Molecular Recognition Mechanism Research Articles

Selective Separation of Thyroid-Hormone-Receptor-Binding Substances Using Molecularly Imprinted Polymers.

To date, an identification protocol for endocrine disruptors that bind to the thyroid hormone receptor (TR) has not been established. A method for screening and identifying TR-binding substances is highly required due to the existence of unknown TR-binding substances from the environment. Here, we conceived a chromatographic method using a molecularly imprinted polymer (MIP) to create a novel screening protocol for the endocrine disruptors. A receptor-imitating MIP was prepared using N-acetylthyroxine (AcetylT4) and 4-vinylpyridine as a pseudo-template and a functional monomer, respectively, based on the existing molecular recognition mechanism of the TR. The receptor-imitating MIP provided molecular recognition ability for all the TR-binding substances that were employed in this study. The prepared MIPs were packed into a high-performance liquid chromatography column for the simultaneous analysis of TR-binding and non-binding substances. The former was strongly retained, while the latter was not. The presence or absence of TR-binding/non-binding activity resulted in successful dichotomous separation. Additionally, the surface imprinting technique was applied to improve the separation performance of the MIP packing material. MIP-coated uniformly sized silica-based particles of 5 μm were successfully prepared, and the MIP-coated silica column enabled more efficient dichotomous separation of TR-binding and non-binding substances.

Recent Advances and Future Prospects of Molecular Imprinting Polymers as a Recognition Sensing System for Food Analysis: A Review

Molecular imprinting polymers (MIPs) have been widely used to produce stable polymeric materials due to their highly selective binding sites to determine the analyte (target molecule) in food products. MIPs begin with a complex compound between the template molecule and the functional monomers that can be polymerized when there is a closely crossed link. MIPs left specific cavities after the removal of templates during washing, which complements the size and shape of the templates. The use of MIPs has contributed to novel advances in materials science, polymer science, natural science, and other multi-disciplinary systems. Optical chemical sensor is an exciting field in MIPs today due to comprehend the unique affirmation limit of associated polymers giving stable polymers with high molecular recognition capabilities. MIPs display a wide extent of relevance, incredible flexibility, security, and high selectivity; their internal affirmation districts can be explicitly gotten together with design molecules to achieve specific affirmation. This review covers the various achievements of sensors used in laboratory analyses. The advancement in the development of MIPs is evaluated with an accentuation on the preparation principle, the discovery process, the molecular recognition mechanism and future perspectives and challenges for MIPs in building an optical chemical sensor.

Investigating the substitution of intermolecular hydrogen bonds on the surface of self-assembled monolayer by scanning electrochemical microscopy

In this work, the substitution of intermolecular hydrogen bonds on the surface of 3-mercaptopropionic acid self-assembled monolayer (MPA SAM) was investigated using scanning electrochemical microscopy (SECM). • The substitution of intermolecular hydrogen bonds on the surface of MPA SAM was investigated by scanning electrochemical microscopy. • The formation of charge-assisted hydrogen bonds was more likely to induce intermolecular hydrogen bonds substitution. • All phenomena were properly characterized by the changes in current of substrate and tip electrodes. • The substitution of intermolecular hydrogen bonds is conductive to understanding the molecular selective recognition process on biofilm surface. Investigating the substitution of intermolecular hydrogen bonds on the simulated biofilm surface at molecular level is beneficial to understand the mechanism of molecular selective recognition occurring on biofilm surface. In this paper, the substitution of intermolecular hydrogen bonds on the surface of simulated biofilm, self-assembled monolayer of 3-mercaptopropionic acid (MPA SAM), is studied using scanning electrochemical microscopy (SECM). When acetic acid exists on MPA SAM surface, the intermolecular hydrogen bonds between acetic acid and MPA SAM are able to replace those between the redox mediator ferrocenemethanol (FcCH 2 OH) and MPA SAM, because the stronger charge-assisted hydrogen bonds (CAHB) formed between acetic acid and MPA SAM have the ability of substitution. The effect of the substitution of intermolecular hydrogen bonds is more significant with increasing the concentration of acetic acid within certain limits. This work may provide ideas for deeper understanding of molecular selective recognition on biofilm surface.

OsPHS1 is required for both male and female gamete development in rice

Meiosis plays an essential role in the production of male and female gametes. Extensive studies have elucidated that homologous chromosome association and pairing are essential for crossing-over and recombination of chromosomal segments. However, the molecular mechanism of chromosome recognition and pairing remains elusive. Here, we identified a rice male–female sterility mutant plant. Cytological observations showed that the development of both pollen and embryo sacs of the mutant were abnormal due to defects in homologous chromosome recognition and pairing during prophase I. Map-based cloning revealed that Os06g0473000 encoding a poor homologous synapsis 1 (PHS1) protein is the candidate target gene, which was confirmed by knockout using CRISPR/Cas9 technology. Sequence analysis revealed a single base mutation (G > A) involving the junction of the fourth exon and intron of OsPHS1, which is predicted to alter splicing, resulting in an Osphs1 mutant. Expression pattern analysis indicated that OsPHS1 expression levels were mainly expressed in panicles at the beginning of meiosis. Subcellular localization analysis demonstrated that the OsPHS1 protein is situated in the nucleus and cytoplasm. Taken together, our results suggest an important role for OsPHS1 in homologous chromosome pairing in both male and female gametogenesis in rice.

MqsR is a noncanonical microbial RNase toxin that is inhibited by antitoxin MqsA via steric blockage of substrate binding

The MqsRA toxin-antitoxin system is a component of the Escherichia coli stress response. Free MqsR, a ribonuclease, cleaves mRNAs containing a 5'-GC-3' sequence causing a global shutdown of translation and the cell to enter a state of dormancy. Despite a general understanding of MqsR function, the molecular mechanism(s) by which MqsR binds and cleaves RNA and how one or more of these activities is inhibited by its cognate antitoxin MqsA is still poorly understood. Here, we used NMR spectroscopy coupled with mRNA cleavage assays to identify the molecular mechanism of MqsR substrate recognition and the MqsR residues that are essential for its catalytic activity. We show that MqsR preferentially binds substrates that contain purines in the-2 and-1 position relative to the MqsR consensus cleavage sequence and that two residues of MqsR, Tyr81, and Lys56 are strictly required for mRNA cleavage. We also show that MqsA inhibits MqsR activity by sterically blocking mRNA substrates from binding while leaving the active site fully accessible to mononucleotides. Together, these data identify the residues of MqsR that mediate RNA cleavage and reveal a novel mechanism that regulates MqsR substrate specificity.

Inhibition of FGFR Signaling by Targeting FGF/FGFR Extracellular Interactions: Towards the Comprehension of the Molecular Mechanism through NMR Approaches.

NMR-based approaches play a pivotal role in providing insight into molecular recognition mechanisms, affording the required atomic-level description and enabling the identification of promising inhibitors of protein–protein interactions. The aberrant activation of the fibroblast growth factor 2 (FGF2)/fibroblast growth factor receptor (FGFR) signaling pathway drives several pathologies, including cancer development, metastasis formation, resistance to therapy, angiogenesis-driven pathologies, vascular diseases, and viral infections. Most FGFR inhibitors targeting the intracellular ATP binding pocket of FGFR have adverse effects, such as limited specificity and relevant toxicity. A viable alternative is represented by targeting the FGF/FGFR extracellular interactions. We previously identified a few small-molecule inhibitors acting extracellularly, targeting FGFR or FGF. We have now built a small library of natural and synthetic molecules that potentially act as inhibitors of FGF2/FGFR interactions to improve our understanding of the molecular mechanisms of inhibitory activity. Here, we provide a comparative analysis of the interaction mode of small molecules with the FGF2/FGFR complex and the single protein domains. DOSY and residue-level NMR analysis afforded insights into the capability of the potential inhibitors to destabilize complex formation, highlighting different mechanisms of inhibition of FGF2-induced cell proliferation.

Redox-labelled electrochemical aptasensors with nanosupported cancer cells

The transfer of redox-labelled bioelectrochemical sensors from proteins to cells is not straightforward because of the cell downward force issue on the surface of the sensors. In this paper, 20-nm-thick nanopillars are introduced to overcome this issue, in a well-controlled manner. We show on both molecular dynamics simulations and experiments that suspending cells a few nanometers above an electrode surface enables redox-labelled tethered DNA aptamer probes to move freely, while remaining at an interaction distance from a target membrane protein, i. e. epithelial cell adhesion molecule (EpCAM), which is typically overexpressed in cancer cells. By this nanopillar configuration, the interaction of aptamer with cancer cells is clearly observable, with 13 cells as the lower limit of detection. Nanoconfinement induced by the gap between the electrode surface and the cell membrane appears to improve the limit of detection and to lower the melting temperature of DNA aptamer hairpins, offering an additional degree of freedom to optimize molecular recognition mechanisms. This novel nanosupported electrochemical DNA cell sensor scheme including Brownian-fluctuating redox species opens new opportunities for the design of all-electrical sensors using redox-labelled probes.

A mechanistic analysis of bacterial recognition and serine protease cascade initiation in larval hemolymph of Manduca sexta

Serine protease cascades have evolved in vertebrates and invertebrates to mediate rapid defense responses. Previous biochemical studies showed that in hemolymph of a caterpillar, Manduca sexta, recognition of fungi by β-1,3-glucan recognition proteins (βGRP1 and βGRP2) or recognition of bacteria by peptidoglycan recognition protein-1 (PGRP1) and microbe binding protein (MBP) results in autoactivation of hemolymph protease-14 precursor (proHP14). HP14 then activates downstream members of a protease cascade leading to the melanization immune response. ProHP14 has a complex domain architecture, with five low-density lipoprotein receptor class A repeats at its amino terminus, followed by a Sushi domain, a Sushi domain variant called Wonton, and a carboxyl-terminal serine protease catalytic domain. Its zymogen form is activated by specific proteolytic cleavage at the amino-terminal end of the protease domain. While a molecular mechanism for recognition and triggering the response to β-1,3-glucan has been delineated, it is unclear how bacterial recognition stimulates proHP14 activation. To fill this knowledge gap, we expressed the two domains of M. sexta MBP and found that the amino-terminal domain binds to diaminopimelic acid-peptidoglycan (DAP-PG). ProHP14 bound to both the carboxyl-terminal domain (MBP-C) and amino-terminal domain (MBP-N) of MBP. In the mixture of DAP-PG, MBP, and larval plasma, inclusion of an HP14 fragment composed of LDLa repeats 2–5 (LDLa2–5) or MBP-C significantly reduced prophenoloxidase activation, likely by competing with the interactions of the full-length proteins, and suggesting that molecular interactions involving these regions of proHP14 and MBP take part in proHP14 activation in response to peptidoglycan. Using a series of N-terminally truncated versions of proHP14, we found that autoactivation required LDLa2–5. The optimal ratio of PGRP1, MBP, and proHP14 is close to 3:2:1. In summary, proHP14 autoactivation by DAP-type peptidoglycan requires binding of DAP-PG by PGRP1 and the MBP N-terminal domain and association of the LDLa2–5 region of proHP14 with the MBP C-terminal domain. These interactions may concentrate the proHP14 zymogen at the bacterial cell wall surface and promote autoactivation.

Molecular recognition mechanism for the galactoside binding in a melibiose transporter MelB

Molecular recognition mechanism for the galactoside binding in a melibiose transporter MelB

Flexibility of flanking DNA is a key determinant of transcription factor affinity for the core motif

Selective gene regulation is mediated by recognition of specific DNA sequences by transcription factors (TFs). The extremely challenging task of searching out specific cognate DNA binding sites among several million putative sites within the eukaryotic genome is achieved by complex molecular recognition mechanisms. Elements of this recognition code include the core binding sequence, the flanking sequence context, and the shape and conformational flexibility of the composite binding site. To unravel the extent to which DNA flexibility modulates TF binding, in this study, we employed experimentally guided molecular dynamics simulations of ternary complex of closely related Hox heterodimers Exd-Ubx and Exd-Scr with DNA. Results demonstrate that flexibility signatures embedded in the flanking sequences impact TF binding at the cognate binding site. A DNA sequence has intrinsic shape and flexibility features. While shape features are localized, our analyses reveal that flexibility features of the flanking sequences percolate several basepairs and allosterically modulate TF binding at the core. We also show that lack of flexibility in the motif context can render the cognate site resistant to protein-induced shape changes and subsequently lower TF binding affinity. Overall, this study suggests that flexibility-guided DNA shape, and not merely the static shape, is a key unexplored component of the complex DNA-TF recognition code.

Computational Strategies to Identify New Drug Candidates against Neuroinflammation.

Increasing application of computational approaches in these last decades has deeply modified the process of discovery and commercialization of new therapeutic entities. This is especially true in the field of neuroinflammation, in which both the peculiar anatomical localization and the presence of the blood-brain barrier make it mandatory to finely tune the candidates' physicochemical properties from the early stages of the discovery pipeline. The aim of this review is, therefore, to provide a general overview of neuroinflammation to the readers, together with the most common computational strategies that can be exploited to discover and design small molecules controlling neuroinflammation, especially those based on the knowledge of the three-dimensional structure of the biological targets of therapeutic interest. The techniques used to describe the molecular recognition mechanisms, such as molecular docking and molecular dynamics, will therefore be discussed, highlighting their advantages and limitations. Finally, we report several case studies in which computational methods have been applied to drug discovery for neuroinflammation, focusing on the research conducted in the last decade.

The engagement of histone lysine methyltransferases with nucleosomes: structural basis, regulatory mechanisms, and therapeutic implications.

Histone lysine methyltransferases (HKMTs) deposit methyl groups onto lysine residues on histones and play important roles in regulating chromatin structure and gene expression. The structures and functions of HKMTs have been extensively investigated in recent decades, significantly advancing our understanding of the dynamic regulation of histone methylation. Here, we review the recent progress in structural studies of representative HKMTs in complex with nucleosomes (H3K4, H3K27, H3K36, H3K79, and H4K20 methyltransferases), with emphasis on the molecular mechanisms of nucleosome recognition and trans-histone crosstalk by these HKMTs. These structural studies inform HKMTs' roles in tumorigenesis and provide the foundations for developing new therapeutic approaches targeting HKMTs in cancers.

Insight into the mechanism of molecular recognition between human Integrin-Linked Kinase and Cpd22 and its implication at atomic level.

AbsractPseudokinases have received increasing attention over the past decade because of their role in different physiological phenomena. Although pseudokinases lack several active-site residues, thereby hindering their catalytic activity, recent discoveries have shown that these proteins can play a role in intracellular signaling thanks to their non-catalytic functions. Integrin-linked kinase (ILK) was discovered more than two decades ago and was subsequently validated as a promising target for neoplastic diseases. Since then, only a few small-molecule inhibitors have been described, with the V-shaped pyrazole Cpd22 being the most interesting and characterized. However, little is known about its detailed mechanism of action at atomic level. In this study, using a combination of computational chemistry methods including PELE calculations, docking, molecular dynamics and experimental surface plasmon resonance, we were able to prove the direct binding of this molecule to ILK, thus providing the basis of its molecular recognition by the protein and the effect over its architecture. Our breakthroughs show that Cpd22 binding stabilizes the ILK domain by binding to the pseudo-active site in a similar way to the ATP, possibly modulating its scaffolding properties as pseudokinase. Moreover, our results explain the experimental observations obtained during Cpd22 development, thus paving the way to the development of new chemical probes and potential drugs.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s10822-022-00466-1.

Crystal structure of the phosphorylated Arabidopsis MKK5 reveals activation mechanism of MAPK kinases.

The mitogen-activated protein kinase (MAPK) signaling pathways are highly conserved in eukaryotes, regulating various cellular processes. The MAPK kinases (MKKs) are dual specificity kinases, serving as convergence and divergence points of the tripartite MAPK cascades. Here, we investigate the biochemical characteristics and three-dimensional structure of MKK5 in Arabidopsis (AtMKK5). The recombinant full-length AtMKK5 is phosphorylated and can activate its physiological substrate AtMPK6. There is a conserved kinase interacting motif (KIM) at the N-terminus of AtMKK5, indispensable for specific recognition of AtMPK6. The kinase domain of AtMKK5 adopts active conformation, of which the extended activation segment is stabilized by the phosphorylated Ser221 and Thr215 residues. In line with sequence divergence from other MKKs, the αD and αK helices are missing in AtMKK5, suggesting that the AtMKK5 may adopt distinct modes of upstream kinase/substrate binding. Our data shed lights on the molecular mechanisms of MKK activation and substrate recognition, which may help design specific inhibitors targeting human and plant MKKs.

Identification and Expression Analysis of Chemosensory Genes in the Antennal Transcriptome of Chrysanthemum Aphid Macrosiphoniella sanborni.

Simple SummaryThe olfactory system is key for insects to receive external chemical signals, and various chemosensory genes are involved in this process. Thus, research focused on the olfactory mechanisms of insects can provide theoretical guidance for the development of effective green pest-control measures. In this study, an antennal transcriptome analysis of the chrysanthemum aphid (Macrosiphoniella sanborni) was conducted to identify putative chemosensory genes. The relative relationships of chemosensory genes between chrysanthemum aphids and other aphid species were analyzed. Then, the wing-specific and odor-specific expression profiles of these candidate genes were examined. This study contributes to our understanding of the olfactory system and lays the foundation for functional studies of the chemoreception mechanism in M. sanborni.As one of the most destructive oligophagous pests, the chrysanthemum aphid (Macrosiphoniella sanborni) has seriously restricted the sustainable development of the chrysanthemum industry. Olfaction plays a critical role in the environmental perception of aphids, but very little is currently known about the chemosensory mechanism of M. sanborni. In this study, four MsanOBPs, four MsanCSPs, eight MsanORs, two MsanIRs and one MsanSNMP were identified among the 28,323 unigenes derived from the antennal transcriptome bioinformatic analysis of M. sanborni adults. Then, comprehensive phylogenetic analyses of these olfactory-related proteins in different aphid species were performed using multiple sequence alignment. Subsequently, the odor-specific and wing-specific expression profiles of these candidate chemosensory genes were investigated using quantitative real-time PCR. The data showed that most of these chemosensory genes exhibited higher expression levels in alate aphids. Among them, MsanOBP9, MsanOR2, MsanOR4, MsanOR43b-1, MsanCSP1, MsanCSP2, MsanCSP4, MsanIR25a and MsanIR40a in alate aphids showed remarkably higher expression levels than in apterous aphids under the effect of the host plant volatiles, indicating that these genes may take part in the specific behaviors of alate adults, such as host recognition, oviposition site selection and so on. This study lays the groundwork for future research into the molecular mechanism of olfactory recognition in M. sanborni.

Molecular recognition between membrane epitopes and nearly free surface silanols explains silica membranolytic activity

Inhaled crystalline silica causes inflammatory lung diseases, but the mechanism for its unique activity compared to other oxides remains unclear, preventing the development of potential therapeutics. Here, the molecular recognition mechanism between membrane epitopes and “nearly free silanols” (NFS), a specific subgroup of surface silanols, is identified and proposed as a novel broad explanation for particle toxicity in general. Silica samples having different bulk and surface properties, specifically different amounts of NFS, are tested with a set of membrane systems of decreasing molecular complexity and different charge. The results demonstrate that NFS content is the primary determinant of membrane disruption causing red blood cell lysis and changes in lipid order in zwitterionic, but not in negatively charged liposomes. NFS-rich silica strongly and irreversibly adsorbs zwitterionic self-assembled phospholipid structures. This selective interaction is corroborated by density functional theory and supports the hypothesis that NFS recognize membrane epitopes that exhibit a positive quaternary amino and negative phosphate group. These new findings define a new paradigm for deciphering particle-biomembrane interactions that will support safer design of materials and what types of treatments might interrupt particle-biomembrane interactions.

Development of nano Bio LC columns for the search of acetylcholinesterase molecular targets.

A novel acetylcholinesterase Nano liquid chromatography capillary column (75 μm i.d. × 50mm length) was developed for the fast screening of acetylcholinesterase inhibitors and the evaluation of their molecular recognition mechanism. Biotinylated acetylcholinesterase was immobilized on a streptavidin Nano liquid chromatography capillary column. Because of the very strong streptavidin-biotin interaction, the acetylcholinesterase immobilization step performed by frontal analysis is very fast (required less than 10min), and the amount of immobilized acetylcholinesterase was in the microgram range (1μg). The yellow anion obtained from the enzymatic reaction detected at 412nm was achieved within 60 s, and the immobilized acetylcholinesterase retained 96% of the initial activity beyond 90 days. This column was successfully applied for the discrimination of weak affinity ligands to acetylcholinesterase from nonbinders, which is the heart of fragment-based drug design. This column was used for the determination of the IC50 values of a series of inhibitor molecules. In addition, it was demonstrated that competitive experiments could be performed with our miniaturized system to confirm the existence and binding pocket of a ligand to acetylcholinesterase contained in a methanol plant extract. The results revealed that our acetylcholinesterase Nano liquid chromatography capillary column developed in this work represents a useful tool for the rapid screening of inhibitor candidates and evaluation of the action mechanism.

Multivalent Interaction of Beta-Catenin With its Intrinsically Disordered Binding Partner Adenomatous Polyposis Coli.

The Wnt signalling pathway plays key roles in cell proliferation, differentiation and fate decisions in embryonic development and maintenance of adult tissues, and the twelve Armadillo (ARM) repeat-containing protein β-catenin acts as the signal transducer in this pathway. Here we investigate the interaction between β-catenin’s ARM repeat domain and the intrinsically disordered protein adenomatous polyposis coli (APC). APC is a giant multivalent scaffold that brings together the different components of the so-called “β-catenin destruction complex”, which drives β-catenin degradation via the ubiquitin-proteasome pathway. Mutations and truncations in APC, resulting in loss of APC function and hence elevated β-catenin levels and upregulation of Wnt signalling, are associated with numerous cancers including colorectal carcinomas. APC has a long intrinsically disordered region (IDR) that contains a series of 15-residue and 20-residue binding regions for β-catenin. Here we explore the multivalent nature of the interaction of β-catenin with the highest affinity APC repeat, both at equilibrium and under kinetic conditions. We use a combination of single-site substitutions, deletions and insertions to dissect the mechanism of molecular recognition and the roles of the three β-catenin-binding subdomains of APC.

Development of a Highly Sensitive and Specific ic-ELISA and Lateral Flow Immunoassay for Diacetoxyscirpenol.

To monitor the contamination of a type A trichothecene, diacetoxyscirpenol (DAS), one monoclonal antibody (mAb) 8A9 with high affinity and specificity was prepared in the present study. The mAb 8A9 showed a 50% inhibition concentration (IC50) of 0.31 μg/L, which is of the highest affinity reported to date. An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and lateral flow immunoassay (LFIA) based on mAb 8A9 were developed and exhibited limits of detection as low as 0.65 μg/kg and 100 μg/kg in rice samples, respectively. The molecular recognition mechanism of mAb 8A9 to DAS was explored by molecular docking. The results showed that the hydrophobic amino acids of mAb 8A9 interacted with DAS by forming hydrogen bonds and a pi-sigma bond, which lead to a highly specific recognition of DAS. In summary, we produced one mAb, developed ELISA and LFIA for DAS detection in rice with significantly sensitivity, specificity, accuracy, and precision.

Thermodynamic and Molecular Recognition Mechanism of Diastereomeric Salt/Cocrystal-Induced Chiral Separation

To better understand the thermodynamics and molecular self-assembly mechanism of diastereomeric salt/cocrystal-induced chiral separation, a series of 1:1 cocrystals and salts consisting of chiral valines (VAL) and tartaric acid derivatives were synthesized via different methods. Powders of these as-screened cocrystals/salts were characterized by PXRD, TGA, DSC, FT-IR, and Raman spectroscopy. The crystal structures of the five cocrystals/salts were determined and analyzed. It was found that both DBTA and DTTA form diastereomeric salt pairs with VAL enantiomers. Interestingly, l-DMTA cocrystallizes with d-VAL and l-VAL via hydrogen bonding and proton transfer, respectively. Considering this particularity, the differential isothermal (10 °C) ternary phase diagrams (TPDs) of d-DMTA and l-VAL (d-VAL) cocrystals (salt) were constructed in the mixed solvent of MeOH/H2O. Moreover, a pure l-VAL:d-DMTA cocrystal and d-VAL:d-DMTA:0.5CH4O:0.25H2O salt were prepared via equimolar slurry conversion at 10 °C. In situ Raman spectroscopy was applied to monitor the molecular assembly process during the incubation of the cocrystal/salt. Molecular dynamics simulation was employed to rationalize the molecular recognition mechanism, demonstrating the excellent chirality preference of l-DMTA toward l-VAL instead of d-VAL. Calculations of density functional theory approved the synergistic instead of antagonistic effects of binding energy and solvation free energy toward chiral separation.