Polysaccharide utilization loci from Bacteroidota encode CE15 enzymes with possible roles in cleaving pectin-lignin bonds.

Lignocellulose is a renewable but complex material exhibiting high recalcitrance to enzymatic hydrolysis, which is attributed, in part, to the presence of covalent linkages between lignin and polysaccharides in the plant cell wall. Glucuronoyl esterases from carbohydrate esterase family 15 (CE15) have been proposed as an aid in reducing this recalcitrance by cleaving ester bonds found between lignin and glucuronoxylan. In the Bacteroidota phylum, some species organize genes related to carbohydrate metabolism in polysaccharide utilization loci (PULs) which encode all necessary proteins to bind, deconstruct, and respond to a target glycan. Bioinformatic analyses identified CE15 members in some PULs that appear to not target the expected glucuronoxylan. Here, five CE15 members from such PULs were investigated with the aim of gaining insights on their biological roles. The selected targets were characterized using glucuronoyl esterase model substrates and with a new synthetic molecule mimicking a putative ester linkage between pectin and lignin. The CE15 enzyme from Phocaeicola vulgatus was structurally determined by X-ray crystallography both with and without carbohydrate ligands with galacturonate binding in a distinct conformation than that of glucuronate. We further explored whether these CE15 enzymes could act akin to pectin methylesterases on pectin-rich biomass but did not find evidence to support the proposed activity. Based on the evidence gathered, the CE15 enzymes in the PULs expected to degrade pectin could be involved in cleavage of uronic acid esters in rhamnogalacturonans.IMPORTANCEThe plant cell wall is a highly complex matrix, and while most of its polymers interact non-covalently, there are also covalent bonds between lignin and carbohydrates. Bonds between xylan and lignin are known, such as the glucuronoyl ester bonds that are cleavable by CE15 enzymes. Our work here indicates that enzymes from CE15 may also have other activities, as we have discovered enzymes in PULs proposed to target other polysaccharides, including pectin. Our study represents the first investigation of such enzymes. Our first hypothesis that the enzymes would act as pectin methylesterases was shown to be false, and we instead propose that they may cleave other esters on complex pectins such as rhamnogalacturonan II. The work presents both the characterization of five novel enzymes and can also provide indirect information about the components of the cell wall itself, which is a highly challenging material to chemically analyze in fine detail.

Family characteristics, phylogenetic reconstruction, and potential applications of the plant BAHD acyltransferase family.

The BAHD acyltransferase family is a class of proteins in plants that can acylate a variety of primary and specialized secondary metabolites. The typically acylated products have greatly improved stability, lipid solubility, and bioavailability and thus show significant differences in their physicochemical properties and pharmacological activities. Here, we review the protein structure, catalytic mechanism, and phylogenetic reconstruction of plant BAHD acyltransferases to describe their family characteristics, acylation reactions, and the processes of potential functional differentiation. Moreover, the potential applications of the BAHD family in human activities are discussed from the perspectives of improving the quality of economic plants, enhancing the efficacy of medicinal plants, improving plant biomass for use in biofuel, and promoting stress resistance of land plants. This review provides a reference for the research and production of plant BAHD acyltransferases.

JAK3 Y841 Autophosphorylation Is Critical for STAT5B Activation, Kinase Domain Stability and Dimer Formation.

Janus tyrosine kinase 3 (JAK3) is primarily expressed in immune cells and is needed for signaling by the common gamma chain (γc) family of cytokines. Abnormal JAK3 signal transduction can manifest as hematological disorders, e.g., leukemia, severe combined immunodeficiency (SCID) and autoimmune disease states. While regulatory JAK3 phosphosites have been well studied, here a functional proteomics approach coupling a JAK3 autokinase assay to mass spectrometry revealed ten previously unreported autophosphorylation sites (Y105, Y190, Y238, Y399, Y633, Y637, Y738, Y762, Y824, and Y841). Of interest, Y841 was determined to be evolutionarily conserved across multiple species and JAK family members, suggesting a broader role for this residue. Phospho-substitution mutants confirmed that Y841 is also required for STAT5 tyrosine phosphorylation. The homologous JAK1 residue Y894 elicited a similar response to mutagenesis, indicating the shared importance for this site in JAK family members. Phospho-specific Y841-JAK3 antibodies recognized activated kinase from various T-cell lines and transforming JAK3 mutants. Computational biophysics analysis linked Y841 phosphorylation to enhanced JAK3 JH1 domain stability across pH environments, as well as to facilitated complementary electrostatic JH1 dimer formation. Interestingly, Y841 is not limited to tyrosine kinases, suggesting it represents a conserved ubiquitous enzymatic function that may hold therapeutic potential across multiple kinase families.

Use of soybean oil to modulate the gel properties of soybean protein isolation-wheat gluten composite with or without CaCl2.

BACKGROUND: Plant protein is widely used in the study of animal protein substitutes and healthy sustainable products. The gel properties are crucial for the production of plant protein foods. Therefore, the present study investigated the use of soybean oil to modulate the gel properties of soybean protein isolation-wheat gluten composite with or without CaCl2 . RESULTS: Oil droplets filled protein network pores under the addition of soybean oil (1-2%). This resulted in an enhanced gel hardness and water holding capacity. Further addition of soybean oil (3-4%), oil droplets and some protein-oil compounds increased the distance between the protein molecule chain. The results of Fourier transform infrared spectroscopy and intermolecular interaction also showed that the disulfide bond and ß-sheet ratio decreased in the gel system, which damaged the overall structure of the gel network. Compared with the addition of 0 m CaCl2 , salt ion reduced the electrostatic repulsion between proteins, and local protein cross-linking was more intense at 0.005 m CaCl2 concentration. In the present study, structural properties and rheological analysis showed that the overall strength of the gel was weakened after the addition of CaCl2 . CONCLUSION: The presence of appropriate amount of soybean oil can fill the gel pores and improve the texture properties and network structure of soy protein isolate-wheat gluten (SPI-WG) composite gel. Excessive soybean oil may hinder protein-protein interaction and adversely affect protein gel. In addition, the presence or absence of CaCl2 significantly affected the gelling properties of SPI-WG composite protein gels. © 2023 Society of Chemical Industry.

Milling of buckwheat hull to cell-scale: Influences on the behaviors of protein and starch in dough and noodles.

Superfine grinding of insoluble dietary fiber (IDF) is a promising method to improve the product quality by regulating the interaction between protein and starch. In this study, the effects of buckwheat-hull IDF powder, at cell-scale (50-10 µm) and tissue-scale (500-100 µm), on the dough rheology and noodle quality were investigated. Results showed that cell-scale IDF with higher exposure of active groups increased the viscoelasticity and deformation resistance of the dough, due to the aggregation of protein-protein and protein-IDF. Compared with the control sample, the addition of tissue-scale or cell-scale IDF significantly increased the starch gelatinization rate (ß, C3-C2) and decreased the starch hot-gel stability. Cell-scale IDF increased the rigid structure (ß-sheet) of protein, thus improving the noodle texture. The decreased cooking quality of cell-scale IDF-fortified noodles was related to the poor stability of rigid gluten matrix and the weakened interaction between water and macromolecules (starch and protein) during cooking.

NIAS-Server 2.0: A versatile complementary tool for structural biology studies.

Increasing the repertoire of available complementary tools to advance the knowledge of protein structures is fundamental for structural biology. The Neighbors Influence of Amino Acids and Secondary Structures (NIAS) is a server that analyzes a protein's conformational preferences of amino acids. NIAS is based on the Angle Probability List, representing the normalized frequency of empirical conformational preferences, such as torsion angles, of different amino acid pairs and their corresponding secondary structure information, as available in the Protein Data Bank. In this work, we announce the updated NIAS server with the data comprising all structures deposited until Sep 2022, 7 years after the initial release. Unlike the original publication, which accounted for only studies conducted with X-ray crystallography, we added data from solid nuclear magnetic resonance (NMR), solution NMR, CullPDB, Electron Microscopy, and Electron Crystallography using multiple filtering parameters. We also provide examples of how NIAS can be applied as a complementary analysis tool for different structural biology works and what are its limitations.

Effect of Four Extraction Processes with Acetone-Ethanol Protocols on the Properties of Egg Yolk Phosphatidylcholine and the Structure of Residual Protein.

In this study, four acetone-ethanol protocols were employed to investigate the effect of extraction processes on the yield and purity of phosphatidylcholine (PC) from dried egg yolk powder and fresh liquid egg yolk, as well as the cholesterol distribution between the oil and PC fraction. Furthermore, the physicochemical (thermo-stability, fatty acid composition, and molecular structure) and emulsifying (zeta potential, particle size, EAI, ESI, and creaming index) properties of the final PC product were also examined. In addition, the structural characteristics of the egg yolk residual protein were highlighted to promote its application in food industries. The results showed that de-oiling with acetone prior to ethanol extraction can achieve high yield (19.92%) and purity (68.62%) of the PC product with low cholesterol content (< 0.12%). The extraction processes exhibited a significant impact on the emulsifying properties of the PC product. The creaming index of PC emulsions was higher than that of egg yolk powder emulsions with high protein concentration, suggesting that PC plays a critical role in the emulsifying stability of egg yolk protein dispersion. The structural characteristics of residual protein, including free sulfhydryl groups and primary, secondary, and ternary structures, showed considerable differentiation related to extraction processes. These findings provide a powerful tool for the dietary utilization of egg yolk PC and protein in future.

Bovine Serum Albumin as a Platform for Designing Biologically Active Nanocarriers-Experimental and Computational Studies.

Due to the specificity of their structure, protein systems are adapted to carry various ligands. The structure of many proteins potentially allows for two types of immobilization of a therapeutic agent, either on the outer surface of the protein or within the protein structure. The existence of two active sites in BSA's structure, the so-called Sudlow I and II, was confirmed. The conducted research involved determining the effectiveness of BSA as a potential carrier of 5-fluorouracil (5FU). 5-fluorouracil is a broad-spectrum anticancer drug targeting solid tumors. The research was carried out to estimate the physicochemical properties of the system using complementary measurement techniques. The optimization of the complex formation conditions made it possible to obtain significant correlations between the form of the drug and the effective localization of the active substance in the structure of the protein molecule. The presence of two amino groups in the 5FU structure contributes to the deprotonation of the molecule at high pH values (pH > 8) and the transition to the anionic form (AN1 and AN3). To investigate the binding affinity of the tautomeric form with BSA, UV-vis absorption, fluorescence quenching, zeta potential, QCM-D, and CD spectroscopic studies were performed. The experimental research was supported by molecular dynamics (MD) simulations and molecular docking. The simulations confirm the potential location of 5FU tautomers inside the BSA structure and on its surface.

Protein Function Analysis through Machine Learning.

Machine learning (ML) has been an important arsenal in computational biology used to elucidate protein function for decades. With the recent burgeoning of novel ML methods and applications, new ML approaches have been incorporated into many areas of computational biology dealing with protein function. We examine how ML has been integrated into a wide range of computational models to improve prediction accuracy and gain a better understanding of protein function. The applications discussed are protein structure prediction, protein engineering using sequence modifications to achieve stability and druggability characteristics, molecular docking in terms of protein-ligand binding, including allosteric effects, protein-protein interactions and protein-centric drug discovery. To quantify the mechanisms underlying protein function, a holistic approach that takes structure, flexibility, stability, and dynamics into account is required, as these aspects become inseparable through their interdependence. Another key component of protein function is conformational dynamics, which often manifest as protein kinetics. Computational methods that use ML to generate representative conformational ensembles and quantify differences in conformational ensembles important for function are included in this review. Future opportunities are highlighted for each of these topics.

PcoB is a defense outer membrane protein that facilitates cellular uptake of copper.

Copper (Cu) is one of the most abundant trace metals in all organisms, involved in a plethora of cellular processes. Yet elevated concentrations of the element are harmful, and interestingly prokaryotes are more sensitive for environmental Cu stress than humans. Various transport systems are present to maintain intracellular Cu homeostasis, including the prokaryotic plasmid-encoded multiprotein pco operon, which is generally assigned as a defense mechanism against elevated Cu concentrations. Here we structurally and functionally characterize the outer membrane component of the Pco system, PcoB, recovering a 2.0 Å structure, revealing a classical ß-barrel architecture. Unexpectedly, we identify a large opening on the extracellular side, linked to a considerably electronegative funnel that becomes narrower towards the periplasm, defining an ion-conducting pathway as also supported by metal binding quantification via inductively coupled plasma mass spectrometry and molecular dynamics (MD) simulations. However, the structure is partially obstructed towards the periplasmic side, and yet flux is permitted in the presence of a Cu gradient as shown by functional characterization in vitro. Complementary in vivo experiments demonstrate that isolated PcoB confers increased sensitivity towards Cu. Aggregated, our findings indicate that PcoB serves to permit Cu import. Thus, it is possible the Pco system physiologically accumulates Cu in the periplasm as a part of an unorthodox defense mechanism against metal stress. These results point to a previously unrecognized principle of maintaining Cu homeostasis and may as such also assist in the understanding and in efforts towards combatting bacterial infections of Pco-harboring pathogens.

Capturing acyltransferase(s) transforming final step in the biosynthesis of a major Iridoid Glycoside, (Picroside-II) in a Himalayan Medicinal Herb, Picrorhiza kurroa.

BACKGROUND: Picrorhiza kurroa has been reported as an age-old ayurvedic hepato-protection to treat hepatic disorders due to the presence of iridoids such as picroside-II (P-II), picroside-I, and kutkoside. The acylation of catalpol and vanilloyl coenzyme A by acyltransferases (ATs) is critical step in P-II biosynthesis. Since accumulation of P-II occurs only in roots, rhizomes and stolons in comparison to leaves uprooting of this critically endangered herb has been the only source of this compound. Recently, we reported that P-II acylation likely happen in roots, while stolons serve as the vital P-II storage compartment. Therefore, developing an alternate engineered platform for P-II biosynthesis require identification of P-II specific AT/s. METHODS AND RESULTS: In that direction, egg-NOG function annotated 815 ATs from de novo RNA sequencing of tissue culture based 'shoots-only' system and nursery grown shoots, roots, and stolons varying in P-II content, were cross-compared in silico to arrive at ATs sequences unique and/or common to stolons and roots. Verification for organ and accession-wise upregulation in gene expression of these ATs by qRT-PCR has shortlisted six putative 'P-II-forming' ATs. Further, six-frame translation, ab initio protein structure modelling and protein-ligand molecular docking of these ATs signified one MBOAT domain containing AT with preferential binding to the vanillic acid CoA thiol ester as well as with P-II, implying that this could be potential AT decorating final structure of P-II. CONCLUSIONS: Organ-wise comparative transcriptome mining coupled with reverse transcription real time qRT-PCR and protein-ligand docking led to the identification of an acyltransferases, contributing to the final structure of P-II.

Inhibition of SARS-CoV-2 replication by zinc gluconate in combination with hinokitiol.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic is currently the major challenge to global public health. Two proteases, papain-like protease (PLpro) and the 3-chymotrypsin-like protease (3CLpro or Mpro), are indispensable for SARS-CoV-2 replication, making them attractive targets for antiviral therapy development. Here we screened a panel of essential metal ions using a proteolytic assay and identified that zinc gluconate, a widely-used zinc supplement, strongly inhibited the proteolytic activities of the two proteases in vitro. Biochemical and crystallographic data reveal that zinc gluconate exhibited the inhibitory function via binding to the protease catalytic site residues. We further show that treatment of zinc gluconate in combination with a small molecule ionophore hinokitiol, could lead to elevated intracellular Zn2+ level and thereby significantly impaired the two protease activities in cellulo. Particularly, this approach could also be applied to rescue SARS-CoV-2 infected mammalian cells, indicative of potential application to combat coronavirus infections. Our studies provide the direct experimental evidence that elevated intracellular zinc concentration directly inhibits SARS-CoV-2 replication and suggest the potential benefits to use the zinc supplements for coronavirus disease 2019 (COVID-19) treatment.

Distribution and speciation of selenium in soybean proteins and its effect on protein structure and functionality.

Although the Se concentration and recovery efficiency of soybean seeds treated with selenate were âˆ¼ 1.8 times those of the selenite treatment, the Se was mainly in the organic form of selenomethionine (>90% of total Se) irrespective of the Se source. The Se concentrations of soybean protein isolate (SPI) and glycinin (11S) were 29.1%-38.6% higher than those of soybean protein concentrate (SPC) and ß-conglycinin (7S) in Se-enriched soybeans, with selenomethionine accounting for > 80% of the Se in all proteins. The content of sulfur-containing methionine in SPI and 11S markedly decreased in Se-enriched soybeans compared with the control. No significant effect of Se was observed on protein content, subunit composition, secondary structure, micromorphology, or functionality. Foliar spray of selenate provides an economical and efficient way to produce Se-enriched soybeans without affecting protein structure and functionality, where SPI and 11S display a high ability to enrich Se (mainly selenomethionine).

NMR Spectroscopy of the Main Protease of SARS-CoV-2 and Fragment-Based Screening Identify Three Protein Hotspots and an Antiviral Fragment.

The main protease (3CLp) of the SARS-CoV-2, the causative agent for the COVID-19 pandemic, is one of the main targets for drug development. To be active, 3CLp relies on a complex interplay between dimerization, active site flexibility, and allosteric regulation. The deciphering of these mechanisms is a crucial step to enable the search for inhibitors. In this context, using NMR spectroscopy, we studied the conformation of dimeric 3CLp from the SARS-CoV-2 and monitored ligand binding, based on NMR signal assignments. We performed a fragment-based screening that led to the identification of 38 fragment hits. Their binding sites showed three hotspots on 3CLp, two in the substrate binding pocket and one at the dimer interface. F01 is a non-covalent inhibitor of the 3CLp and has antiviral activity in SARS-CoV-2 infected cells. This study sheds light on the complex structure-function relationships of 3CLp and constitutes a strong basis to assist in developing potent 3CLp inhibitors.

Molecular cloning and structural analysis of key enzymes in Tetrastigma hemsleyanum for resveratrol biosynthesis.

Resveratrol (RES), a plant antitoxin, has antioxidant, anti-inflammatory, anti-cancer and cardiovascular protection effects. It has been reported that RES can be stably detected in a Chinese herbal medicinal plant Tetrastigma hemsleyanum. At present, the research of T. hemsleyanum mainly focused on the discovery of new compounds and pharmacology. However, there were few studies on the molecular mechanism of the synthesis of secondary metabolites in T. hemsleyanum. In this experiment, four key enzymes (ThPAL/ThC4H/Th4CL/ThRS) involved in the RES biosynthesis pathway were cloned and obtained. They contained an open reading frame (ORF) of 2139 bp, 1518 bp, 1716 bp and 1035 bp, encoding 712, 505, 571 and 344 amino acids, separately. Various bioinformatics tools were used to analyze these deduced protein domains, secondary structures, three-dimensional (3D) structures and phylogenetic trees. Subsequently, quantitative primers were designed to conduct the tissue-specific expression. Quantitative results displayed that the four genes were expressed in all tested tissues, and their expression in root tubers was more stable. Moreover, the subcellular localization of the four genes was studied by constructed recombinant green fluorescent expression vectors. Herein, by digging out the key enzyme genes in the biosynthesis of RES in T. hemsleyanum, this experiment tried to reveal the expression patterns of these key enzyme genes. It also provided the basis for the research on the molecular level, which will help people further illuminate and clarify the biosynthesis and regulation mechanism of secondary metabolites in T. hemsleyanum.

Hydrogen Sulfide Oxidation by Sulfide Quinone Oxidoreductase.

Hydrogen sulfide (H2 S) is an environmental toxin and a heritage of ancient microbial metabolism that has stimulated new interest following its discovery as a neuromodulator. While many physiological responses have been attributed to low H2 S levels, higher levels inhibit complex IV in the electron transport chain. To prevent respiratory poisoning, a dedicated set of enzymes that make up the mitochondrial sulfide oxidation pathway exists to clear H2 S. The committed step in this pathway is catalyzed by sulfide quinone oxidoreductase (SQOR), which couples sulfide oxidation to coenzyme Q10 reduction in the electron transport chain. The SQOR reaction prevents H2 S accumulation and generates highly reactive persulfide species as products; these can be further oxidized or can modify cysteine residues in proteins by persulfidation. Here, we review the kinetic and structural characteristics of human SQOR, and how its unconventional redox cofactor configuration and substrate promiscuity lead to sulfide clearance and potentially expand the signaling potential of H2 S. This dual role of SQOR makes it a promising target for H2 S-based therapeutics.

Structural features of myofibrillar fish protein interacting with phosphatidylcholine liposomes.

The interactions between liposomes and fish myofibrillar protein (surimi ground salted protein, SURP) were evaluated. Liposomes prepared with ultrapure phosphatidylcholine (UPC) or partially purified phosphatidylcholine (PPC) were dispersed at different weight ratio on SURP. Changes in protein stability and structure were evaluated using FTIR, intrinsic fluorescence and free sulfhydryl groups, and changes in liposome properties were studied by dynamic light scattering and electron microscopy. PPC promoted denaturation and aggregation of SURP, reflected in secondary structure loss, exposure of tyrosine residues and increment of free sulfhydryl. UPC produced partial unfolding and changes in the secondary structure of SURP from α-helical to ß-strand. Liposome size increased by about 40% and showed modified surface charge after SURP exposure, indicating the formation of protein corona. Surface charge and composition of liposomes influence SURP stability and could exert different effects on the myofibrillar protein network, which is important for liposome applications in surimi products.

Molecular Structure and Functional Analysis of Pyocin S8 from Pseudomonas aeruginosa Reveals the Essential Requirement of a Glutamate Residue in the H-N-H Motif for DNase Activity.

Multidrug resistance (MDR) is a serious threat to public health, making the development of new antimicrobials an urgent necessity. Pyocins are protein antibiotics produced by Pseudomonas aeruginosa strains to kill closely related cells during intraspecific competition. Here, we report an in-depth biochemical, microbicidal, and structural characterization of a new S-type pyocin, named S8. Initially, we described the domain organization and secondary structure of S8. Subsequently, we observed that a recombinant S8 composed of the killing subunit in complex with the immunity (ImS8) protein killed the strain PAO1. Furthermore, mutation of a highly conserved glutamic acid to alanine (Glu100Ala) completely inhibited this antimicrobial activity. The integrity of the H-N-H motif is probably essential in the killing activity of S8, as Glu100 is a highly conserved residue of this motif. Next, we observed that S8 is a metal-dependent endonuclease, as EDTA treatment abolished its ability to cleave supercoiled pUC18 plasmid. Supplementation of apo S8 with Ni2+ strongly induced this DNase activity, whereas Mn2+ and Mg2+ exhibited moderate effects and Zn2+ was inhibitory. Additionally, S8 bound Zn2+ with a higher affinity than Ni2+ and the Glu100Ala mutation decreased the affinity of S8 for these metals, as shown by isothermal titration calorimetry (ITC). Finally, we describe the crystal structure of the Glu100Ala S8 DNase-ImS8 complex at 1.38 Å, which gave us new insights into the endonuclease activity of S8. Our results reinforce the possibility of using pyocin S8 as an alternative therapy for infections caused by MDR strains, while leaving commensal human microbiota intact.IMPORTANCE Pyocins are proteins produced by Pseudomonas aeruginosa strains that participate in intraspecific competition and host-pathogen interactions. They were first described in the 1950s and since then have gained attention as possible new antibiotics. However, there is still only scarce information about the molecular mechanisms by which these molecules induce cell death. Here, we show that the metal-dependent endonuclease activity of pyocin S8 is involved with its antimicrobial action against strain PAO1. We also describe that this killing activity is dependent on a conserved Glu residue within the H-N-H motif. The potency and selectivity of pyocin S8 toward a narrow spectrum of P. aeruginosa strains make this protein an attractive antimicrobial alternative for combatting MDR strains, while leaving commensal human microbiota intact.

Origins and features of pectate lyases and their applications in industry.

Pectate lyase treatment can be an alternative strategy of the chemical processing, which causes severe environmental pollution, and has been broadly studied and applied for diverse industrial applications including textile industry, beverage industry, pulp processing, pectic wastewater pretreatment, and oil extraction. This review gave a brief description of the origins, enzymatic characterizations, structure, and applications of pectate lyases (Pels). Most of the reported pectate lyases are originated from microorganisms with a small number of them from plants and animals. Due to the diverse environments that these microorganisms exist, Pels present diversified features, especially for the range of optimal pH and temperature. The diversified biochemical properties of Pels define their applications in different industries, and the applications of alkaline Pels on cotton bioscouring and ramie degumming in textile industry were focused in this review. This review also discussed the perspectives of the development and applications of Pels. KEY POINTS: • The first review on pectate lyase focusing on biotechnological applications. • Origins, features, structures, applications of pectate lyases reviewed. • Applications of alkaline Pels in textile industry demonstrated. • Perspectives on future development and applications of Pels discussed.

Virtual Screening of Human Class-A GPCRs Using Ligand Profiles Built on Multiple Ligand-Receptor Interactions.

G protein-coupled receptors (GPCRs) are a large family of integral membrane proteins responsible for cellular signal transductions. Identification of therapeutic compounds to regulate physiological processes is an important first step of drug discovery. We proposed MAGELLAN, a novel hierarchical virtual-screening (VS) pipeline, which starts with low-resolution protein structure prediction and structure-based binding-site identification, followed by homologous GPCR detections through structure and orthosteric binding-site comparisons. Ligand profiles constructed from the homologous ligand-GPCR complexes are then used to thread through compound databases for VS. The pipeline was first tested in a large-scale retrospective screening experiment against 224 human Class A GPCRs, where MAGELLAN achieved a median enrichment factor (EF) of 14.38, significantly higher than that using individual ligand profiles. Next, MAGELLAN was examined on 5 and 20 GPCRs from two public VS databases (DUD-E and GPCR-Bench) and resulted in an average EF of 9.75 and 13.70, respectively, which compare favorably with other state-of-the-art docking- and ligand-based methods, including AutoDock Vina (with EF = 1.48/3.16 in DUD-E and GPCR-Bench), DOCK 6 (2.12/3.47 in DUD-E and GPCR-Bench), PoLi (2.2 in DUD-E), and FINDSITECcomb2.0 (2.90 in DUD-E). Detailed data analyses show that the major advantage of MAGELLAN is attributed to the power of ligand profiling, which integrates complementary methods for ligand-GPCR interaction recognition and thus significantly improves the coverage and sensitivity of VS models. Finally, cases studies on opioid and motilin receptors show that new connections between functionally related GPCRs can be visualized in the minimum spanning tree built on the similarities of predicted ligand-binding ensembles, suggesting a novel use of MAGELLAN for GPCR deorphanization.