Residues In Pocket Research Articles

Development of New N-{4-[(7-Chloro-5-methylpyrrolo[2,1-f] [1,2,4]triazin-4-yl)oxy]-3-fluorophenyl}benzenesulfonamide Analogues: Exploring Anticancer Potential through MerTK Inhibition

AbstractMer proto-oncogene tyrosine-protein kinase (MerTK), a part of the TAM (TYRO3, AXL, and MerTK) family, is directly correlated with metastasis and various types of cancers. The inhibition of this receptor is a promising strategy for more-effective chemotherapy. Considering the pharmacophoric features of the active domain of MerTK and the structural characteristics of the investigational drug BMS794833, we designed five new N-{4-[(7-chloro-5-methylpyrrolo[2,1-f][1,2,4]triazin-4-yl)oxy]-3-fluorophenyl}benzenesulfonamide analogues. In cytotoxicity studies, one of the analogues displayed a significantly higher cytotoxicity than cisplatin. It showed IC50 values of 2.09, 1.96, and 3.08 μM against A549, MCF-7, and MDA-MB-231 cancer cell lines, respectively. In drug metabolism and pharmacokinetic studies, it was the most stable analogue and displayed a moderate MerTK inhibitory potential. Molecular-docking studies were performed to corroborate the MerTK inhibition, and the same analogue achieved the most significant docking score (–12.33 kcal/mol). Docking interactions demonstrated that the imine and amine group of the 3-chloropyridine moiety of BMS794833 formed hydrogen bonds with the main chain of the ATP pocket residue Met674, while the oxygen atoms of the 4-oxo-1,4-dihydropyridine-3-carboxamide moiety established hydrogen bonds with the Lys619 and Asp741 amino acid residues of the allosteric pocket of MerTK protein. These promising results provide evidence that the N-{4-[(7-chloro-5-methylpyrrolo[2,1-f][1,2,4]triazin-4-yl)oxy]-3-fluorophenyl}benzenesulfonamide pharmacophore can give potential insights into the development of new MerTK inhibitors.

Discovery of Curcuminoids as Pancreatic Lipase Inhibitors from Medicine-and-Food Homology Plants.

Researchers are increasingly interested in discovering new pancreatic lipase inhibitors as anti-obesity ingredients. Medicine-and-food homology plants contain a diverse set of natural bioactive compounds with promising development potential. This study screened and identified potent pancreatic lipase inhibitors from 20 commonly consumed medicine-and-food homology plants using affinity ultrafiltration combined with spectroscopy and docking simulations. The results showed that turmeric exhibited the highest pancreatic lipase-inhibitory activity, and curcumin, demethoxycurcumin, and bisdemethoxycurcumin were discovered to be potent pancreatic lipase inhibitors within the turmeric extract, with IC50 values of 0.52 ± 0.04, 1.12 ± 0.05, and 3.30 ± 0.08 mg/mL, respectively. In addition, the enzymatic kinetics analyses demonstrated that the inhibition type of the three curcuminoids was the reversible competitive model, and curcumin exhibited a higher binding affinity and greater impact on the secondary structure of pancreatic lipase than found with demethoxycurcumin or bisdemethoxycurcumin, as observed through fluorescence spectroscopy and circular dichroism. Furthermore, docking simulations supported the above experimental findings, and revealed that the three curcuminoids might interact with amino acid residues in the binding pocket of pancreatic lipase through non-covalent actions, such as hydrogen bonding and π-π stacking, thereby inhibiting the pancreatic lipase. Collectively, these findings suggest that the bioactive compounds of turmeric, in particular curcumin, can be promising dietary pancreatic lipase inhibitors for the prevention and management of obesity.

Heme pocket hydrogen bonding residue interactions within the Pectobacterium Diguanylate cyclase-containing globin coupled sensor: A resonance Raman study

Heme-based sensor proteins are used by organisms to control signaling and physiological effects in response to their gaseous environment. Globin-coupled sensors (GCS) are oxygen-sensing proteins that are widely distributed in bacteria. These proteins consist of a heme globin domain linked by a middle domain to various output domains, including diguanylate cyclase domains, which are responsible for synthesizing c-di-GMP, a bacterial second messenger crucial for regulating biofilm formation. To understand the roles of heme pocket residues in controlling activity of the diguanylate cyclase domain, variants of the Pectobacterium carotovorum GCS (PccGCS) were characterized by enzyme kinetics and resonance Raman (rR) spectroscopy. Results of these studies have identified roles for hydrogen bonding and heme edge residues in modulating heme pocket conformation and flexibility. Better understanding of the ligand-dependent GCS signaling mechanism and the residues involved may allow for future development of methods to control O2-dependent c-di-GMP production.

Unraveling the structure and function of a novel SegC protein interacting with the SegAB chromosome segregation complex in Archaea.

Genome segregation is a fundamental process that preserves the genetic integrity of all organisms, but the mechanisms driving genome segregation in archaea remain enigmatic. This study delved into the unknown function of SegC (SSO0033), a novel protein thought to be involved in chromosome segregation in archaea. Using fluorescence polarization DNA binding assays, we discovered the ability of SegC to bind DNA without any sequence preference. Furthermore, we determined the crystal structure of SegC at 2.8 Å resolution, revealing the multimeric configuration and forming a large positively charged surface that can bind DNA. SegC has a tertiary structure folding similar to those of the ThDP-binding fold superfamily, but SegC shares only 5-15% sequence identity with those proteins. Unexpectedly, we found that SegC has nucleotide triphosphatase (NTPase) activity. We also determined the SegC-ADP complex structure, identifying the NTP binding pocket and relative SegC residues involved in the interaction. Interestingly, images from negative-stain electron microscopy revealed that SegC forms filamentous structures in the presence of DNA and NTPs. Further, more uniform and larger SegC-filaments are observed, when SegA-ATP was added. Notably, the introduction of SegB disrupts these oligomers, with ATP being essential for regulating filament formation. These findings provide insights into the functional and structural role of SegC in archaeal chromosome segregation.

In silico investigation on the inhibitory potential of natural polyphenolics against Lanosterol 14α-Demethylase to discover novel antifungal lead compounds

Lanosterol 14α-Demethylase is considered one of the main targets for the development of novel therapeutics against fungal diseases. In this research, more than 100 natural polyphenolic compounds from honeybee propolis were introduced to blind and targeted molecular docking protocols to investigate their antifungal potential against the fungal enzyme. Finally, the compounds with the lowest binding energies were subjected to 250 ns molecular dynamics (MD) simulations. Accordingly, it turns out that three compounds, Dehydroabietic acid (DAA), 3-Hydroxy-2,2-dimethyl-8-prenylchromane-6-propenoic acid (3HPP), and (2R,3R)-6[1-(4′-Hydroxy-3′-methoxyphenyl) (HYM), bound to the catalytic pocket with the lowest binding energies of −10.4, −10.0, −9.8 kcal/mol, respectively. According to MD simulations results, binding of these three compounds to the receptor changed the RMSF magnitudes of residues in the catalytic pocket. Also, DAA and 3HPP significantly changed the structural dynamics of the protein, especially in regions vital for substrate binding and enzymatic activity, suggesting potential disruption of the enzyme's function and hindrance to fungal growth. These compounds caused notable decreases in Rg and SASA of the protein, indicating increased structural compactness. Furthermore, DAA and 3HPP significantly impacted the protein's mobility patterns, potentially impeding its function. Binding free energy calculations also revealed DAA's superior affinity, making it the top candidate among the studied ligands. According to their predicted pharmacokinetics and toxicity properties, DAA and 3HPP, with good pharmacokinetic properties and low toxicities, caused the highest instability in the protein structure, so they can be nominated as the most effective antifungal agents against Lanosterol 14α-Demethylase to further investigations to prove their efficiencies.

Dynamic Comparison of Binding Pocket of Berberine Protein Crystals

Introduction: Berberine is an important isoquinoline alkaloid that has various pharmacological properties. The comparison of the binding pocket similarity of berberine is regarded as the starting point for deciphering various activities. Eight berberine protein crystals were clustered and studied by molecular dynamics (MD) simulations to investigate common features of berberine binding pockets. Method: Root Mean Square Deviation (RMSD) results showed that berberine was able to bind to each protein in a stable manner. Residue analysis showed that the stability of residue composition of different protein pockets varied. This is also consistent with the results of the pocket similarity analysis: PS-score curves of most proteins fluctuated to varying degrees. The binding pocket of 3BTI in homogeneous protein analysis exhibited high stability (PS-scoremean = 0.703 and PS-scoremin = 0.5664). Pocket similarity analysis between two heterologous proteins showed that most of PS-score values were in the interval of 0.3-0.35, and PS-score values of 3D6Y were relatively high when compared with the other three proteins. Results: Pocket residue matching analysis showed that GLU145/VAL147/ILE182/TYR229/GLU253 in 3D6Y can be matched structurally to the corresponding residues in 1JUM, 2QVD, and 5Y0V, respectively, which can be considered as an important pocket feature for the berberine binding. Nevertheless, the obtained matched residues are limited to the category of pocket structural similarity. Conclusion: This was the first study in which dynamic comparison of berberine binding pockets were used to discover pocket patterns. These results were of great significance for the polypharmacological study, the identification of potential off-targets, and the repurposing of berberine. conclusion: This was the first study in which dynamic comparisons of berberine binding pockets were used to discover interaction patterns. These results were of great significance for the polypharmacological study, the identification of potential off-targets, and the repurposing of berberine.

Contribution of Electrostatic CH3-π Interactions to Recognition of Histone Asymmetric Dimethylarginine by the SPIN1 Triple Tudor Domain.

Methylation of arginine (Arg) residues on histones creates a new binding epitope, enabling recognition by aromatic cage binding pockets in Tudor domains; these protein-protein interactions (PPIs) govern gene expression. Despite their biological importance, the molecular details of methylated Arg recognition are poorly understood. While the desolvation, hydrogen bonding, and guanidinium stacking of methylated Arg have been explored in model systems and proposed to contribute to binding, direct interactions between the methyl groups and the aromatic residues in the binding pocket have not previously been investigated. Herein, we mechanistically study the CH3-π interactions between the SPIN1 triple Tudor domain and histone asymmetric dimethylarginine. We find that these CH3-π interactions are electrostatically tunable, exhibiting cation-π character, albeit attenuated relative to cation-π interactions with quaternary ammonium ions, offering key insight into how methylation of Arg alters its binding epitope to enable new PPIs.

Insights into the DNA and RNA Interactions of Human Topoisomerase III Beta Using Molecular Dynamics Simulations.

Human topoisomerase III beta (hTOP3B) is the only topoisomerase in the human cell that can act on both DNA and RNA substrates. Recent findings have emphasized the physiological importance of hTOP3B and consolidated it as a valuable drug target for antiviral and anticancer therapeutics. Although type IA topoisomerases of different organisms have been studied over the years, the step-by-step interaction of hTOP3B and nucleic acid substrates is still not well understood. Due to the lack of hTOP3B-RNA structures as well as DNA/RNA covalent complexes, computational investigations have been limited. In our study, we utilized molecular dynamics (MD) simulations to study the interactions between hTOP3B and nucleic acids to get a closer look into the residues that play a role in binding DNA or RNA and facilitate catalysis, along with the differences and similarities when hTOP3B interacts with DNA compared to RNA. For this, we generated multiple models of hTOP3B complexed with DNA and RNA sequences using the hTOP3B crystal structure and 8-mer single-stranded DNA and RNA sequences. These models include both covalent and noncovalent complexes, which are then subjected to MD simulations and analyzed. Our findings highlight the complexes' stability, sequence preference, and interactions of the binding pocket residues with different nucleotides. Our work demonstrates that hTOP3B forms stable complexes with both DNA and RNA and provides a better understanding of the enzyme's interaction with different nucleic acid substrate sequences.

Effect of Lipid Bilayer Anchoring on the Conformational Properties of the Cytochrome P450 2D6 Binding Site.

Human cytochrome P450 (CYP) proteins metabolize 75% of small-molecule pharmaceuticals, which makes structure-based modeling of CYP metabolism and inhibition, bolstered by the current availability of X-ray crystal structures of CYP globular catalytic domains, an attractive prospect. Accounting for this broad metabolic capacity is a combination of the existence of multiple different CYP proteins and the capacity of a single CYP protein to metabolize multiple different small molecules. It is thought that structural plasticity and flexibility contribute to this latter property; therefore, incorporating diverse conformational states of a particular CYP is likely an important consideration in structure-based CYP metabolism and inhibition modeling. While all-atom explicit-solvent molecular dynamics simulations can be used to generate conformational ensembles under biologically relevant conditions, existing CYP crystal structures are of the globular domain only, whereas human CYPs contain N-terminal transmembrane and linker peptides that anchor the globular catalytic domain to the endoplasmic reticulum. To determine whether this can cause significant differences in the sampled binding site conformations, microsecond scale all-atom explicit-solvent molecular dynamics simulations of the CYP2D6 globular domain in an aqueous environment were compared with those of the full-length protein anchored in a POPC lipid bilayer. While bilayer-anchoring damped some structural fluctuations in the globular domain relative to the aqueous simulations, none of the affected residues included binding site pocket residues. Furthermore, clustering of molecular dynamics snapshots based on either pairwise binding site pocket RMSD or volume differences demonstrated a lack of separation of snapshots from the two simulation conditions into different clusters. These results suggest the substantially simpler and computationally cheaper aqueous simulation approach can be used to generate a relevant conformational ensemble of the CYP2D6 binding site for structure-based metabolism and inhibition modeling.

Anti-Human Immunodeficiency Virus-1 Property of Thai Herbal Extract Kerra™.

Kerra™, a Thai traditional herbal medicine derived from the "Tak-Ka-Si-La Scripture" and composed of nine medicinal plants, has demonstrated potential antiviral properties against HIV. This study investigated the inhibitory effects of Kerra™ on HIV-1 reverse transcriptase (RT) and its ability to prevent pseudo-HIV viral infection in HEK293 cells. The results showed that Kerra™ extract achieved a 95.73 ± 4.24% relative inhibition of HIV-1 RT, with an IC50 value of 42.66 ± 8.74 µg/mL. Docking studies revealed that key phytochemicals in Kerra™, such as oleamide, formononetin, and biochanin A, interact with several residues in the RT non-nucleoside binding pocket, contributing to their inhibitory effects. Furthermore, Kerra™ was able to reduce pseudo-HIV infection in HEK293 cells at a concentration of 10 µg/mL, suggesting its potential as a supplementary treatment for HIV.

Residues in the fructose-binding pocket are required for ketohexokinase-A activity

Excessive fructose consumption is a primary contributor to the global surges in obesity, cancer, and metabolic syndrome. Fructolysis is not robustly regulated and is initiated by ketohexokinase (KHK). In this study, we determined the crystal structure of KHK-A, one of two human isozymes of KHK, in the apo-state at 1.85 Å resolution, and we investigated the roles of residues in the fructose-binding pocket by mutational analysis. Introducing alanine at D15, N42, or N45 inactivated KHK-A, whereas mutating R141 or K174 reduced activity and thermodynamic stability. Kinetic studies revealed that the R141A and K174A mutations reduced fructose affinity by 2- to 4-fold compared to WT KHK-A, without affecting ATP affinity. Molecular dynamics simulations provided mechanistic insights into the potential roles of the mutated residues in ligand coordination and the maintenance of an open state in one monomer and a closed state in the other. Protein–protein interactome analysis indicated distinct expression patterns and downregulation of partner proteins in different tumor tissues, warranting a re-evaluation of KHK’s role in cancer development and progression. The connections between different cancer genes and the KHK signaling pathway suggest that KHK is a potential target for preventing cancer metastasis. This study enhances our understanding of KHK-A's structure and function and offers valuable insights into potential targets for developing treatments for obesity, cancer, and metabolic syndrome.

Podophyllotoxin derivatives-tubulin complex reveals a potential binding site of tubulin polymerization inhibitors in α-tubulin adjacent to colchicine site

The colchicine site of β-tubulin has been proven to be essential binding sites of microtubule polymerization inhibitors. Recent studies implied that GTP pocket of α-tubulin adjacent to colchicine sites is a potential binding site for developing tubulin polymerization inhibitors. However, the structural basis for which type of structural fragments was more beneficial for enhancing the affinity of α-tubulin is still unclear. Here, podophyllotoxin derivatives-tubulin complex crystals indicated that heterocyclic with the highly electronegative and small steric hindrance was conducive to change configuration and enhance the affinity of the residues in GTP pocket of α-tubulin. Triazole with lone-pairs electrons and small steric hindrance exhibited the strongest affinity for enhancing affinity of podophyllotoxin derivatives by forming two hydrogen bonds with αT5 Ser178. Pyrimidine with the secondary strong affinity could bind Asn101 to make the αH7 configuration deflection, which reduces the stability of tubulin result in its depolymerization. Conversely, 4β-quinoline-podophyllotoxin with the weakest affinity did not interact with α-tubulin. The molecular dynamics simulation and protein thermal shift results showed that 4β-triazole-podophyllotoxin-tubulin was the most stable mainly due to two hydrogen bonds and the higher van der Waals force. This work provided a structural basis of the potential binding sites for extending the α/β-tubulin dual-binding sites inhibitors design strategy.

The efficacy of the food-grade antimicrobial xanthorrhizol against Staphylococcus aureus is associated with McsL channel expression.

The emergence and spread of multidrug-resistant Staphylococcus aureus strains demonstrates the urgent need for new antimicrobials. Xanthorrhizol, a plant-derived sesquiterpenoid compound, has a rapid killing effect on methicillin-susceptible strains and methicillin-resistant strains of S. aureus achieving the complete killing of staphylococcal cells within 2 min using 64 μg/mL xanthorrhizol. However, the mechanism of its action is not yet fully understood. The S. aureus cells treated with xanthorrhizol were studied using optical diffraction tomography. Activity of xanthorrhizol against the wild-type and mscL null mutant of S. aureus ATCC 29213 strain was evaluated in the time-kill assay. Molecular docking was conducted to predict the binding of xanthorrhizol to the SaMscL protein. Xanthorrhizol treatment of S. aureus cells revealed a decrease in cell volume, dry weight, and refractive index (RI), indicating efflux of the cell cytoplasm, which is consistent with the spontaneous activation of the mechanosensitive MscL channel. S. aureus ATCC 29213ΔmscL was significantly more resistant to xanthorrhizol than was the wild-type strain. Xanthorrhizol had an enhanced inhibitory effect on the growth and viability of exponentially growing S. aureus ATCC 29213ΔmscL cells overexpressing the SaMscL protein and led to a noticeable decrease in their viability in the stationary growth phase. The amino acid residues F5, V14, M23, A79, and V84 were predicted to be the residues of the binding pocket for xanthorrhizol. We also showed that xanthorrhizol increased the efflux of solutes such as K+ and glutamate from S. aureus ATCC 29213ΔmscL cells overexpressing SaMscL. Xanthorrhizol enhanced the antibacterial activity of the antibiotic dihydrostreptomycin, which targets the MscL protein. Our findings indicate that xanthorrhizol targets the SaMscL protein in S. aureus cells and may have important implications for the development of a safe antimicrobial agent.

Unveiling the Mechanism of Phenamacril Resistance in F. graminearum: Computational and Experimental Insights into the C423A Mutation in FgMyoI.

Phenamacril (PHA) is a highly selective fungicide for controlling fusarium head blight (FHB) mainly caused by F. graminearum and F. asiaticum. However, the C423A mutation in myosin I of F. graminearum (FgMyoI) leads to natural resistance to PHA. Here, based on the computational approaches and biochemical validation, we elucidate the atomic-level mechanism behind the natural resistance of F. graminearum to the fungicide PHA due to the C423A mutation in FgMyoI. The mutation leads to a rearrangement of pocket residues, resulting in increased size and flexibility of the binding pocket, which impairs the stable binding of PHA. MST experiments confirm that the mutant protein FgMyoIC423A exhibits significantly reduced affinity for PHA compared to wild-type FgMyoI and the nonresistant C423K mutant. This decreased binding affinity likely underlies the development of PHA resistance in F. graminearum. Conversely, the nonresistant C423K mutant retains sensitivity to PHA due to the introduction of a strong hydrogen bond donor, which facilitates stable binding of PHA in the pocket. These findings shed light on the molecular basis of PHA resistance and provide new directions for the creation of new myosin inhibitors.

Engineering of a Baeyer-Villiger monooxygenase to Improve Substrate Scope, Stereoselectivity and Regioselectivity.

Baeyer-Villiger monooxygenases belong to a family of flavin-binding proteins that catalyze the Baeyer-Villiger (BV) oxidation of ketones to produce lactones or esters, which are important intermediates in pharmaceuticals or sustainable materials. Phenylacetone monooxygenase (PAMO) from Thermobifida fusca with moderate thermostability catalyzes the oxidation of aryl ketone substrates, but is limited by high specificity and narrow substrate scope. In the present study, we applied loop optimization by loop swapping followed by focused saturation mutagenesis in order to evolve PAMO mutants capable of catalyzing the regioselective BV oxidation of cyclohexanone and cyclobutanone derivatives with formation of either normal or abnormal esters or lactones. We further modulated PAMO to increase enantioselectivity. Crystal structure studies indicate that rotation occurs in the NADP-binding domain and that the high B-factor region is predominantly distributed in the catalytic pocket residues. Computational analyses further revealed dynamic character in the catalytic pocket and reshaped hydrogen bond interaction networks, which is more favorable for substrate binding. Our study provides useful insights for studying enzyme-substrate adaptations.

Binding characteristics of the doxepin E/Z-isomers to the histamine H1 receptor revealed by receptor-bound ligand analysis and molecular dynamics study.

Doxepin is an antihistamine and tricyclic antidepressant that binds to the histamine H1 receptor (H1R) with high affinity. Doxepin is an 85:15 mixture of the E- and Z-isomers. The Z-isomer is well known to be more effective than the E-isomer, whereas based on the crystal structure of the H1R/doxepin complex, the hydroxyl group of Thr1123.37 is close enough to form a hydrogen bond with the oxygen atom of the E-isomer. The detailed binding characteristics and reasons for the differences remain unclear. In this study, we analyzed doxepin isomers bound to the receptor following extraction from a purified H1R protein complexed with doxepin. The ratio of the E- and Z-isomers bound to wild-type (WT) H1R was 55:45, indicating that the Z-isomer was bound to WT H1R with an approximately 5.2-fold higher affinity than the E-isomer. For the T1123.37V mutant, the E/Z ratio was 89:11, indicating that both isomers have similar affinities. Free energy calculations using molecular dynamics (MD) simulations also reproduced the experimental results of the relative binding free energy differences between the isomers for WT and T1123.37V. Furthermore, MD simulations revealed that the hydroxyl group of T1123.37 did not form hydrogen bonds with the E-isomer, but with the adjacent residues in the binding pocket. Analysis of the receptor-bound doxepin and MD simulations suggested that the hydroxyl group of T1123.37 contributes to the formation of a chemical environment in the binding pocket, which is slightly more favorable for the Z-isomer without hydrogen bonding with doxepin.

Unveiling the potential of Traditional Chinese Medicines in combating NorA-mediated S. aureus drug resistance. A molecular dynamic study

Drug resistance Staphylococcus aureus (S. aureus) is emerging as the most prevalent pathogen in surgical wounds and orthopedic surgeries. NorA is a major drug efflux involved in the drug resistance, reducing intracellular drug concentrations. The current study aimed to find some potential inhibitors derived from Traditional Chinese Medicines (TCM) against NorA using a 500 ns molecular dynamics (MD) simulation. Root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), free energy, dynamic cross correlation matrix (DCCM) was computed. Three TCM compound; piperine, demethoxycurcumin, and tectorigenin exhibited many hydrogen bonds and hydrophobic contacts in the binding pockets residues, especially with ionizable residues (Arg 98, Glu 222, Asp 307, and Arg 310), forming two different charged regions. RMSD of NorA-piperine complex shows some initial instability at 5.2 Å, followed by an increase to 5.8 Å at 222 ns, and a further rise to 6.1 Å by the end of the 500 ns simulation period. Similarly, the NorA-demethoxycurcumin and NorA-tectorigenin complexes display elevated terminal RMSD of 7.9 Å each. RMSF, Rg, free energy, and binding pockets interactions confirmed the potential activity of piperine, demethoxycurcumin, and tectorigenin against NorA inhibition. In apo state NorA exhibited dispersion within the ranges of −100 to 150 (PC1: 39.97 %) while in complexed with ligands it illustrated significant differences in the motion patterns on PC1 (46.04 % and 27.39 %). The NorA-ligands DCCM displayed predominantly negatively correlated motions, particularly at the terminal resides from 200 to 370 where the pocket binding residues are located. These compounds also demonstrated drug-likeness and fitting best in the binding pocket residues (Asn > 137, Phe > 140, Phe > 303 Asp > 307, Arg > 310, Glu > 222, and Thr > 223). In conclusion, the compounds piperine, demethoxycurcumin, and tectorigenin interact with NorA through hydrogen bonds and hydrophobic contacts, particularly with key ionizable residues, Arg 98, Glu 222, Asp 307, and Arg 310, indicating potential inhibitory activity.

Structure and Substrate Recognition of the Escherichia coli Transport Protein NupG from the Nucleoside: H+ Symporter (NHS) Family

The nucleoside transporter NupG is one of the two principal transport proteins in the inner membrane of Escherichia coli that enable the organism to scavenge nucleosides from its external environment. NupG functions in a symport manner driven by the proton motive force and is a member of the Nucleoside:H+ Symporter (NHS) subfamily of the Major Facilitator Superfamily (MFS) of transporters. NupG has broad substrate specificity, transporting all naturally occurring purine and pyrimidine nucleosides. In studies over many years the nupG gene has been cloned and amplified, and the NupG protein has been purified, subjected to biochemical, biophysical and computational analysis, and its X-ray structure determined in the apo state at 3.0 Å resolution. The NupG structure had a typical MFS fold with twelve transmembrane spanning α-helices and distinct N- and C-terminal domains linked by a flexible loop. Preliminary site-directed mutagenesis and molecular docking studies on NupG identified nine putative nucleoside binding pocket residues (R136, T140, F143, Q225, N228, Q261, E264, Y318, F322) and a mutant (D323A) with 20-fold enhanced uridine binding activity. Further biochemical and structural investigations are necessary to better understand the substrate recognition and molecular mechanism of E. coli NHS family proteins (NupG, XapB, YegT).

Evaluation of antifungal potentials of Albizia kalkora extract as a natural fungicide: In vitro and computational studies

The antifungal bioactivity potential of the organic extract of silk tree (Albizia kalkora) was investigated in the current study. The crude extracts of A. kalkora and methanol, n-hexane, chloroform, and ethyl acetate fractions were prepared. The antifungal activity of obtained fractions of A. kalkora was studied at different concentrations ranging from 0.39-50 µg/mL. Dimethyl sulfoxide (DMSO) was taken as a toxicity control, whereas thiophanate methyl (TM) as a positive control. All the fractions significantly reduced the FOL growth (methanolic: 9.49–94.93 %, n-hexane: 11.12–100 %, chloroform: 20.96–91.41 %, and ethyl acetate: 18.75–96.70 %). The n-hexane fraction showed 6.25 µg/mL MIC as compared to TM with 64 µg/mL MIC. The non-polar (n-hexane) fraction showed maximum antifungal bioactivity against FOL in comparison with chloroform, methanol, and ethyl acetate fractions. GC/MS analysis exhibited that the n-hexane fraction contained hexadecanoic acid, 9,12,15-octadecatrienoic acid, 9,12-octadecadienoic acid, bis(2-ethylhexyl) phthalate, methyl stearate, and [1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylic acid. The results of in vitro antifungal inhibition were further reinforced by molecular docking analysis. Five virulence proteins of FOL i.e., pH-responsive PacC transcription factor (PACC), MeaB, TOR; target of rapamycin (FMK1), Signal transducing MAP kinase kinase (STE-STE7), and High Osmolarity Glycerol 1(HOG1) were docked with identified phytocompounds in the n-hexane fraction by GC/MS analysis. MEAB showed maximum binding affinities with zinnimide (−12.03 kcal/mol), HOG1 and FMK1with α-Tocospiro-B (−11.51 kcal/mol) and (−10.55 kcal/mol) respectively, STE-STE7 with docosanoic acid (−11.31 kcal/mol), and PACC with heptadecanoic acid (−9.88 kcal/mol) respectively with strong hydrophobic or hydrophilic interactions with active pocket residues. In conclusion, the n-hexane fraction of the A. kalkora can be used to manage FOL.

Discovery of periplasmic solute binding proteins with specificity for ketone bodies: β-hydroxybutyrate binding proteins.

Polyhydroxyalkanoates are a class of biodegradable, thermoplastic polymers which represent a major carbon source for various bacteria. Proteins which mediate the translocation of polyhydroxyalkanoate breakdown products, such as β-hydroxybutyrate (BHB)-a ketone body which in humans serves as an important biomarker, have not been well characterized. In our investigation to screen a solute-binding protein (SBP) which can act as a suitable recognition element for BHB, we uncovered insights at the intersection of bacterial metabolism and diagnostics. Herein, we identify SBPs associated with putative ATP-binding cassette transporters that specifically recognize BHB, with the potential to serve as recognition elements for continuous quantification of this analyte. Through bioinformatic analysis, we identified candidate SBPs from known metabolizers of polyhydroxybutyrate-including proteins from Cupriavidus necator, Ensifer meliloti, Paucimonas lemoignei, and Thermus thermophilus. After recombinant expression in Escherichia coli, we demonstrated with intrinsic tryptophan fluorescence spectroscopy that four candidate proteins interacted with BHB, ranging from nanomolar to micromolar affinity. Tt.2, an intrinsically thermostable protein from Thermus thermophilus, was observed to have the tightest binding and specificity for BHB, which was confirmed by isothermal calorimetry. Structural analyses facilitated by AlphaFold2, along with molecular docking and dynamics simulations, were used to hypothesize key residues in the binding pocket and to model the conformational dynamics of substrate unbinding. Overall, this study provides strong evidence identifying the cognate ligands of SBPs which we hypothesize to be involved in prokaryotic cellular translocation of polyhydroxyalkanoate breakdown products, while highlighting these proteins' promising biotechnological application.