Structural Homology Modeling Research Articles

Small angle X-ray scattering and in silico based structure and function analysis of a novel xylobiohydrolase (AcGH30A) from Acetivibrio clariflavus

Xylobiohydrolase plays a crucial role in the hydrolysis of xylan, a complex polysaccharide present in the cell walls of plants. This study focuses on the solution structure and substrate binding analysis of a novel xylobiohydrolase, AcGH30A, from Acetivibrio clariflavus. Secondary structure analysis of AcGH30A in an aqueous environment using Circular Dichroism and in silico modeling revealed an α/β/α sandwich structure with a central β-barrel comprising eight β-strands. Superposition of the homology-modelled structure of AcGH30A with its closest homolog showed that the active-site contains Glu175 and Glu268 as the catalytic residues. Molecular docking confirmed xylobiose as the preferred ligand, showcasing polar interactions with the catalytic amino acids, indicating its xylobiohydrolase activity. AcGH30A displayed a high binding affinity with xylobiose with an association constant (Ka ) of 7.83 × 105 M−1, as determined by isothermal titration calorimetry. Molecular dynamics (MD) simulations of AcGH30A and AcGH30A-xylobiose complex in solution showed reduced RMSD, Rg and SASA values, confirming the stability and compactness of the complex. MD simulations further highlighted the crucial role of Glu175 in hydrogen bonding with the ligand, which acts as an acid or base. Small-angle X-ray scattering (SAXS) analysis of AcGH30A showed its molecular shape as an earbud with a globular structure existing in a monodispersed state, which was corroborated by dynamic light scattering (DLS). The hydrodynamic radius (Rh ) of AcGH30A, determined by DLS, was 3.7 nm. This study significantly contributed valuable insights into the structure and functional aspects of AcGH30A.

Mechanisms of Polyethylene Terephthalate Pellet Fragmentation into Nanoplastics and Assimilable Carbons by Wastewater Comamonas.

Comamonadaceae bacteria are enriched on poly(ethylene terephthalate) (PET) microplastics in wastewaters and urban rivers, but the PET-degrading mechanisms remain unclear. Here, we investigated these mechanisms with Comamonas testosteroniKF-1, a wastewater isolate, by combining microscopy, spectroscopy, proteomics, protein modeling, and genetic engineering. Compared to minor dents on PET films, scanning electron microscopy revealed significant fragmentation of PET pellets, resulting in a 3.5-fold increase in the abundance of small nanoparticles (<100 nm) during 30-day cultivation. Infrared spectroscopy captured primarily hydrolytic cleavage in the fragmented pellet particles. Solution analysis further demonstrated double hydrolysis of a PET oligomer, bis(2-hydroxyethyl) terephthalate, to the bioavailable monomer terephthalate. Supplementation with acetate, a common wastewater co-substrate, promoted cell growth and PET fragmentation. Of the multiple hydrolases encoded in the genome, intracellular proteomics detected only one, which was found in both acetate-only and PET-only conditions. Homology modeling of this hydrolase structure illustrated substrate binding analogous to reported PET hydrolases, despite dissimilar sequences. Mutants lacking this hydrolase gene were incapable of PET oligomer hydrolysis and had a 21% decrease in PET fragmentation; re-insertion of the gene restored both functions. Thus, we have identified constitutive production of a key PET-degrading hydrolase in wastewater Comamonas, which could be exploited for plastic bioconversion.

The Y44 residue point mutation in CXCL14 reveals the significance of CXCL14 protein stability in anti-tumor activity

Abstract Objectives CXCL14, a member of the CXC chemokine family, plays a significant role in tumor development, progression, and metastasis, making it a potential target for cancer diagnosis and treatment. This study aims to investigate the impact of a point mutation in the (41VSRYR45) motif of CXCL14 on its anti-tumor activity. Methods Phylogenetic analysis, tertiary structure prediction, homology modeling, and cell culture experiments were employed to assess the effects of CXCL14 mutations on protein stability and anti-tumor activity. Phylogenetic analysis identified conserved regions critical for function, while computational tools predicted structural changes due to mutations. Homology modeling provided structural insights, and cell culture experiments involved transfecting HeLa cells with wild-type or mutant CXCL14 plasmids, assessing stability and anti-tumor effects. Additionally, in vivo, xenograft experiments using nude mice were conducted to evaluate the anti-tumor efficacy of CXCL14 variants. Results The conserved Y44 site within the (41VSRYR45) motif was identified as a key ubiquitination site. Mutations Y44V and Y44F enhanced CXCL14 stability, reducing HeLa cell proliferation and migration while increasing apoptosis. In vivo, tumor xenograft experiments confirmed the stronger inhibitory effect of these CXCL14 variants on tumor growth compared to the wild-type protein. Conclusion The Y44 site in CXCL14 is crucial for its stability and anti-tumor activity. Mutations Y44V and Y44F enhance CXCL14 stability and anti-tumor effects, suggesting that targeting this site could be a promising therapeutic strategy for cervical cancer.

Discovery of a novel mutation F184S (c.551T>C) in GATA4 gene causing congenital heart disease in a consanguineous Saudi family

Background & aimCongenital heart disease (CHD) is the most common cause of non-infectious deaths in infants worldwide. However, the molecular mechanisms underlying CHD remain unclear. Approximately 30 % of the causes are believed to be genetic mutations and chromosomal abnormalities. In this study, we aimed to identify the genetic causes of CHD in consanguineous families. MethodsFourth-generation pedigrees with CHD were recruited. The main cardiac features of the patient included absence of the right pulmonary artery and a large dilated left pulmonary artery. To determine the underlying genetic cause, whole-exome sequencing was performed and subsequently confirmed using Sanger sequencing and different online databases to study the pathogenesis of the identified gene mutation. An in-silico homology model was created using the Alphafold homology model structure of GATA4 (AF-P43694-F1). The missense3D online program was used to evaluate the structural alterations. ResultsWe identified a deleterious mutation c.551T > C (p.Phe184Ser) in GATA4. GATA4 is a highly conserved zinc-finger transcription factor, and its continuous expression is essential for cardiogenesis during embryogenesis. The in-silico model suggested a compromised binding efficiency with other proteins. Several variant interpretation algorithms indicated that the F184S missense variant in GATA4 is damaging, whereas HOPE analysis indicated the functional impairment of DNA binding of transcription factors and zinc-ion binding activities of GATA4. ConclusionThe variant identified in GATA4 appears to cause recessive CHD in the family. In silico analysis suggested that this variant was damaging and caused multiple structural and functional aberrations. This study may support prenatal screening of the fetus in this family to prevent diseases in new generations.

UPC2 mutations and development of azole resistance in Candida albicans hospital isolates from Lebanon

ObjectivesThis study evaluated the role of Upc2 in the development of azole resistance in Candida albicans isolates from Lebanese hospitalized patients and determined a correlation between resistance and virulence. MethodsThe UPC2 gene which codes for an ergosterol biosynthesis regulator was sequenced and analysed in two azole-resistant and one azole-susceptible C. albicans isolates. An amino acid substitution screening was carried out on Upc2 with a focus on its ligand binding domain (LBD) known to interact with ergosterol. Then, Upc2 protein secondary structure prediction and homology modelling were conducted, followed by total plasma membrane ergosterol and cell wall chitin quantifications. For virulence, mouse models of systemic infection were generated and an agar adhesion and invasion test was performed. ResultsAzole-resistant isolates harboured novel amino acid substitutions in the LBD of Upc2 and changes in protein secondary structures were observed. In addition, these isolates exhibited a significant increase in plasma membrane ergosterol content. Resistance and virulence were inversely correlated while increased cell wall chitin concentration does not seem to be linked to resistance since even though we observed an increase in chitin concentration, it was not statistically significant. ConclusionsThe azole-resistant C. albicans isolates harboured novel amino acid substitutions in the LBD of Upc2 which are speculated to induce an increase in plasma membrane ergosterol content, preventing the binding of azoles to their target, resulting in resistance.

Identification of Potent SFRP1 Inhibitors for Colorectal Cancer using a Comprehensive Computational Approach.

The incidence of CRC has increased worldwide over the past decade. The statistics report from WHO highlights the increased severity and fatality rate of CRC among the populations. Wnt/β-catenin is recognized as the resource for cell regeneration and cancer signaling pathways driven by frizzled receptor cofactors. Aberrant regulation of Wnt/β- catenin suppression is an important challenge in treating CRC management. The SFRP1 comprises a cysteine-rich region that is homologous to the putative Wnt-binding sites of Frizzled proteins, with the potential to impede and alter the cascade of Wnt signaling. Indirect regulation, like targeting Wnt antagonist SFRP1, is an alternative strategy to suppress the cancer signals by enhancing the apoptotic activity. Hence, this study aimed to approach the SFRP1 protein as a therapeutic target to inhibit Wnt signaling in colorectal cancer. Further, it aimed to identify the lead compounds against the SFRP1 protein, which inhibit the oncogenic expression of CRC, which might be possible using computational approaches, recognizing the importance of the SFRP1 protein role in CRC. The homology-modeled SFRP1 structure was refined, and virtual screening was performed against the anti-cancer drugs and natural drug databases to find the best hit molecules. The molecular docking, MD, and MMGBSA analysis confirmed the firm binding of SFRP1 complexes to identify the potent CRC inhibitors. The amino acid residues Arg5, Arg11, Ala13, Lys 245, Lys274, Phe147, Pro99, and Ser277 are essential for ligand binding and show similar interactions for SFRP1 complexes. The ADME/T profile for top hits is acceptable in range and obtains the drug-likeness property. The 100ns run for MD simulation confirms the stability of protein complexes. Overall, the findings of this study reveal that the lead compounds screened are capable of inhibiting SFRP1 against CRC. Targeting SFRP1 paves the way for new platforms in the field of cancer and the therapeutic sector for new approachable finds.

Efficient Biodegradation of the Neonicotinoid Insecticide Flonicamid by Pseudaminobacter salicylatoxidans CGMCC 1.17248: Kinetics, Pathways, and Enzyme Properties.

Nitrile-containing insecticides can be converted into their amide derivatives by Pseudaminobacter salicylatoxidans. N-(4-trifluoromethylnicotinoyl) glycinamide (TFNG-AM) is converted to 4-(trifluoromethyl) nicotinoyl glycine (TFNG) using nitrile hydratase/amidase. However, the amidase that catalyzes this bioconversion has not yet been fully elucidated. In this study, it was discovered that flonicamid (FLO) is degraded by P. salicylatoxidans into the acid metabolite TFNG via the intermediate TFNG-AM. A half-life of 18.7 h was observed for P. salicylatoxidans resting cells, which transformed 82.8% of the available FLO in 48 h. The resulting amide metabolite, TFNG-AM, was almost all converted to TFNG within 19 d. A novel amidase-encoding gene was cloned and overexpressed in Escherichia coli. The enzyme, PmsiA, hydrolyzed TFNG-AM to TFNG. Despite being categorized as a member of the amidase signature enzyme superfamily, PsmiA only shares 20-30% identity with the 14 previously identified members of this family, indicating that PsmiA represents a novel class of enzyme. Homology structural modeling and molecular docking analyses suggested that key residues Glu247 and Met242 may significantly impact the catalytic activity of PsmiA. This study contributes to our understanding of the biodegradation process of nitrile-containing insecticides and the relationship between the structure and function of metabolic enzymes.

Peripheral Linker Mediates Acyl Carrier Protein's Recognition of Dehydratase and Stabilizes Type-I Mycobacterium tuberculosis Fatty Acid Synthase.

Incomplete structural details of Mycobacterium tuberculosis (Mtb) fatty acid synthase-I (FAS-I) at near-atomic resolution have limited our understanding of the shuttling mechanism of its mobile acyl carrier protein (ACP). Here, we have performed atomistic molecular dynamics simulation of Mtb FAS-I with a homology-modeled structure of ACP stalled at dehydratase (DH) and identified key residues that mediate anchoring of the recognition helix of ACP near DH. The observed distance between catalytic residues of ACP and DH agrees with that reported for fungal FAS-I. Further, the conformation of the peripheral linker is found to be crucial in stabilizing ACP near DH. Correlated interdomain motion is observed between DH, enoyl reductase, and malonyl/palmitoyl transferase, consistent with prior experimental reports of fungal and Mtb FAS-I.

Exploration of Fingerprints and Data Mining-based Prediction of Some Bioactive Compounds from Allium sativum as Histone Deacetylase 9 (HDAC9) Inhibitors.

Histone deacetylase 9 (HDAC9) is an important member of the class IIa family of histone deacetylases. It is well established that over-expression of HDAC9 causes various types of cancers including gastric cancer, breast cancer, ovarian cancer, liver cancer, lung cancer, lymphoblastic leukaemia, etc. The important role of HDAC9 is also recognized in the development of bone, cardiac muscles, and innate immunity. Thus, it will be beneficial to find out the important structural attributes of HDAC9 inhibitors for developing selective HDAC9 inhibitors with higher potency. The classification QSAR-based methods namely Bayesian classification and recursive partitioning method were applied to a dataset consisting of HADC9 inhibitors. The structural features strongly suggested that sulphur-containing compounds can be a good choice for HDAC9 inhibition. For this reason, these models were applied further to screen some natural compounds from Allium sativum. The screened compounds were further accessed for the ADME properties and docked in the homology-modelled structure of HDAC9 in order to find important amino acids for the interaction. The best-docked compound was considered for molecular dynamics (MD) simulation study. The classification models have identified good and bad fingerprints for HDAC9 inhibition. The screened compounds like ajoene, 1,2 vinyl dithiine, diallyl disulphide and diallyl trisulphide had been identified as compounds having potent HDAC9 inhibitory activity. The results from ADME and molecular docking study of these compounds show the binding interaction inside the active site of the HDAC9. The best-docked compound ajoene shows satisfactory results in terms of different validation parameters of MD simulation study. This in-silico modelling study has identified the natural potential lead (s) from Allium sativum. Specifically, the ajoene with the best in-silico features can be considered for further in-vitro and in-vivo investigation to establish as potential HDAC9 inhibitors.

Homology Modeling of Alpha-Glucosidase from Candida albicans: Sequence Analysis and Structural Validation Studies in silico

The alpha-glucosidase enzyme of Candida albicans plays a vital role in the pathogenesis of candidiasis, a serious fatal disease in immune-compromised patients. The unavailability of the threedimensional crystallographic structure of this enzyme creates a hindrance in developing novel and potent inhibitors. Here, an attempt has been made to design a stable three-dimensional conformer of alpha-glucosidase through in silico analysis which may be helpful for the designing of effective drugs. For this purpose, the oligo-1-6-glucosidase enzyme is used as a template for homology modeling of the alpha-glucosidase structure by Molecular Operating Environment 2011-12 software. The generated model was validated through ERRAT and Ramachandran tools, whereas its stability was studied through the molecular dynamics simulation technique. The obtained results indicate that model of alpha-glucosidase has stable secondary and tertiary arrangements. This finding may spur new directions for the rational designing and development of new antifungal inhibitors. Nevertheless, additional experimental investigations and validation are needed to confirm the in silico results of this study.

Identification of early flowering mutant gene in Phalaenopsis amabilis (L.) Blume for sgRNA construction in CRISPR/Cas9 genome editing system.

Phalaenopsis amabilis (L.) Blume commonly called Moth Orchid (Orchidaceae) is a natural orchid species designated as the National Flower of Indonesia for its beautiful flower shape and long-lasting flowering period. Basically, P. amabilis has a long vegetative phase that cause late flowering, about 2 to 3 years for flowering, hence a method to shorten vegetative period is desired. The latest technological approach that can be used to accelerate flowering of P. amabilis is the CRISPR/Cas9 genome editing method to inactivate the GAI (Gibberellic Acid Insensitive) gene as a mutant gene that can accelerate the regulation of FLOWERING TIME (FT) genes flowering biosynthesis pathway. The approach that needs to be taken is to silence the GAI gene with a knockout system which begins with identifying and characterizing the GAI target gene in the P. amabilis which will be used as a single guide RNA. CRISPR/Cas9 mediated knockout efficiency is highly dependent on the properties of the sgRNA used. SgRNA consists of a target sequence, determining its specificity performance. We executed phylogenetic clustering for the PaGAI protein with closely related orchid species such as Dendrobium capra, Dendrobium cultivars and Cymbidium sinensis. SWISS-Model as tool webserver for protein structure homology modeling. Results show that P. amabilis has a specific domain with the occurrence of point mutations in the two conservative domains. Therefore, a single guide RNA reconstruction needs to be implemented.

In silico Docking Analysis of the FDA-Approved Drugs on Envelope Protein of SARS CoV-2 Omicron Variant

The current pandemic situation is created by the highly evolving SARS coronavirus 2 which is having several mutations in its structural proteins. The structural proteins of SARS CoV-2 include spike (S), Envelope (E), Membrane (M), and Nucleocapsid (N) which are primarily responsible for the infection, transmission, and pathogenesis of the virus. Envelope protein is the smallest of the four proteins containing 75 amino acids with a molecular weight of about 8 kDa. The major functions of the hydrophobic envelope protein include envelope formation, budding, replication, and release of the virion. The presence of mutation on the envelope protein results in improper formation of the pentameric structure and also hinders other functional properties. Our computational analysis majorly focuses on several FDA-approved inhibitory compounds that bind to SARS CoV-2 envelope protein that help in the inhibition of virion formation. The percentage of similarity of the envelope protein between SARS CoV and SARS CoV-2 is approximately 96 percent. The homology-modeled structure of the SARS CoV E protein was downloaded from Protein Model Database (PMDB) and the mutation which was found to be consistent among most of the SARS CoV-2 variants was selected as T9I which is present in the N-terminal region. This mutation was introduced into the SARS CoV Envelope protein and was remodeled. The ligands which were approved by FDA were selected for docking analysis to understand their binding capabilities with the envelope protein. Ligands such as Beta-D-Fucose, Mycophenolic Acid, Castanospermine, 1-Deoxynojirimycin, Nafcillin, Guaifenesin, Nabumetone, Cinametic Acid, Lauric acid were used in our study. The docking simulations revealed that Lauric acid, Nafcillin, Nabumetone, and Mycophenolic acid have high binding energy with the SARS CoV-2 wild type (Wuhan) and mutant E protein of the SARS CoV-2 (Omicron) variant. This Insilico data gives insights to test these high binding compounds in invitro studies to prove their efficacy and the protein-protein interactions of envelope protein with its other partnering proteins. These pharmaceutical compounds are a potential alternative in the future for a novel drug development to treat several emerging variants of SARS CoV-2.

An attacin antimicrobial peptide, Hill_BB_C10074, from Hermetia illucens with anti-Pseudomonas aeruginosa activity

BackgroundThere is a global need to develop new therapies to treat infectious diseases and tackle the rise in antimicrobial resistance. To date, the larvae of the Black Solider Fly, Hermetia illucens, have the largest repertoire of antimicrobial peptides derived from insects. Antimicrobial peptides are of particular interest in the exploration of alternative antimicrobials due to their potent action and reduced propensity to induce resistance compared with more traditional antibiotics.ResultsThe predicted attacin from H. illucens, Hill_BB_C10074, was first identified in the transcriptome of H. illucens populations that had been fed a plant-oil based diet. In this study, recombinant Hill_BB_C10074 (500 µg/mL), was found to possess potent antimicrobial activity against the serious Gram-negative pathogen, Pseudomonas aeruginosa. Sequence and structural homology modelling predicted that Hill_BB_C10074 formed a homotrimeric complex that may form pores in the Gram-negative bacterial outer membrane. In vitro experiments defined the antimicrobial action of Hill_BB_C10074 against P. aeruginosa and transmission electron microscopy and electrochemical impedance spectroscopy confirmed the outer membrane disruptive power of Hill_BB_C10074 which was greater than the clinically relevant antibiotic, polymyxin B.ConclusionsCombining predictive tools with in vitro approaches, we have characterised Hill_BB_C10074 as an important insect antimicrobial peptide and promising candidate for the future development of clinical antimicrobials.

Characterization of the Novel Leaderless Bacteriocin, Bawcin, from Bacillus wiedmannii

The rise of drug-resistant bacteria is a major threat to public health, highlighting the urgent need for new antimicrobial compounds and treatments. Bacteriocins, which are ribosomally synthesized antimicrobial peptides produced by bacteria, hold promise as alternatives to conventional antibiotics. In this study, we identified and characterized a novel leaderless bacteriocin, bawcin, the first bacteriocin to be characterized from a Bacillus wiedmannii species. Chemically synthesized and purified bawcin was shown to be active against a broad range of Gram-positive bacteria, including foodborne pathogens Staphylococcus aureus, Bacillus cereus, and Listeria monocytogenes. Stability screening revealed that bawcin is stable over a wide range of pH (2.0–10.0), temperature conditions (25–100 °C), and against the proteases, papain and pepsin. Lastly, three-dimensional structure homology modeling suggests that bawcin contains a saposin-fold with amphipathic helices and a highly cationic surface that may be critical for membrane interaction and the subsequent cell death of its targets. This study provides the foundational understanding of the activity and properties of bawcin, offering valuable insights into its applications across different antimicrobial uses, including as a natural preservative in food and livestock industries.

EasyModel: a user-friendly web-based interface based on MODELLER

Three-dimensional protein structures are invaluable sources of information for the functional annotation of protein molecules. Describing the function of a protein sequence is one of the most common problems in biology. Generally, this problem can be facilitated by studying the tertiary structure of proteins. In the lack of protein structures, comparative modeling often provides a useful three-dimensional model of the protein associated with at least one known protein structure. Comparative modeling predicts the tertiary structure of a certain protein sequence (target) mainly based on its homological sequence to the sequence of one or more proteins with known structures (templates). MODELLER is one of the most widely used tools for homology or comparative modeling of three-dimensional protein structures. However, most users find it challenging to start with MODELLER as it is a command line based and requires knowledge of basic Python scripting to use it efficiently. In this study, a web-based interface has been designed to predict the tertiary structure of proteins based on Modeller, which does the comparative modeling automatically, and uses PHP and Python programming languages. This tool is called “EasyModel” and is available at http://bioinf.modares.ac.ir/software/easymodel/. EasyModel provides a straightforward graphical interface for Modeller that can be used in only one browser.

Recursive genome engineering decodes the evolutionary origin of an essential thymidylate kinase activity in Pseudomonas putida KT2440.

Investigating fundamental aspects of metabolism is vital for advancing our understanding of the diverse biochemical capabilities and biotechnological applications of bacteria. The origin of the essential thymidylate kinase function in the model bacterium Pseudomonas putida KT2440, seemingly interrupted due to the presence of a large genomic island that disrupts the cognate gene, eluded a satisfactory explanation thus far. This is a first-case example of an essential metabolic function, likely acquired by horizontal gene transfer, which "landed" in a locus encoding the same activity. As such, foreign DNA encoding an essential dNMPK could immediately adjust to the recipient host-instead of long-term accommodation and adaptation. Understanding how these functions evolve is a major biological question, and the work presented here is a decisive step toward this direction. Furthermore, identifying essential and accessory genes facilitates removing those deemed irrelevant in industrial settings-yielding genome-reduced cell factories with enhanced properties and genetic stability.

Design of a structure-activity relationship model of vitamin K epoxide reductase (VKORC1) inhibitors combining chemical synthesis of new compounds, enzymatic assays and molecular modelling

Vitamin K antagonists (VKAs) anticoagulants have been used since the 1950s as medicines and rodenticides. These molecules are mainly 4-hydroxycoumarin derivatives and act by inhibiting the vitamin K epoxide reductase (VKORC1), an endoplasmic reticulum membrane resident enzyme. However, many VKORC1 mutations have been reported over the last decade, inducing VKAs resistances and thus treatments failures. Although studies have reported experimental and computational investigations of VKAs based on VKORC1 structural homology models, the development of new effective anticoagulants has been quite complex due to the lack of structural data and reliable structure–activity relationships. However, the recent publication of VKORC1 crystal structure provides new information for further studies. Based on these findings, we combined chemical synthesis, enzymatic assays and molecular modelling methods to design a structure–activity relationship (SAR) model. Our results proved that the lipophilicity, the membrane permeability of inhibitors and their affinity towards human VKORC1 enzyme are the main characteristics for potent anticoagulants. Our SAR model managed to rank compounds according to their ability to inhibit the human VKORC1. Such a tool might constitute an alternative to evaluate new molecules potency before their chemical synthesis and biological assessment and might assist the development of new VKAs.

Molecular Modeling Insights into Metal-Organic Frameworks (MOFs) as a Potential Matrix for Immobilization of Lipase: An In Silico Study.

CRL is a highly versatile enzyme that finds extensive utility in numerous industries, which is attributed to its selectivity and catalytic efficiency, which have been impeded by the impracticality of its implementation, leading to a loss of native catalytic activity and non-reusability. Enzyme immobilization is a necessary step for enabling its reuse, and it provides methods for regulating the biocatalyst's functional efficacy in a synthetic setting. MOFs represent a novel category of porous materials possessing distinct superlative features that make MOFs an optimal host matrix for developing enzyme-MOF composites. In this study, we employed molecular modeling approaches, for instance, molecular docking and MD simulation, to explore the interactions between CRL and a specific MOF, ZIF-8. The present study involved conducting secondary structural analysis and homology modeling of CRL, followed by docking ZIF-8 with CRL. The results of the molecular docking analysis indicate that ZIF-8 was situated within the active site pocket of CRL, where it formed hydrogen bonds with Val-81, Phe-87, Ser-91, Asp-231, Thr-132, Lue-297, Phe-296, Phe-344, Thr-347, and Ser-450. The MD simulation analysis revealed that the CRL and ZIF-8 docked complex exhibited stability over the entire simulation period, and all interactions presented in the initial docked complex were maintained throughout the simulation. The findings derived from this investigation could promote comprehension of the molecular mechanisms underlying the interaction between CRL and ZIF-8 as well as the development of immobilized CRL for diverse industrial purposes.

Discovery and structure-activity relationships (SAR) of a novel class of 2-substituted N-piperidinyl indole-based nociceptin opioid receptor ligands

The development of SAR around substituted N-piperidinyl indole-based nociceptin opioid receptor (NOP) ligands led to the discovery of a novel series of 2-substituted N-piperidinyl indoles that provide both selective NOP full agonists and bifunctional NOP full agonists–μ opioid (MOP) receptor partial agonists. 2-substituted N-piperidinyl indoles have improved potency at the NOP receptor and are NOP full agonists, compared to our previously reported 3-substituted N-piperidinyl indoles that are selective NOP partial agonists. SAR in this series of 2-substituted N-piperidinyl indoles shows that 2-substitution versus 3-substitution on the indole moiety affects their intrinsic activity and opioid receptor selectivity. Molecular docking of these 2-substituted N-piperidinyl indoles in an active-state NOP homology model and MOP receptor structures provides a rationale for the differences observed in the binding, functional profiles and selectivity of 2-substituted versus 3-substituted N-piperidinyl indoles.

Outer membrane protein OMP76 of Riemerella anatipestifer contributes to complement evasion and virulence by binding to duck complement factor vitronectin

ABSTRACT Riemerella anatipestifer is an important bacterial pathogen in poultry. Pathogenic bacteria recruit host complement factors to resist the bactericidal effect of serum complement. Vitronectin (Vn) is a complementary regulatory protein that inhibits the formation of the membrane attack complex (MAC). Microbes use outer membrane proteins (OMPs) to hijack Vn for complement evasion. However, the mechanism by which R. anatipestifer achieves evasion is unclear. This study aimed to characterise OMPs of R. anatipestifer which interact with duck Vn (dVn) during complement evasion. Far-western assays and comparison of wild-type and mutant strains that were treated with dVn and duck serum demonstrated particularly strong binding of OMP76 to dVn. These data were confirmed with Escherichia coli strains expressing and not expressing OMP76. Combining tertiary structure analysis and homology modelling, truncated and knocked-out fragments of OMP76 showed that a cluster of critical amino acids in an extracellular loop of OMP76 mediate the interaction with dVn. Moreover, binding of dVn to R. anatipestifer inhibited MAC deposition on the bacterial surface thereby enhancing survival in duck serum. Virulence of the mutant strain ΔOMP76 was attenuated significantly relative to the wild-type strain. Furthermore, adhesion and invasion abilities of ΔOMP76 decreased, and histopathological changes showed that ΔOMP76 was less virulent in ducklings. Thus, OMP76 is a key virulence factor of R. anatipestifer. The identification of OMP76-mediated evasion of complement by recruitment of dVn contributes significantly to the understanding of the molecular mechanism by which R. anatipestifer escapes host innate immunity and provides a new target for the development of subunit vaccines.