Major Binding Site Research Articles

Antimicrobial Potency of Nor-Pyochelin Analogues and Their Cation Complexes against Multidrug-Resistant Pathogens.

The opportunistic pathogen Pseudomonas aeruginosa develops increasing resistance toward even the most potent antibiotics. Like other bacteria, the pathogen produces a number of virulence factors including metallophores, which constitute an important group. Pseudomonads produce the iron-chelating metallophore (siderophore) pyochelin, which, in addition to its iron-scavenging ability, is an effector for the transcriptional regulator PchR in its FeIII-bound form (ferripyochelin). In the present study, docking studies predicted a major ferripyochelin binding site in PchR, which prompted the exploration of nor-pyochelin analogues to produce tight binding to PchR, and thereby upregulation of the pyochelin metabolism. In addition, we investigated the effects of using the analogues to bind the antimicrobial cations GaIII and InIII. Selected analogues of nor-pyochelin were synthesized, and their GaIII- and InIII-based complexes were assessed for antimicrobial activity. The results indicate that the GaIII complexes inhibit the pathogens under iron-limited conditions, while the InIII-based systems are more effective in iron-rich media. Several of the GaIII complexes were shown to be highly effective against a multidrug-resistant P. aeruginosa clinical isolate, with minimum inhibitory concentrations (MICs) of ≤1 μg/mL. Similarly, two of the InIII-based systems were particularly effective against the isolate, with an MIC of 8 μg/mL. These results show high promise in comparison with other, traditionally potent antibiotics, as the compounds generally indicated low cytotoxicity toward mammalian cells. Preliminary mechanistic investigations using pseudomonal transposon mutants suggested that the inhibitory effects of the InIII-based systems could be due to acute iron deficiency as a result of InIII-bound bacterioferritin.

A cobalt coordination complex binds on a unique binding site between domain-I and domain-III of serum albumin

Serum albumin binding influences physiological effects and pharmacokinetics of drugs. Major binding sites in serum albumin are sudlow sites I and II, located on domains IIA and IIIA, respectively. However, groove between domain I and III as a binding site is not well studied. In this work, we studied the binding of a cobalt coordination complex (DCoP) with bovine serum albumin (BSA) using multi-spectroscopic methods and molecular docking. DCoP strongly interacts with BSA with association constant of ∼7 × 105M−1. The binding was found to significantly alter protein's conformation and unfolding pathway. Results of this study shows that DCoP binds at the groove formed between domains III and I. We conclude that the groove is a rare binding site for large molecules and coordination complexes. The knowledge of unique binding site of serum albumin enlightens the comprehensive role of protein as a carrier biomacromolecule.

Complex Formation and Hydrolytic Processes of Protected Peptides Containing the -SXH- Motif in the Presence of Nickel(II) Ion.

Interactions between metal ions and proteins are considered reversible, such as the coordination of a metal ion to a protein or enzyme, but irreversible processes like the oxidative reactions, aggregation or hydrolytic processes may occur. In the presence of Ni(II)-ions selective hydrolysis of the peptides containing the -SXH- or -TXH- motif was observed. Since the side chain of histidine serves as the metal ion binding site for many native proteins, and very often histidine is present in a -SXH- or -TXH- sequence, to study the complex formation and hydrolytic processes in presence of nickel(II) ion four peptides were synthesised: Ac-SKHM-NH2, A3SSH-NH2, A4SSH-NH2, AAAϵKSH-NH2. The Ni(II)-induced hydrolysis of Ac-SKHM-NH2 peptide occurs rapidly in alkaline medium already at room temperature. In two peptides containing -SSH- sequence on the C-termini, the N-terminal part is the major binding site for the nickel(II) ion, but the formation of dinuclear complexes was also observed. In the [Ni2LH-6]2- complex of hexapeptide, the coordination sphere of the metal ions is saturated with deprotonated Ser-O-, which does not result in hydrolysis of the peptide. For A4SSH-NH2, both Ni(II) ions fulfill the conditions for hydrolysis, which was confirmed by HPLC analyses at pH ≈8.2 and 25 °C.

Competitive Binding of Viral Nuclear Localization Signal Peptide and Inhibitor Ligands to Importin-α Nuclear Transport Protein.

Venezuelan equine encephalitis virus (VEEV) is a highly virulent pathogen whose nuclear localization signal (NLS) sequence from capsid protein binds to the host importin-α transport protein and blocks nuclear import. We studied the molecular mechanisms by which two small ligands, termed I1 and I2, interfere with the binding of VEEV's NLS peptide to importin-α protein. To this end, we performed all-atom replica exchange molecular dynamics simulations probing the competitive binding of the VEEV coreNLS peptide and I1 or I2 ligand to the importin-α major NLS binding site. As a reference, we used our previous simulations, which examined noncompetitive binding of the coreNLS peptide or the inhibitors to importin-α. We found that both inhibitors completely abrogate the native binding of the coreNLS peptide, forcing it to adopt a manifold of nonnative loosely bound poses within the importin-α major NLS binding site. Both inhibitors primarily destabilize the native coreNLS binding by masking its amino acids rather than competing with it for binding to importin-α. Because I2, in contrast to I1, binds off-site localizing on the edge of the major NLS binding site, it inhibits fewer coreNLS native binding interactions than I1. Structural analysis is supported by computations of the free energies of the coreNLS peptide binding to importin-α with or without competition from the inhibitors. Specifically, both inhibitors reduce the free energy gain from coreNLS binding, with I1 causing significantly larger loss than I2. To test our simulations, we performed AlphaScreen experiments measuring IC50 values for both inhibitors. Consistent with in silico results, the IC50 value for I1 was found to be lower than that for I2. We hypothesize that the inhibitory action of I1 and I2 ligands might be specific to the NLS from VEEV's capsid protein.

The 5'-terminal stem-loop RNA element of SARS-CoV-2 features highly dynamic structural elements that are sensitive to differences in cellular pH.

We present the nuclear magnetic resonance spectroscopy (NMR) solution structure of the 5'-terminal stem loop 5_SL1 (SL1) of the SARS-CoV-2 genome. SL1 contains two A-form helical elements and two regions with non-canonical structure, namely an apical pyrimidine-rich loop and an asymmetric internal loop with one and two nucleotides at the 5'- and 3'-terminal part of the sequence, respectively. The conformational ensemble representing the averaged solution structure of SL1 was validated using NMR residual dipolar coupling (RDC) and small-angle X-ray scattering (SAXS) data. We show that the internal loop is the major binding site for fragments of low molecular weight. This internal loop of SL1 can be stabilized by an A12-C28 interaction that promotes the transient formation of an A+•C base pair. As a consequence, the pKa of the internal loop adenosine A12 is shifted to 5.8, compared to a pKa of 3.63 of free adenosine. Furthermore, applying a recently developed pH-differential mutational profiling (PD-MaP) approach, we not only recapitulated our NMR findings of SL1 but also unveiled multiple sites potentially sensitive to pH across the 5'-UTR of SARS-CoV-2.

Moss biomass as effective biosorbents for heavy metals in contaminated water

The study explored batch adsorption of Cd(II) and Pb(II) ions using moss biomass from Barbula consanguinea and Hyophila involuta, assessing removal efficiency concerning various parameters. Both moss species showed high removal rates for Cd(II) (87 % for B. consanguinea and 89 % for H. involuta) and Pb(II) (93 % for B. consanguinea and 94 % for H. involuta) from contaminated water, reaching equilibrium within 30 min. While Cd(II) removal was pH-independent, Pb(II) removal showed pH-dependence, peaking at pH 5.0–5.5. Adsorption isotherm analysis indicated that the Langmuir, Freundlich, Elovich, Sips, and Redlich-Peterson models best described Cd(II) and Pb(II) adsorption onto both moss species (except for Cd(II) adsorption onto H. involuta), with R2 > 0.98. This confirms a heterogeneous surface with both monolayer and multilayer adsorption sites. The pseudo-second-order kinetic model confirmed chemisorption on moss biomass from both species. FTIR spectra identified major binding sites such as phenols, alkaloids, amines, alkenes, nitro compounds, and low-molecular-weight carbohydrates. EDS analysis validated the bonding of Cd(II) and Pb(II) ions to the biomass surface by displacing Ca(II) ions. According to the Langmuir model, moss biomass exhibited selective adsorption, favoring Pb(II) over Cd(II). B. consanguinea showed a higher adsorption capacity than H. involuta, which is attributed to its higher negative zeta potential. This study underscores the novelty of moss biomass for heavy metal removal in wastewater treatment, highlighting its sustainability, effectiveness, cost-efficiency, versatility, and eco-friendliness.

Lactobacillus helveticus-Derived Whey-Calcium Chelate Promotes Calcium Absorption and Bone Health of Rats Fed a Low-Calcium Diet.

This study investigated the characteristics of Lactobacillus helveticus-derived whey-calcium chelate (LHWCC) and its effect on the calcium absorption and bone health of rats. Fourier-transform infrared spectroscopy showed that carboxyl oxygen atoms, amino nitrogen atoms, and phosphate ions were the major binding sites with calcium in LHWCC, which has a sustained release effect in simulated in vitro digestion. LHWCC had beneficial effects on serum biochemical parameters, bone biomechanics, and the morphological indexes of the bones of calcium-deficient rats when fed at a dose of 40 mg Ca/kg BW for 7 weeks. In contrast to the inorganic calcium supplement, LHWCC significantly upregulated the gene expression of transient receptor potential cation V5 (TRPV5), TRPV6, PepT1, calcium-binding protein-D9k (Calbindin-D9k), and a calcium pump (plasma membrane Ca-ATPase, PMCA1b), leading to promotion of the calcium absorption rate, whereas Ca3(PO4)2 only upregulated the TRPV6 channel in vivo. These findings illustrate the potential of LHWCC as an organic calcium supplement.

Screening of the Antagonistic Activity of Potential Bisphenol A Alternatives toward the Androgen Receptor Using Machine Learning and Molecular Dynamics Simulation.

Over the past few decades, extensive research has indicated that exposure to bisphenol A (BPA) increases the health risks in humans. Toxicological studies have demonstrated that BPA can bind to the androgen receptor (AR), resulting in endocrine-disrupting effects. In recent investigations, many alternatives to BPA have been detected in various environmental media as major pollutants. However, related experimental evaluations of BPA alternatives have not been systematically implemented for the assessment of chemical safety and the effects of structural characteristics on the antagonistic activity of the AR. To promote the green development of BPA alternatives, high-throughput toxicological screening is fundamental for prioritizing chemical tests. Therefore, we proposed a hybrid deep learning architecture that combines molecular descriptors and molecular graphs to predict AR antagonistic activity. Compared to previous models, this hybrid architecture can extract substantial chemical information from various molecular representations to improve the model's generalization ability for BPA alternatives. Our predictions suggest that lignin-derivable bisguaiacols, as alternatives to BPA, are likely to be nonantagonist for AR compared to bisphenol analogues. Additionally, molecular dynamics (MD) simulations identified the dihydrotestosterone-bound pocket, rather than the surface, as the major binding site of bisphenol analogues. The conformational changes of key helix H12 from an agonistic to an antagonistic conformation can be evaluated qualitatively by accelerated MD simulations to explain the underlying mechanism. Overall, our computational study is helpful for toxicological screening of BPA alternatives and the design of environmentally friendly BPA alternatives.

A functional and structural comparative analysis of large tumor antigens reveals evolution of different importin α-dependent nuclear localization signals.

Nucleocytoplasmic transport regulates the passage of proteins between the nucleus and cytoplasm. In the best characterized pathway, importin (IMP) α bridges cargoes bearing basic, classical nuclear localization signals (cNLSs) to IMPβ1, which mediates transport through the nuclear pore complex. IMPα recognizes three types of cNLSs via two binding sites: the major binding site accommodates monopartite cNLSs, the minor binding site recognizes atypical cNLSs, while bipartite cNLSs simultaneously interact with both major and minor sites. Despite the growing knowledge regarding IMPα-cNLS interactions, our understanding of the evolution of cNLSs is limited. We combined bioinformatic, biochemical, functional, and structural approaches to study this phenomenon, using polyomaviruses (PyVs) large tumor antigens (LTAs) as a model. We characterized functional cNLSs from all human (H)PyV LTAs, located between the LXCXE motif and origin binding domain. Surprisingly, the prototypical SV40 monopartite NLS is not well conserved; HPyV LTA NLSs are extremely heterogenous in terms of structural organization, IMPα isoform binding, and nuclear targeting abilities, thus influencing the nuclear accumulation properties of full-length proteins. While several LTAs possess bipartite cNLSs, merkel cell PyV contains a hybrid bipartite cNLS whose upstream stretch of basic amino acids can function as an atypical cNLS, specifically binding to the IMPα minor site upon deletion of the downstream amino acids after viral integration in the host genome. Therefore, duplication of a monopartite cNLS and subsequent accumulation of point mutations, optimizing interaction with distinct IMPα binding sites, led to the evolution of bipartite and atypical NLSs binding at the minor site.

Structural and functional characterization of siadenovirus core protein VII nuclear localization demonstrates the existence of multiple nuclear transport pathways.

Adenovirus protein VII (pVII) plays a crucial role in the nuclear localization of genomic DNA following viral infection and contains nuclear localization signal (NLS) sequences for the importin (IMP)-mediated nuclear import pathway. However, functional analysis of pVII in adenoviruses to date has failed to fully determine the underlying mechanisms responsible for nuclear import of pVII. Therefore, in the present study, we extended our analysis by examining the nuclear trafficking of adenovirus pVII from a non-human species, psittacine siadenovirus F (PsSiAdV). We identified a putative classical (c)NLS at pVII residues 120-128 (120PGGFKRRRL128). Fluorescence polarization and electrophoretic mobility shift assays demonstrated direct, high-affinity interaction with both IMPα2 and IMPα3 but not IMPβ. Structural analysis of the pVII-NLS/IMPα2 complex confirmed a classical interaction, with the major binding site of IMPα occupied by K124 of pVII-NLS. Quantitative confocal laser scanning microscopy showed that PsSiAdV pVII-NLS can confer IMPα/β-dependent nuclear localization to GFP. PsSiAdV pVII also localized in the nucleus when expressed in the absence of other viral proteins. Importantly, in contrast to what has been reported for HAdV pVII, PsSiAdV pVII does not localize to the nucleolus. In addition, our study demonstrated that inhibition of the IMPα/β nuclear import pathway did not prevent PsSiAdV pVII nuclear targeting, indicating the existence of alternative pathways for nuclear localization, similar to what has been previously shown for human adenovirus pVII. Further examination of other potential NLS signals, characterization of alternative nuclear import pathways, and investigation of pVII nuclear targeting across different adenovirus species is recommended to fully elucidate the role of varying nuclear import pathways in the nuclear localization of pVII.

Small molecule WDR5 inhibitors down-regulate lncRNA expression.

WD repeat domain 5 (WDR5) plays an important role as a scaffold protein in both protein-protein and RNA-protein complexes involved in epigenetic gene regulation. In particular, some of these lncRNAs were reported to regulate the expression of genes in cis as well as themselves through binding WDR5. In this report, we investigate the two known binding sites of WDR5 in relation to lncRNA binding and expression. The WBM binding site mediates both protein-protein and lncRNA-protein interactions while the WIN site, which is on the opposite side of the protein, is only known to mediate protein-protein interactions. To dissect the function of different binding sites on WDR5, we characterized them with selective peptide ligands using fluorescence polarization and used these to demonstrate the selectivity of small molecule inhibitors of these two major binding sites. RNA immunoprecipitation experiments were performed to show that lncRNA-WDR5 complex formation could be interrupted using a WBM site inhibitor. Finally, we demonstrated that WDR5 regulated lncRNAs are down regulated with different sensitivity toward the corresponding inhibitors, demonstrating the potential of targeting lncRNA-protein interactions to reduce oncogenic lncRNA expression.

Structural basis of nuclear transport for NEIL DNA glycosylases mediated by importin-alpha

NEIL glycosylases, including NEIL1, NEIL2, and NEIL3, play a crucial role in the base excision DNA repair pathway (BER). The classical importin pathway mediated by importin α/β and cargo proteins containing nuclear localization sequences (NLS) is the most common transport mechanism of DNA repair proteins to the nucleus. Previous studies have identified putative NLSs located at the C-terminus of NEIL3 and NEIL1. Crystallographic, bioinformatics, calorimetric (ITC), and fluorescence assays were used to investigate the interaction between NEIL1 and NEIL3 putative NLSs and importin-α (Impα). Our findings showed that NEIL3 contains a typical cNLS, with medium affinity for the major binding site of Impα. In contrast, crystallographic analysis of NEIL1 NLS revealed its binding to Impα, but with high B-factors and a lack of electron density at the linker region. ITC and fluorescence assays indicated no detectable affinity between NEIL1 NLS and Impα. These data suggest that NEIL1 NLS is a non-classical NLS with low affinity to Impα. Additionally, we compared the binding mode of NEIL3 and NEIL1 with Mus musculus Impα to human isoforms HsImpα1 and HsImpα3, which revealed interesting binding differences for HsImpα3 variant. NEIL3 is a classical medium affinity monopartite NLS, while NEIL1 is likely to be an unclassical low-affinity bipartite NLS. The base excision repair pathway is one of the primary systems involved in repairing DNA. Thus, understanding the mechanisms of nuclear transport of NEIL proteins is crucial for comprehending the role of these proteins in DNA repair and disease development.

Checkpoint Inhibitor PD-1H/VISTA Affects Myeloma Bone Disease By Osteoclast Cytoskeleton Remodeling

Introduction We have recently described the critical role of checkpoint inhibitor PD-1H/VISTA in osteoclast activation and multiple myeloma bone disease. Highly expressed in osteoclast precursors and mature osteoclasts, PD-1H functions as the receptor for osteoclastogenic factor MMP-13 and mediates MMP-13 induced osteoclast activation and myeloma bone disease ( Nat Commun14, 4271 (2023)). Despite the extensive functional study of PD-1H in immune disease models, the PD-1H interacting cellular complex is yet to be defined. Here we report how PD-1H interacts with cytoskeleton proteins contributing to osteoclast remodeling in multiple myeloma bone disease. Methods and Results To identify interacting proteins of PD-1H, PD-1H-His 6 recombinant protein was overexpressed in mouse mononuclear BMCs, and PD-1H-His 6 binding protein complex was pulled down by Ni-NTA agarose beads followed by mass spectrum analysis (Figure A). LIM and SH3 domain protein 1 (LASP1) was one of the most abundant cytoskeleton proteins identified in the PD-1H complex. We therefore first confirmed its binding to PD-1H by two-way co-IP assays (Figure B and C). Co-localization of LASP1 and PD-1H were further confirmed by immunofluorescence assay when co-expressed in HEK293 cells (Figure D). LASP1 is an F-actin-binding protein that stabilizes F-actin bundles and is involved in podosome regulation in macrophages (PLoS ONE 7(4): e35340). It has four domains: LIM, two nebulin-like repeats (N1 and N2) and SH3 domains. While nebulin-like repeats mediate binding to F-actin, SH3 domain is the major protein-protein binding site (PLoS ONE 7(4): e35340). In co-IP assay using PD-1H and domain deleted LASP1, deletion of LIM (△LIM) or either one or both nebulin-like repeats (△N1, △N2 and △N1/2) did not affect the binding to PD-1H. Instead, deletion of SH3 (△SH3) in LASP1 lead to loss of PD-1H binding, demonstrating that PD-1H binds to LASP1 at the SH3 domain (Figure E). Next, the association of LASP1 and PD-1H in the osteoclast cytoskeleton was demonstrated by confocal immunofluorescence microscopy. LASP1 co-localized with F-actin (Figure F) and PD-1H (Figure G) at the sealing ring in mature osteoclasts. Interestingly, although MMP-13 stimulation increased the size of osteoclasts and the sealing ring, MMP-13 did not change the co-localization pattern of LASP1 with PD-1H or F-actin. Conclusions Taken together, this study reveals the novel mechanism of PD-1H regulation of osteoclasts cytoskeleton reorganization, which is critical for osteoclast bone resorption activity. PD-1H directly associates with LASP1 at its C-terminal SH3 domain. Although LASP1 was reported to regulates podosomes function in macrophages, its role in osteoclast cytoskeletons remain largely obscure. The future study will be focused on delineating the role of LASP1 in osteoclast function, and its role in MMP-13/PD-1H signaling regulated myeloma bone disease.

Binding of viral nuclear localization signal peptides to importin-α nuclear transport protein

Using all-atom replica-exchange molecular dynamics simulations, we mapped the mechanisms of binding of the nuclear localization signal (NLS) sequence from Venezuelan equine encephalitis virus (VEEV) capsid protein to importin-α (impα) transport protein. Our objective was to identify the VEEV NLS sequence fragment that confers native, experimentally resolved binding to impα as well as to study associated binding energetics and conformational ensembles. The two selected VEEV NLS peptide fragments, KKPK and KKPKKE, show strikingly different binding mechanisms. The minNLS peptide KKPK binds non-natively and nonspecifically by adopting five diverse conformational clusters with low similarity to the x-ray structure 3VE6 of NLS-impα complex. Despite the prevalence of non-native interactions, the minNLS peptide still largely binds to the impα major NLS binding site. In contrast, the coreNLS peptide KKPKKE binds specifically and natively, adopting a largely homogeneous binding ensemble with a dominant, highly native-like conformational cluster. The coreNLS peptide retains most of native binding interactions, including π-cation contacts and a tryptophan cage. While KKPK binding is governed by a complex multistate free energy landscape featuring transitions between multiple binding poses, the coreNLS peptide free energy map is simple, exhibiting a single dominant native-like bound basin. We argue that the origin of the coreNLS peptide binding specificity is several electrostatic interactions formed by the two C-terminal amino acids, Lys10 and Glu11, with impα. The coreNLS sequence is then sufficient for native binding, but none of the amino acids flanking minNLS, including Lys10 and Glu11, are strictly necessary for the native pose. Our analyses indicate that the VEEV coreNLS sequence is virtually unique among human and viral proteins interacting with impα making it a potential target for VEEV-specific inhibitors.

Henipavirus Matrix Protein Employs a Non-Classical Nuclear Localization Signal Binding Mechanism.

Nipah virus (NiV) and Hendra virus (HeV) are highly pathogenic species from the Henipavirus genus within the paramyxovirus family and are harbored by Pteropus Flying Fox species. Henipaviruses cause severe respiratory disease, neural symptoms, and encephalitis in various animals and humans, with human mortality rates exceeding 70% in some NiV outbreaks. The henipavirus matrix protein (M), which drives viral assembly and budding of the virion, also performs non-structural functions as a type I interferon antagonist. Interestingly, M also undergoes nuclear trafficking that mediates critical monoubiquitination for downstream cell sorting, membrane association, and budding processes. Based on the NiV and HeV M X-ray crystal structures and cell-based assays, M possesses a putative monopartite nuclear localization signal (NLS) (residues 82KRKKIR87; NLS1 HeV), positioned on an exposed flexible loop and typical of how many NLSs bind importin alpha (IMPα), and a putative bipartite NLS (244RR-10X-KRK258; NLS2 HeV), positioned within an α-helix that is far less typical. Here, we employed X-ray crystallography to determine the binding interface of these M NLSs and IMPα. The interaction of both NLS peptides with IMPα was established, with NLS1 binding the IMPα major binding site, and NLS2 binding as a non-classical NLS to the minor site. Co-immunoprecipitation (co-IP) and immunofluorescence assays (IFA) confirm the critical role of NLS2, and specifically K258. Additionally, localization studies demonstrated a supportive role for NLS1 in M nuclear localization. These studies provide additional insight into the critical mechanisms of M nucleocytoplasmic transport, the study of which can provide a greater understanding of viral pathogenesis and uncover a potential target for novel therapeutics for henipaviral diseases.

Identification and functional characterization of a bipartite nuclear localization signal in ANKRD11

ANKRD11 gene encodes for the large nuclear protein essential for multiple system development including the nervous system. However, the molecular basis for the proper nuclear localization of ANKRD11 has not yet been elucidated. In this study, we have identified a functional bipartite nuclear localization signal (bNLS) between residues 53 and 87 of ANKRD11. Using biochemical approaches, we discovered two major binding sites in this bipartite NLS for Importin α1. Through site-directed mutagenesis and functional analysis, we further found that this bipartite NLS is sufficient for nuclear import of overexpressing GFP in HeLa cells and necessary for nuclear localization of ANKRD11. Importantly, our study provides a possible pathogenic mechanism for certain clinical variants located within the bipartite nuclear localization signal of ANKRD11.

Zeb2 DNA-Binding Sites in Neuroprogenitor Cells Reveal Autoregulation and Affirm Neurodevelopmental Defects, Including in Mowat-Wilson Syndrome.

Functional perturbation and action mechanism studies have shown that the transcription factor Zeb2 controls cell fate decisions, differentiation, and/or maturation in multiple cell lineages in embryos and after birth. In cultured embryonic stem cells (ESCs), Zeb2's mRNA/protein upregulation is necessary for the exit from primed pluripotency and for entering general and neural differentiation. We edited mouse ESCs to produce Flag-V5 epitope-tagged Zeb2 protein from one endogenous allele. Using chromatin immunoprecipitation coupled with sequencing (ChIP-seq), we mapped 2432 DNA-binding sites for this tagged Zeb2 in ESC-derived neuroprogenitor cells (NPCs). A new, major binding site maps promoter-proximal to Zeb2 itself. The homozygous deletion of this site demonstrates that autoregulation of Zeb2 is necessary to elicit the appropriate Zeb2-dependent effects in ESC-to-NPC differentiation. We have also cross-referenced all the mapped Zeb2 binding sites with previously obtained transcriptome data from Zeb2 perturbations in ESC-derived NPCs, GABAergic interneurons from the ventral forebrain of mouse embryos, and stem/progenitor cells from the post-natal ventricular-subventricular zone (V-SVZ) in mouse forebrain, respectively. Despite the different characteristics of each of these neurogenic systems, we found interesting target gene overlaps. In addition, our study also contributes to explaining developmental disorders, including Mowat-Wilson syndrome caused by ZEB2 deficiency, and also other monogenic syndromes.

Two odorant receptors regulate 1-octen-3-ol induced oviposition behavior in the oriental fruit fly

The oriental fruit fly Bactrocera dorsalis (Hendel) is a notorious pest of fruit crops. Gravid females locate suitable oviposition sites by detecting host plant volatiles. Here, we demonstrate that 1-octen-3-ol, a volatile from mango, guides the oviposition behavior of female flies. Two odorant receptors (BdorOR7a-6 and BdorOR13a) are identified as key receptors for 1-octen-3-ol perception by qPCR analysis, heterologous expression in Xenopus laevis oocytes and HEK 293 cells followed by in vitro binding assays, as well as CRISPR/Cas9 genome editing in B. dorsalis. Molecular docking and site-directed mutagenesis are used to determine major binding sites for 1-octen-3-ol. Our results demonstrate the potential of 1-octen-3-ol to attract gravid females and molecular mechanism of its perception in B. dorsalis. BdorOR7a-6 and BdorOR13a can therefore be used as molecular targets for the development of female attractants. Furthermore, our site-directed mutagenesis data will facilitate the chemical engineering of 1-octen-3-ol to generate more efficient attractants.

Cover Image:

Xu et al. (pages 235–248) demonstrate that OsCERK1 directly recognizes chitin molecules, which play a key role in mediating rice immunity against fungal pathogens and symbiotic relationships with AM fungi in rice. Structural information on chitin recognition by OsCERK1 can facilitate engineering of chitin receptors to enhance rice disease resistance. The cover image shows the crystal structure of OsCERK1‐ECD in complex with (NAG)6, in which the second LysM domain (LysM2) of OsCERK1‐ECD is the major binding site of the chitin oligomer.

Deciphering the Binding of the Nuclear Localization Sequence of Myc Protein to the Nuclear Carrier Importin α3

The oncoprotein Myc is a transcription factor regulating global gene expression and modulating cell proliferation, apoptosis, and metabolism. Myc has a nuclear localization sequence (NLS) comprising residues Pro320 to Asp328, to allow for nuclear translocation. We designed a peptide comprising such region and the flanking residues (Ala310-Asn339), NLS-Myc, to study, in vitro and in silico, the ability to bind importin α3 (Impα3) and its truncated species (ΔImpα3) depleted of the importin binding domain (IBB), by using fluorescence, circular dichroism (CD), biolayer interferometry (BLI), nuclear magnetic resonance (NMR), and molecular simulations. NLS-Myc interacted with both importin species, with affinity constants of ~0.5 µM (for Impα3) and ~60 nM (for ΔImpα3), as measured by BLI. The molecular simulations predicted that the anchoring of NLS-Myc took place in the major binding site of Impα3 for the NLS of cargo proteins. Besides clarifying the conformational behavior of the isolated NLS of Myc in solution, our results identified some unique properties in the binding of this localization sequence to the nuclear carrier Impα3, such as a difference in the kinetics of its release mechanism depending on the presence or absence of the IBB domain.