cytochrome c (Cyt c) was demonstrated in recent study. In order to investigate the proper binding conformation between the human AChE (hAChE) and human Cyt c (hCyt c), macro-molecular docking simulation was performed using DOT 2.0 program. The hCyt c was bound to peripheral anionic site (PAS) on hAChE and binding mode of the docked conformation was very similar to the reported crystal structure of the AChE and fasciculin-II (Fas-II) complex. Two 10 ns molecular dynamics (MD) simulations were carried out to refine the binding mode of docked structure and to observe the differences of the binding conformations between the absent (Apo) and presence (Holo) of heme group. The key hydrogen bonding residues between hAChE and hCyt c proteins were found in Apo and Holo systems, as well as each Tyr341 and Trp286 residue of hAChE was participated in cation-pi (π) interactions with Lys79 of hCyt c in Apo and Holo systems, respectively. From the present study, although the final structures of the Apo and Holo systems have similar binding pattern, several differences were investigated in flexibilities, interface interactions, and interface accessible surface areas. Based on these results, we were able to predict the reasonable binding conformation which is indispensable for apoptosome formation.

Peroxisome proliferator-activated receptors (PPARs) are members of nuclear receptors and their activation induces regulation of fatty acid storage and glucose metabolism. Therefore, the PPARγ is a major target for the treatment of type 2 diabetes mellitus. In order to generate pharmacophore model, 1080 known agonists database was constructed and a training set was selected. The Hypo7, selected from 10 hypotheses, contains four features: three hydrogen-bond acceptors (HBA) and one general hydrophobic (HY). This pharmacophore model was validated by using 862 test set compounds with a correlation coefficient of 0.903 between actual and estimated activity. Secondly, CatScramble method was used to verify the model. Hence, the validated Hypo7 was utilized for searching new lead compounds over 238,819 and 54,620 chemical structures in NCI and Maybridge database, respectively. Then the leads were selected by screening based on the pharmacophore model, predictive activity, and Lipinski’s rules. Candidates were obtained and subsequently the binding affinities to PPARγ were investigated by the molecular docking simulations. Finally the best two compounds were presented and would be useful to treat type 2 diabetes.

The behavior of peptide or protein solutes in saline aqueous solution is a fundamental topic in physical chemistry. Addition of ions can strongly alter the thermodynamic and physical properties of peptide molecules in solution. In order to study the effects of added ionic salts on protein conformation and dynamics, we have used the molecular dynamics (MD) simulations to investigate the behavior of Staphylococcus aureus Hfq protein under two different ionic concentrations: 0.1 M NaCl and 1.0 M NaCl in presence and absence of RNA (a hepta-oligoribonucleotide AU5G). Hfq, a global regulator of gene expression is highly conserved and abundant RNA-binding protein. It is already reported that in vivo the increase of ionic strength results in a drastic reduction of Hfq affinity for Qβ RNA and reduces the tendency of aggregation of Escherichia coli host factor hexamers. Our results revealed the crucial role of 0.1 M NaCl Hfq system on the bases with strong hydrogen bonding interactions and by stabilizing the aromatic stacking of Tyr42 residue of the adjacent subunits/monomers with the adenine and uridine nucleobases. An increase in RNA pore diameter and weakened compactness of the Hfq-RNA complex was clearly observed in 1.0 M NaCl Hfq system with bound RNA. Aggregation of monomers in Hfq and the interaction of Hfq with RNA are greatly affected due to the presence of high ionic strength. Higher the ionic concentration, weaker is the aggregation and interaction. Our results were compatible with the experimental data and this is the first theoretical report for the experimental study done in 1980 by Uhlenbeck group for the present system.

Substituted imidazolidin-2-ones deduced as potential inhibitors of IleRS by docking simulations were synthesized from an aziridine-2-carboxaldehyde. Reductive amination of an aziridine-2-carboxaldehyde with dipeptides for the sub- stituents at N1 and followed by aziridine-ring expansion with triphosgene afforded 4-chloromethylimidazolidin-2-ones whose chloride were further manipulated towards phenylurea, pyrimidin-2-yl-urea or benzenesulfonamide at C4.

Aminoacyl-tRNA synthetases (aaRSs) play vital roles in protein biosynthesis of living organisms and are interesting antibacterial drug targets. In order to find out new inhibitor candidate molecules as antibacterial agent, the binding modes of the candidate molecules were investigated at the active sites of aaRSs by molecular docking study. The docking simulations were performed with 48 compounds from four different scaffolds into the eight different aaRSs. The results show that scaffolds 3 and 4 compounds have consistently better binding capabilities, specifically for HisRS (E. coli) and IIeRS (S. aureus). The binding modes of the best compounds with the proteins were well compatible with those of two ligands in crystal structures. Therefore, we expect that the final compounds we present may have reasonable aaRS inhibitory activity.

Human histamine H 1 receptor (HHR1) is a G protein-coupled receptor and a primary target for antiallergic therapy. Here, the ligand-based three-dimensional pharmacophore models were built from a set of known HHR1 inverse agonists using HypoGen module of CATALYST software. All ten generated pharmacophore models consist of five essential features: hydrogen bond acceptor, ring aromatic, positive ionizable and two hydrophobic functions. Best model had a correlation coefficient of 0.854 for training set compounds and it was validated with an external test set with a high correlation value of 0.925. Using this model Maybridge database containing 60,000 compounds was screened for potential leads. A rigorous screening for drug-like compounds unveiled RH01692 and SPB00834, two novel molecules for HHR1 with good CATALYST fit and estimated activity values. The new lead molecules were docked into the active site of constructed HHR 1 homology model based on recently crystallized squid rhodopsin as template. Both the hit compounds were found to have critical interactions with Glu177, Phe432 and other important amino acids. The interpretations of this study may effectively be deployed in designing of novel HHR1 inverse agonists.

Human cytochrome P450 (CYP) 3A4 extensively contributes to metabolize 50% of the marketed drugs. Recently, a CYP3A4 structure with two molecules of ketoconazole (2KT) was identified. However, channels for egresses of these inhibitors are unexplored. Thus, we applied molecular dynamics simulations followed by channel analyses. Two simulations of empty and 2KT-bound CYP3A4 results revealed the multiple ligand-induced conformational changes in channel forming regions, which appear to be important for the regulation of channels. In addition, we observed that the channel-3 entrance is closed due to the large structural deviation of the key residues from Phe-cluster. F215 and F220 are known as entrance blockers of channel-2 in metyrapone-bound CYP3A4. Currently, F220 blocks the channel-3 along with F213 and F241. Therefore, it suggested that channel-1 and 2 could potentially serve as egress routes for 2KT. It is also supported by the results from MOLAxis analyses, in which the frequency of channel occurrence and bottleneck radius during simulation favor channel-1 and 2. Several bottleneck residues of these channels may have critical roles in 2KT egresses, especially S119. Our modeling study for multiple ligand-channeling of CYP3A4 could be very helpful to gain new insights into channel selectivity of CYP3A4.

Obg is a ribosome-associated GTPase essential for bacterial viability and is conserved in most organisms, from bacteria to eukaryotes. Obg is also expressed in plants, which predicts an important role for this molecule in plant viability; however, the functions of the plant Obg homologs have not been reported. Here, we first identified Arabidopsis AtObgC as a plant chloroplast-targeting Obg and elucidated its molecular biological and physiological properties. AtObgC encodes a plant-specific Obg GTPase that contains an N-terminal region for chloroplast targeting and has intrinsic GTP hydrolysis activity. A targeting assay using a few AtObgC N-terminal truncation mutants revealed that AtObgC localizes to chloroplasts and its transit peptide consists of more than 50 amino acid residues. Interestingly, GFP-fused full-length AtObgC exhibited a punctate staining pattern in chloroplasts of Arabidopsis protoplasts, which suggests a dimerization or multimerization of AtObgC. Moreover, its Obg fold was indispensable for the generation of the punctate staining pattern, and thus, was supposed to be important for such oligomerization of AtObgC by mediating the protein-protein interaction. In addition, the T-DNA insertion AtObgC null mutant exhibited an embryonic lethal phenotype that disturbed the early stage of embryogenesis. Altogether, our results provide a significant implication that AtObgC as a chloroplast targeting GTPase plays an important role at the early embryogenesis by exerting its function in chloroplast protein synthesis.

The main role of aminoacyl-tRNA synthetases (aaRSs) is to transfer the cognate amino acids to the 3'-end of their tRNA by strictly discriminating from non-cognate amino acids. Some aaRSs accomplish this via proofreading and editing mechanisms, among which valyl-tRNA synthetase (ValRS) hydrolyses the non-cognate amino acid, threonine. In ValRS, existence of pre-transfer editing process is still unclear, although crystal structure of editing site with pre-transfer substrate analog (Thr-AMS) was released. In the case of isoleucyl-tRNA synthetase (IleRS), editing mechanism is well studied and mutational analyses revealed the existence of post- and pre-transfer editing mechanisms. Our aim is to investigate the possibility of pre-transfer editing process by performing molecular dynamics (MD) simulation studies. Simulations were carried out for ValRS with pre-transfer substrates (Thr-AMP/Val-AMP) and post-transfer substrates (Thr-A76/Val-A76) to understand their binding pattern. Two important point mutation studies were performed to observe their effect on editing process. This study also intends to compare and contrast the pre-transfer editing with post-transfer editing of ValRS. Interestingly, the MD simulation results revealed that non-cognate substrates (Thr-AMP/Thr-A76) bind more strongly than the cognate substrates (Val-AMP/Val-A76) in both pre- and post-transfer editing respectively. The editing site mutations (Lys270Ala and Asp279Ala) severely affected the binding ability of pre-transfer substrate (Thr-AMP) by different ways. Even though pre- and post-transfer substrates bind to the same site, specific differences were observed which has led us to believe the existence of the pre-transfer editing process in ValRS.

Silent information regulator 2 (Sir2) or sirtuins are NAD(+)-dependent deacetylases, which hydrolyze the acetyl-lysine residues. In mammals, sirtuins are classified into seven different classes (SIRT1-7). SIRT1 was reported to be involved in age related disorders like obesity, metabolic syndrome, type II diabetes mellitus and Parkinson’s disease. Activation of SIRT1 is one of the promising approaches to treat these age related diseases. In this study, we have used HipHop module of CATALYST to identify a series of pharmacophore models to screen SIRT1 enhancing molecules. Three molecules from Sirtris Pharmaceuticals were selected as training set and 607 sirtuin activator molecules were used as test set. Five different hypotheses were developed and then validated using the training set and the test set. The results showed that the best pharmacophore model has four features, ring aromatic, positive ionization and two hydrogen-bond acceptors. The best hypothesis from our study, Hypo2, screened high number of active molecules from the test set. Thus, we suggest that this four feature pharmacophore model could be helpful to screen novel SIRT1 activator molecules. Hypo2-virtual screening against Maybridge database reveals seven molecules, which contains all the critical features. Moreover, two new scaffolds were identified from this study. These scaffolds may be a potent lead for the SIRT1 activation.