Molecular Recognition Mechanism Research Articles

Elucidating the Interaction and Stability of Withanone and Withaferin‐A with Human Serum Albumin, Lysozyme and Hemoglobin Using Computational Biophysical Modeling

AbstractWithania somnifera consists of various bioactive compounds, of which withanone (WT−N) and withaferin‐A (WT−A) are known to have many therapeutic properties. To understand drug metabolism and transportation methods, binding of endogenous and exogenous ingredients with transport proteins is of utmost importance. Therefore, to perceive the molecular recognition mechanism of WT−N and WT−A, the association of human serum albumin, human hemoglobin and human lysozyme with WT−N and WT−A were examined, using various in silico techniques. For our study, we have employed both quantum mechanics and molecular mechanics approaches. Resultantly, drug‐like properties and reactivity were calculated with WT−N and WT−A being the most reactive among all the withanolides. The protein HSA in presence of WT−N showed more stability than that of WT−A. However, HL and Hb showed better stability with WT−A as compared to WT−N. Several non‐covalent interactions contributed the utmost part in the binding of WT−N and WT−A with the proteins. The MD simulation studies also showed evidence in the conformational changes and other dynamical properties of the three proteins with both the ligands. Finally, MM‐PBSA results confirmed the free binding energy of the complexes. Our study has provided a substantial recognition of bioactive molecules WT−N and WT−A binding with the carrier proteins, which can significantly affect the pharmacokinetics of these molecules.

Flagella at the Host-Microbe Interface: Key Functions Intersect With Redundant Responses.

Many bacteria and other microbes achieve locomotion via flagella, which are organelles that function as a swimming motor. Depending on the environment, flagellar motility can serve a variety of beneficial functions and confer a fitness advantage. For example, within a mammalian host, flagellar motility can provide bacteria the ability to resist clearance by flow, facilitate access to host epithelial cells, and enable travel to nutrient niches. From the host’s perspective, the mobility that flagella impart to bacteria can be associated with harmful activities that can disrupt homeostasis, such as invasion of epithelial cells, translocation across epithelial barriers, and biofilm formation, which ultimately can decrease a host’s reproductive fitness from a perspective of natural selection. Thus, over an evolutionary timescale, the host developed a repertoire of innate and adaptive immune countermeasures that target and mitigate this microbial threat. These countermeasures are wide-ranging and include structural components of the mucosa that maintain spatial segregation of bacteria from the epithelium, mechanisms of molecular recognition and inducible responses to flagellin, and secreted effector molecules of the innate and adaptive immune systems that directly inhibit flagellar motility. While much of our understanding of the dynamics of host-microbe interaction regarding flagella is derived from studies of enteric bacterial pathogens where flagella are a recognized virulence factor, newer studies have delved into host interaction with flagellated members of the commensal microbiota during homeostasis. Even though many aspects of flagellar motility may seem innocuous, the host’s redundant efforts to stop bacteria in their tracks highlights the importance of this host-microbe interaction.

Crystal structure of Acetyl-CoA carboxylase (AccB) from Streptomyces antibioticus and insights into the substrate-binding through in silico mutagenesis and biophysical investigations

Acetyl-CoA carboxylase (ACC) is crucial for polyketides biosynthesis and acts as an essential metabolic checkpoint. It is also an attractive drug target against obesity, cancer, microbial infections, and diabetes. However, the lack of knowledge, particularly sequence-structure function relationship to narrate ligand-enzyme binding, has hindered the progress of ACC-specific therapeutics and unnatural “natural” polyketides. Structural characterization of such enzymes will boost the opportunity to understand the substrate binding, designing new inhibitors and information regarding the molecular rules which control the substrate specificity of ACCs. To understand the substrate specificity, we determined the crystal structure of AccB (Carboxyl-transferase, CT) from Streptomyces antibioticus with a resolution of 2.3 Å and molecular modeling approaches were employed to unveil the molecular mechanism of acetyl-CoA recognition and processing. The CT domain of S. antibioticus shares a similar structural organization with the previous structures and the two steps reaction was confirmed by enzymatic assay. Furthermore, to reveal the key hotspots required for the substrate recognition and processing, in silico mutagenesis validated only three key residues (V223, Q346, and Q514) that help in the fixation of the substrate. Moreover, we also presented atomic level knowledge on the mechanism of the substrate binding, which unveiled the terminal loop (500–514) function as an opening and closing switch and pushes the substrate inside the cavity for stable binding. A significant decline in the hydrogen bonding half-life was observed upon the alanine substitution. Consequently, the presented structural data highlighted the potential key interacting residues for substrate recognition and will also help to re-design ACCs active site for proficient substrate specificity to produce diverse polyketides.

Insight into the Phylogeny and Binding Ability of WRKY Transcription Factors.

WRKY transcription factors (TFs), which make up one of the largest families of TFs in the plant kingdom, are key players in modulating gene expression relating to embryogenesis, senescence, pathogen resistance, and abiotic stress responses. However, the phylogeny and grouping of WRKY TFs and how their binding ability is affected by the flanking regions of W-box sequences remain unclear. In this study, we reconstructed the phylogeny of WRKY across the plant kingdom and characterized the DNA-binding profile of Arabidopsis thaliana WRKY (WRKY54) based on its W-box recognition sequence. We found that WRKY TFs could be separated into five clades, and that the functional zinc-finger motif at the C-terminal of WRKY appeared after several nucleotide substitutions had occurred at the 3′-end of the zinc-finger region in chlorophytes. In addition, we found that W-box flanking regions affect the binding ability of WRKY54 based on the results of a fluorescence-based electrophoretic mobility shift assay (fEMSA) and quartz crystal microbalance (QCM) analysis. The great abundance of WRKY TFs in plants implicates their involvement in diverse molecular regulatory networks, and the flanking regions of W-box sequences may contribute to their molecular recognition mechanism. This phylogeny and our findings on the molecular recognition mechanism of WRKY TFs should be helpful for further research in this area.

Molecular recognition of formylpeptides and diverse agonists by the formylpeptide receptors FPR1 and FPR2

The formylpeptide receptors (FPRs) mediate pattern recognition of formylated peptides derived from invading pathogens or mitochondria from dead host cells. They can also sense other structurally distinct native peptides and even lipid mediators to either promote or resolve inflammation. Pharmacological targeting of FPRs represents a novel therapeutic approach in treating inflammatory diseases. However, the molecular mechanisms underlying FPR ligand recognition are elusive. We report cryo-EM structures of Gi-coupled FPR1 and FPR2 bound to a formylpeptide and Gi-coupled FPR2 bound to two synthetic peptide and small-molecule agonists. Together with mutagenesis data, our structures reveal the molecular mechanism of formylpeptide recognition by FPRs and structural variations of FPR1 and FPR2 leading to their different ligand preferences. Structural analysis also suggests that diverse FPR agonists sample a conserved activation chamber at the bottom of ligand-binding pockets to activate FPRs. Our results provide a basis for rational drug design on FPRs.

Development of Fluorescence Polarization Immunoassay With scFv to Detect Fumonisin Bs in Maize and Simultaneous Study of Their Molecular Recognition Mechanism.

In this study, a fluorescence polarization immunoassay (FPIA) was developed based on the single-chain variable fragments (scFvs) for fumonisin Bs (FBs). The scFvs were prepared from FBs-specific monoclonal antibody secreting hybridomas (4F5 and 4B9). The established FPIA could determine the sum of fumonisin B1 (FB1) and fumonisin B2 (FB2) within a short time. The IC50 of FPIA for the detection of FB1 and FB2 were 29.36 ng/ml and 1,477.82 ng/ml with 4F5 scFv, and 125.16 ng/ml and 30.44 ng/ml with 4B9 scFv, so the 4B9 scFv was selected for detection of FB1 and FB2 in maize samples with a limit of detection of 441.54 μg/kg and 344.933 μg/kg. The recoveries ranged from 84.7 to 104.1% with a coefficient of variation less than 14.1% in spiked samples, and the result of the FPIA method was in good consistency with that of HPLC-MS/MS. To supply a better understanding of the immunoassay results, the interactions mechanism of scFvs-FBs was further revealed by the homology modelling, molecular docking, and molecular dynamic simulation. It was indicated that six complementarity-determining regions (CDRs) were involved in 4B9 scFv recognition, forming a narrow binding cavity, and FB1/FB2 could be inserted into this binding cavity stably through strong hydrogen bonds and other interactions. While in 4F5 scFv, only the FB1 stably inserted in the binding pocket formed by four CDRs through strong hydrogen bonds, and FB2 did not fit the binding cavity due to the lack of hydroxyl at C10, which is the key recognition site of 4F5 scFv. Also, the binding energy of FB2-4B9 scFv complex is higher than the FB2-4F5 scFv complex. This study established a FPIA method with scFv for the detection of FB1 and FB1 in maize, and systematically predicted recognition mechanism of FBs and scFvs, which provided a reference for the better understanding of the immunoassay mechanism.

Molecular interactions of trichoderma β-1,4-glucosidase (ThBglT12) with mycelial cell wall components of phytopathogenic Macrophomina phaseolina

Efficacy of a β-1,4-glucosidase from Trichoderma harzianum T12 (ThBglT12) in disrupting the cell wall of the phytopathogenic fungus M. phaseolina (Macrophomina phaseolina) was studied, as the underlying molecular mechanisms of cell wall recognition remains elusive. In this study, the binding location identified by a consensus of residues predicted by COACH tool, blind docking, and multiple sequence alignment revealed that molecular recognition by ThBglT12 occurred through interactions between the α-1,3-glucan, β-1,3-glucan, β-1,3/1,4-glucan, and chitin components of M. phaseolina, with corresponding binding energies of −7.4, −7.6, −7.5 and −7.8 kcal/mol. The residue consensus verified the participation of Glu172, Tyr304, Trp345, Glu373, Glu430, and Trp431 in the active site pocket of ThBglT12 to bind the ligands, of which Trp345 was the common interacting residue. Root mean square deviation (RMSD), root mean square fluctuation (RMSF), total energy, and minimum distance calculation from molecular dynamics (MD) simulation further confirmed the stability and the closeness of the binding ligands into the ThBglT12 active site pocket. The h-bond occupancy by Glu373 and Trp431 instated the role of the nucleophile for substrate recognition and specificity, crucial for cleaving the β-1,4 linkage. Further investigation showed that the proximity of Glu373 to the anomeric carbon of β-1,3/1,4-glucan (3.5 Å) and chitin (5.5 Å) indicates the nucleophiles’ readiness to form enzyme-substrate intermediates. Plus, the neighboring water molecule appeared to be correctly positioned and oriented towards the anomeric carbon to hydrolyze the β-1,3/1,4-glucan and chitin, in less than 4.0 Å. In a nutshell, the study verified that the ThBglT12 is a good alternative fungicide to inhibit the growth of M. phaseolina. Communicated by Ramaswamy H. Sarma

The proteome reveals the involvement of serine/threonine kinase in the recognition of self- incompatibility in almond

The self-incompatibility recognition mechanism determines whether the gametophyte is successfully fertilized between pollen tube SCF (SKP1–CUL1–F-box-RBX1) protein and pistil S-RNase protein during fertilization is unclear. In this study, the pistils of two almond cultivars ‘Wanfeng’ and ‘Nonpareil’ were used as the experimental materials after self- and nonself/cross-pollination, and pistils from the stamen-removed flowers were used as controls. We used fluorescence microscopy to observe the development of pollen tubes after pollination and 4D-LFQ to detect the protein expression profiles of ‘Wanfeng’ and ‘Nonpareil’ pistils and in controls. The results showed that it took 24–36 h for the development of the pollen tube to 1/3 of the pistil, and a total of 7684 differentially accumulated proteins (DAPs) were identified in the pistil after pollinating for 36 h, of which 7022 were quantifiable. Bioinformatics analysis based on the function of DAPs, identified RNA polymerases (4 DAPs), autophagy (3 DAPs), oxidative phosphorylation (3 DAPs), and homologous recombination (2 DAPs) pathways associated with the self-incompatibility process. These results were confirmed by parallel reaction monitoring (PRM), protein interaction and bioinformatics analysis. Taken together, these results provide the involvement of serine/threonine kinase protein in the reaction of pollen tube recognition the nonself- and the self-S-RNase protein. SignificanceGametophytic self-incompatibility (GSI) is controlled by the highly polymorphic S locus or S haplotype, with two linked self-incompatibility genes, one encoding the S-RNase protein of the pistil S-determinant and the other encoding the F-box/SLF/SFB (S haplotype-specific F-box protein) protein of the pollen S-determinant. The recognition mechanism between pollen tube SCF protein and pistil S-RNase protein is divided into nonself- and self-recognition hypothesis mechanisms. At present, two hypothetical mechanisms cannot explain the recognition between pollen and pistil well, so the mechanism of gametophytic self-incompatibility recognition is still not fully revealed. In this experiment, we investigated the molecular mechanism of pollen-pistil recognition in self-incompatibility using self- and nonself-pollinated pistils of almond cultivars ‘Wanfeng’ and ‘Nonpareil’. Based on our results, we proposed a potential involvement of the MARK2 (serine/threonine kinase) protein in the reaction of pollen tube recognition of the nonself- and the self-S-RNase protein. It provides a new way to reveal how almond pollen tubes recognize the self and nonself S-RNase enzyme protein.

The biological information flow: From cell theory to a new evolutionary synthesis

Traditionally ignored in comparison with the energy flow, the interest on the information flow and in general on the informational perspective is nowadays manifest in biological fields. Herein we will revisit classical ideas on biomolecular information processing, basically from Efim Liberman and Michael Conrad, weaving them together with new views on the information flow as captured by cellular signaling systems and with the development of biological complexity. A consistent new explanatory framework looms—potentially contributing to a new cell theory. It would incorporate fundamental conceptualizations on the mechanisms of molecular recognition and informational architectures, the life cycle and the characterization of meaning, and finally the emergence of biological complexity. Concerning the evolutionary process, this informational approach depicts an indefinite series of recursion processes performed in the open-ended environment of the real world, potentially affected by multiple contingencies modifying the informational architectures involved in recursion. Consequently, a large variety of molecular tools and systems would have been incorporated speeding up the variability of genomes and facilitating their displacement in sequence space. To the extent to which the comparison with human mobility in physical space may hold, a power law could be hypothesized interconnecting the variability outcomes of the different evolutionary ‘vehicles’ or variation modes. Contributing to a renewed discussion on the evolutionary process is another essential goal of the present work.

Multi-analyte sensing strategies towards wearable and intelligent devices.

Continuous efforts to produce functional nanomaterials and flexible/stretchable devices have promoted cumbersome, laboratorial, detection processes toward wearable and portable intelligent sensing approaches. Responding to the challenges of the multiple analytes, mixtures, and complex components of practical samples, sensing array and multivariate analysis techniques have a significant advantage in terms of superior analytical capabilities, i.e., they are convenient, rapid, sensitive and have high-throughput for multi-analyte identification in food safety, clinical diagnoses, and environmental monitoring. Besides traditional molecular design and recognition mechanisms, materials with micro/nano structures also contribute to strong signals, sensitive responses, and novel properties. In this review, through a new perspective of signal amplification for responsive discrimination, we summarize progress in developing sensing arrays based on diverse micro/nanomaterials and their integrated devices for multi-analyte discrimination. An overview of strategies for constructing sensing arrays through various micro or nano building blocks, including 0D nanoparticle assembly and modification, 1D nanowires and fibers, 2D graphene and textiles, is schematized. Then, portable and wearable devices integrating colorimetric sensors or flexible electrochemical electrodes with the newest microelectronic units and circuit boards are presented. Meanwhile, the latest artificial intelligence (AI) algorithms are introduced for massive data analysis in complex biological and environmental systems. With future developments in facile and accurate discrimination for multi-analyte research, extended applications will gear up in various fields.

REGULARITIES OF CHANGES IN THERMODYNAMIC PARAMETERS INDUCED BY THE COMPLEXES FORMATION OF URACIL WITH SOME AROMATIC AMINO ACIDS IN A BUFFER SOLUTION AT PH 7.4

The increasing interest in the physicochemical studies of nucleic acid bases and their derivatives is due mainly to their biological and pharmaceutical importance. Despite increasing data on the interaction of DNA with proteins, the molecular mechanism of recognition of particles in the resulting DNA-protein complexes is not well understood yet. The investigation of the nature and the forces that stabilize nucleic acid structures and their complexes with other molecules is not only of fundamental significance but may also find practical applications. In this paper, we discussed the interaction of model compounds of nucleic acids and proteins using the example of complexes of uracil with aromatic amino acids: L-histidine, L-tryptophan, and L-phenylalanine in aqueous buffer solutions with physiological value of pH to be 7.4. The available literature data on the thermochemical, volume, and heat capacity properties of these systems were used. It should be noted that a change in the acidity of the medium induces a shift in the equilibrium of ionic forms, tautomers, and this leads to a variation in the reactivity of the reagents. The obtained characteristics of the complex formation process differ markedly from the results obtained for an aqueous solution without the use of buffer. A generalization of data on the change in thermodynamic parameters (equilibrium constants, Gibbs energy, enthalpy, entropy, apparent molar volume, and apparent molar heat capacity) under the formation of uracil complexes with above aromatic amino acids depending on their physicochemical properties is given. The dependence of the thermodynamic parameters of the formation of complexes on the acid-base properties of amino acids as a characteristic of the electron density on the carboxylate group has been established. Changes in the standard apparent molar heat capacity and in the standard apparent molar volume of uracil under the addition of amino acids correlate with the hydrophobicity of the added amino acids.

Chemical design of high performance SPR biosensor based on a dielectric nanoparticle assembly supported onto a gold thin film

The SPR system is a very efficient tool to investigate original and highly efficient biosensors and to study molecular recognition mechanisms.

Crystal structure of endocrine-disrupting chemical bisphenol A and estrogen-related receptor γ.

The human estrogen-related receptor γ (ERRγ) is an orphan nuclear receptor. The ERRγ behaves as a constitutive activator of transcription and plays a key role in controlling mitochondrial energy production and energy metabolism. Bisphenol A (BPA) is used mainly in producing polycarbonate plastics and epoxy resins, but it is known as an endocrine disruptor and strongly binds to ERRγ. We determined the crystal structure of ERRγ in complex with BPA. Our structure revealed the molecular mechanism of BPA recognition by ERRγ, in which BPA is well anchored to its ligand-binding pocket. Our structure is the first report of the complex between a nuclear receptor and endocrine disruptor BPA. This structural analysis had a profound impact on subsequent studies of endocrine disruptors.

Crystal structure of human METTL6, the m3C methyltransferase

In tRNA, the epigenetic m3C modification at position 32 in the anticodon loop is highly conserved in eukaryotes, which maintains the folding and basepairing functions of the anticodon. However, the responsible enzymes METTL2 and METTL6 were identified only in recent years. The loss of human METTL6 (hMETTL6) affects the translational process and proteostasis in cells, while in mESCs cells, it leads to defective pluripotency potential. Despite its important functions, the catalytic mechanism of the C32 methylation by this enzyme is poorly understood. Here we present the 1.9 Å high-resolution crystal structure of hMETTL6 bound by SAH. The key residues interacting with the ligand were identified and their roles were confirmed by ITC. We generated a docking model for the hMETTL6-SAH-CMP ternary complex. Interestingly, the CMP molecule binds into a cavity in a positive patch with the base ring pointing to the inside, suggesting a flipped-base mechanism for methylation. We further generated a model for the quaternary complex with tRNASer as a component, which reasonably explained the biochemical behaviors of hMETTL6. Taken together, our crystallographic and biochemical studies provide important insight into the molecular recognition mechanism by METTL6 and may aid in the METTL-based rational drug design in the future.

Unraveling the Molecular Mechanism of Recognition of Human Interferon-Stimulated Gene Product 15 by Coronavirus Papain-Like Proteases: A Multiscale Simulation Study.

The papain-like protease (PLpro) of the coronavirus (CoV) family plays an essential role in processing the viral polyprotein and immune evasion. Additional proteolytic activities of PLpro include deubiquitination and deISGylation, which can reverse the post-translational modification of cellular proteins conjugated with ubiquitin or (Ub) or Ub-like interferon-stimulated gene product 15 (ISG15). These activities regulate innate immune responses against viral infection. Thus, PLpro is a potential antiviral target. Here, we have described the structural and energetic basis of recognition of PLpro by the human ISG15 protein (hISG15) using atomistic molecular dynamics simulation across the CoV family, i.e., MERS-CoV (MCoV), SARS-CoV (SCoV), and SARS-CoV-2 (SCoV2). The cumulative simulation length for all trajectories was 32.0 μs. In the absence of the complete crystal structure of complexes, protein–protein docking was used. A mutation (R167E) was introduced across all three PLpro to study the effect of mutation on the protein–protein binding. Our study reveals that the apo-ISG15 protein remains closed while it adopts an open conformation when bound to PLpro, although the degree of openness varies across the CoV family. The binding free energy analysis suggests that hISG15 binds more strongly with SCoV2-PLpro compared to SCoV or MCoV. The intermolecular electrostatic interaction drives the hISG15-PLpro complexation. Our study showed that SCoV or MCoV-PLpro binds more strongly with the C-domain of hISG15, while SCoV2-PLpro binds more favorably the N-domain of hISG15. Overall, our study explains the molecular basis of differential deISGylating activities of PLpro among the CoV family and the specificity of SCoV2-PLpro toward hISG15.

Molecular Recognition Insights of Sialic Acid Glycans by Distinct Receptors Unveiled by NMR and Molecular Modeling.

All cells are decorated with a highly dense and complex structure of glycan chains, which are mostly attached to proteins and lipids. In this context, sialic acids are a family of nine-carbon acidic monosaccharides typically found at the terminal position of glycan chains, modulating several physiological and pathological processes. Sialic acids have many structural and modulatory roles due to their negative charge and hydrophilicity. In addition, the recognition of sialic acid glycans by mammalian cell lectins, such as siglecs, has been described as an important immunological checkpoint. Furthermore, sialic acid glycans also play a pivotal role in host–pathogen interactions. Various pathogen receptors exposed on the surface of viruses and bacteria are responsible for the binding to sialic acid sugars located on the surface of host cells, becoming a critical point of contact in the infection process. Understanding the molecular mechanism of sialic acid glycans recognition by sialic acid-binding proteins, present on the surface of pathogens or human cells, is essential to realize the biological mechanism of these events and paves the way for the rational development of strategies to modulate sialic acid-protein interactions in diseases. In this perspective, nuclear magnetic resonance (NMR) spectroscopy, assisted with molecular modeling protocols, is a versatile and powerful technique to investigate the structural and dynamic aspects of glycoconjugates and their interactions in solution at the atomic level. NMR provides the corresponding ligand and protein epitopes, essential for designing and developing potential glycan-based therapies. In this review, we critically discuss the current state of knowledge about the structural features behind the molecular recognition of sialic acid glycans by different receptors, naturally present on human cells or pathogens, disclosed by NMR spectroscopy and molecular modeling protocols.

Probing the molecular basis for sulfonamides recognition in surface molecularly imprinted polymers using computational and experimental approaches

The computer-aided design of surface molecularly imprinted polymers (SMIPs) of sulfadimethoxine (SDM) using methacrylic acid (MAA), 4-vinylpyridine (4-VP) or 4-aminostyrene (AS) as functional monomers was performed using Gaussian software, and the corresponding materials were innovatively prepared by surface-initiated supplemental activator and reducing agent atom transfer radical polymerization (SI-SARA ATRP) using an Fe(0)/Cu(II) catalytic system. Sulfonamides were selected as template molecules because of their stable nuclear parent and regular side chain changes, which could help explore the molecular mechanism of imprinting and recognition from the perspective of structural differences. The imprinted systems were calculated using Gaussian software to obtain parameters related to intermolecular interactions. The morphology and physicochemical properties of SMIPs were determined by scanning electron microscope (SEM), oxygen bomb combustion-ion chromatography (OBC-IC) and thermogravimetric analyzer (TGA). The relevant adsorption mechanism was evaluated using the Langmuir–Freundlich (LF) isotherm. The adsorption capacities (Q) of the three SMIPs for nine other sulfonamides were evaluated by experiments, and the weak interaction energy between other sulfonamide molecules and the molecularly imprinted pores generated by SDM was simulated. Compared with the two other functional monomers, MAA demonstrated advantages in Q, pore uniformity, and imprinting factor. Weak interaction energies between the drug and functional monomer, as the main influencing factor, and steric hindrance as a secondary factor, influenced the Q, difficulty of template elution, and selective adsorption of the polymers for the nine other sulfonamides. Quantum chemical calculation data revealed that the number of hydrogen bonds generated, the participation of other weak interactions, and the charge of hydrogen bond donors had great influence on the weak interaction energy.

Toward Understanding the Molecular Recognition of Fungal Chitin and Activation of the Plant Defense Mechanism in Horticultural Crops.

Large volumes of fruit and vegetable production are lost during postharvest handling due to attacks by necrotrophic fungi. One of the promising alternatives proposed for the control of postharvest diseases is the induction of natural defense responses, which can be activated by recognizing molecules present in pathogens, such as chitin. Chitin is one of the most important components of the fungal cell wall and is recognized through plant membrane receptors. These receptors belong to the receptor-like kinase (RLK) family, which possesses a transmembrane domain and/or receptor-like protein (RLP) that requires binding to another RLK receptor to recognize chitin. In addition, these receptors have extracellular LysM motifs that participate in the perception of chitin oligosaccharides. These receptors have been widely studied in Arabidopsis thaliana (A. thaliana) and Oryza sativa (O. sativa); however, it is not clear how the molecular recognition and plant defense mechanisms of chitin oligosaccharides occur in other plant species or fruits. This review includes recent findings on the molecular recognition of chitin oligosaccharides and how they activate defense mechanisms in plants. In addition, we highlight some of the current advances in chitin perception in horticultural crops.

Detection of salinomycin and lasalocid in chicken liver by icELISA based on functional bispecific single-chain antibody (scDb) and interpretation of molecular recognition mechanism

Salinomycin (SAL) and lasalocid (LAS) are widely used as ionophore antibiotics for coccidiosis control. However, their common use as feed additives has led to the occurrence of feed cross-contamination, which has toxic effects on non-target animals. There have been few reports on multiple-residue detection for SAL and LAS in recent years. In this study, two single-chain antibody fragments (scFvs) capable of specifically recognizing SAL and LAS were constructed. Using LAS-scFv and SAL-scFv as parent antibodies, a complete bispecific single-chain diabody (scDb) against both LAS and SAL was built using splicing by overlap extension polymerase chain reaction (SOE-PCR). In addition, the key amino acid sites and interaction energy of antibody variable regions for small-molecule recognition were preliminarily studied by homology modeling and molecular docking. Finally, IC50 values of 12.9 and 8.6ng/mL, with a linear range of 6.9-24.0 and 4.7-16.0ng/mL, were obtained for LAS-scFv and SAL-scFv, respectively. An indirect competitive enzyme-linked immunosorbent assay (icELISA) method was established using scDb to obtain an IC50 of 3.5ng/mL for LAS and 4.1ng/mL for SAL, which showed better sensitivity and specificity than those of the parent scFv antibodies. The recoveries of LAS and SAL in chicken liver were 89.2-92.7%(CV<4.7%) and 88.6-90.2% (CV<6.8%)), respectively.

Molecular ruler mechanism and interfacial catalysis of the integral membrane acyltransferase PatA.

Glycolipids are prominent components of bacterial membranes that play critical roles not only in maintaining the structural integrity of the cell but also in modulating host-pathogen interactions. PatA is an essential acyltransferase involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. We demonstrate by electron spin resonance spectroscopy and surface plasmon resonance that PatA is an integral membrane acyltransferase tightly anchored to anionic lipid bilayers, using a two-helix structural motif and electrostatic interactions. PatA dictates the acyl chain composition of the glycolipid by using an acyl chain selectivity “ruler.” We established this by a combination of structural biology, enzymatic activity, and binding measurements on chemically synthesized nonhydrolyzable acyl–coenzyme A (CoA) derivatives. We propose an interfacial catalytic mechanism that allows PatA to acylate hydrophobic PIMs anchored in the inner membrane of mycobacteria, through the use of water-soluble acyl-CoA donors.