Types Of Binding Sites Research Articles

NMR study of the structure and dynamics of the BRCT domain from the kinetochore protein KKT4

KKT4 is a multi-domain kinetochore protein specific to kinetoplastids, such as Trypanosoma brucei. It lacks significant sequence similarity to known kinetochore proteins in other eukaryotes. Our recent X-ray structure of the C-terminal region of KKT4 shows that it has a tandem BRCT (BRCA1 C Terminus) domain fold with a sulfate ion bound in a typical binding site for a phosphorylated serine or threonine. Here we present the 1H, 13C and 15N resonance assignments for the BRCT domain of KKT4 (KKT4463–645) from T. brucei. We show that the BRCT domain can bind phosphate ions in solution using residues involved in sulfate ion binding in the X-ray structure. We have used these assignments to characterise the secondary structure and backbone dynamics of the BRCT domain in solution. Mutating the residues involved in phosphate ion binding in T. brucei KKT4 BRCT results in growth defects confirming the importance of the BRCT phosphopeptide-binding activity in vivo. These results may facilitate rational drug design efforts in the future to combat diseases caused by kinetoplastid parasites.

Hydroxychloroquine Attenuates hERG Channel by Promoting the Membrane Channel Degradation: Computational Simulation and Experimental Evidence for QT-Interval Prolongation with Hydroxychloroquine Treatment.

The coronavirus disease 2019 (COVID-19) pandemic has led to millions of confirmed cases and deaths worldwide and has no approved therapy. Currently, more than 700 drugs are tested in the COVID-19 clinical trials, and full evaluation of their cardiotoxicity risks is in high demand. We mainly focused on hydroxychloroquine (HCQ), one of the most concerned drugs for COVID-19 therapy, and investigated the effects and underlying mechanisms of HCQ on hERG channel via molecular docking simulations. We further applied the HEK293 cell line stably expressing hERG-wild-type channel (hERG-HEK) and HEK293 cells transiently expressing hERG-p.Y652A or hERG-p.F656A mutants to validate our predictions. Western blot analysis was used to determine the hERG channel, and the whole-cell patch clamp was utilized to record hERG current (IhERG). HCQ reduced the mature hERG protein in a time- and concentration-dependent manner. Correspondingly, chronic and acute treatment of HCQ decreased the hERG current. Treatment with brefeldin A (BFA) and HCQ combination reduced hERG protein to a greater extent than BFA alone. Moreover, disruption of the typical hERG binding site (hERG-p.Y652A or hERG-p.F656A) rescued HCQ-mediated hERG protein and IhERG reduction. HCQ can reduce the mature hERG channel expression and IhERG via enhancing channel degradation. The QT prolongation effect of HCQ is mediated by typical hERG binding sites involving residues Tyr652 and Phe656.

How does a small molecule bind at a cryptic binding site?

Protein-protein interactions (PPIs) are ubiquitous biomolecular processes that are central to virtually all aspects of cellular function. Identifying small molecules that modulate specific disease-related PPIs is a strategy with enormous promise for drug discovery. The design of drugs to disrupt PPIs is challenging, however, because many potential drug-binding sites at PPI interfaces are “cryptic”: When unoccupied by a ligand, cryptic sites are often flat and featureless, and thus not readily recognizable in crystal structures, with the geometric and chemical characteristics of typical small-molecule binding sites only emerging upon ligand binding. The rational design of small molecules to inhibit specific PPIs would benefit from a better understanding of how such molecules bind at PPI interfaces. To this end, we have conducted unbiased, all-atom MD simulations of the binding of four small-molecule inhibitors (SP4206 and three SP4206 analogs) to interleukin 2 (IL2)—which performs its function by forming a PPI with its receptor—without incorporating any prior structural information about the ligands’ binding. In multiple binding events, a small molecule settled into a stable binding pose at the PPI interface of IL2, resulting in a protein–small-molecule binding site and pose virtually identical to that observed in an existing crystal structure of the IL2-SP4206 complex. Binding of the small molecule stabilized the IL2 binding groove, which when the small molecule was not bound emerged only transiently and incompletely. Moreover, free energy perturbation (FEP) calculations successfully distinguished between the native and non-native IL2–small-molecule binding poses found in the simulations, suggesting that binding simulations in combination with FEP may provide an effective tool for identifying cryptic binding sites and determining the binding poses of small molecules designed to disrupt PPI interfaces by binding to such sites.

Structural basis for the constitutive activity and immunomodulatory properties of the Epstein-Barr virus-encoded G protein-coupled receptor BILF1

Epstein-Barr virus (EBV) encodes a G protein-coupled receptor (GPCR) termed BILF1 that is essential for EBV-mediated immunosuppression and oncogenesis. BILF1 couples with inhibitory G protein (Gi), the major intracellular signaling effector for human chemokine receptors, and exhibits constitutive signaling activity; the ligand(s) for BILF1 are unknown. We studied the origins of BILF1's constitutive activity through structure determination of BILF1 bound to the inhibitory G protein (Gi) heterotrimer. The 3.2-Å resolution cryo-electron microscopy structure revealed an extracellular loop within BILF1 that blocked the typical chemokine binding site, suggesting ligand-autonomous receptor activation. Rather, amino acid substitutions within BILF1 transmembrane regions at hallmark ligand-activated class A GPCR "microswitches" stabilized a constitutively active BILF1 conformation for Gi coupling in a ligand-independent fashion. Thus, the constitutive activity of BILF1 promotes immunosuppression and virulence independent of ligand availability, with implications for the function of GPCRs encoded by related viruses and for therapeutic targeting of EBV.

Identification of Glutathione S-Transferase Genes in Hami Melon (Cucumis melo var. saccharinus) and Their Expression Analysis Under Cold Stress.

As a group of multifunctional enzymes, glutathione S-transferases (GSTs) participate in oxidative stress resistance and cellular detoxification. Here, we identified 39 CmGST genes with typical binding sites from the Hami melon genome, and they can be classified into seven subfamilies. Their molecular information, chromosomal locations, phylogenetic relationships, synteny relationships, gene structures, protein–protein interactions, structure of 3-D models, and expression levels under cold stress were analyzed. Expression analysis indicates that cold-tolerant Jia Shi-310 (JS) had higher GST enzyme activities and expression levels of 28 stress-related genes under cold stress. Some CmGSTs belonging to Tau, Phi, and DHAR classes play significant roles under cold stress, and they could be regarded as candidate genes for further studies. The present study systematically investigated the characterization of the Hami melon GST gene family, extending our understanding of Hami melon GST mediated stress-response mechanisms in this worldwide fruit.

Development and evaluation of poly adenosine 5'-diphosphate-ribose polymerase 1 immobilization-based receptor chromatography.

Yanghe decoction is a traditional Chinese medicine prescription and has been used for breast cancer treatment for many years. However, the effective ingredients in the decoction have not been identified. The expression of poly(ADP-ribose) polymerase-1 is highly related to breast cancer. Using poly(ADP-ribose) polymerase-1 as a probe, we expressed the haloalkane dehalogenase-tagged protein in BL21(DE3) E. coli, immobilized it on hexachlorocaproic acid-modified macroporous silica gel, and established a poly(ADP-ribose) polymerase-1 chromatographic model. The feasibility of the model was verified by testing the retention behaviors of five drugs on the protein column. We applied the model in screening the bioactive components in yanghe decoction. Rutin, liquiritin, and a compound ([M-H]- 681.7) were identified to be the potential bioactive ingredients. We studied the binding property between rutin and poly(ADP-ribose) polymerase-1 by injection amount dependent method, competitive studies, and molecular docking. We found that rutin can bind to the protein through the typical inhibitor binding site of the protein. Therefore, the chromatographic model is a useful tool to screen bioactive compounds from traditional Chinese medicine. The method is fast, reliable, and applicable to other functional proteins that can screen the potential lead compounds for the treatment of the related diseases.

Structure of the FERM domain of a neural scaffold protein FRMPD4 implicated in X-linked intellectual disability.

Scaffold proteins play crucial roles in orchestrating synaptic signaling and plasticity in the excitatory synapses by providing a structural link between glutamatergic receptors, signaling molecules, and neuronal cytoskeletons. FRMPD4 is a neural scaffold protein that binds to metabotropic glutamate receptors via its FERM domain. Here, we determine the crystal structure of the FERM domain of FRMPD4 at 2.49 Å resolution. The structure reveals that the canonical target binding groove of FRMPD4 FERM is occupied by a conserved fragment C-terminal to the FERM domain, suggesting that the FRMPD4-mGluR interaction may adopt a distinct binding mode. In addition, FRMPD4 FERM does not contain a typical phosphoinositide binding site at the F1/F3 cleft found in ERM family FERM domains, but it possesses a conserved basic residue cluster on the F2 lobe which could bind to lipid effectively. Finally, analysis of mutations that are associated with X-linked intellectual disability suggests that they may compromise the biological function of FRMPD4 by destabilizing the FERM structure.

An overview of tubulin modulators deposited in protein data bank

Microtubules are cytoskeletal polymers of tubulin and composed of α- and β-tubulin heterodimers, which are regarded as one of the most important, promising and successful targets for chemotherapeutic agents to treat cancer. With more and more tubulin-modulator co-crystal complex structures being solved, the mechanism of action (MOA) of microtubule targeting agents (MTAs) are well understood, which in turn inspired the development of more efficient tubulin modulators. By analyzing the reported tubulin modulators (most of them are inhibitors) that have been co-crystalized with tubulin and deposited in protein data bank (PDB), two new discovered inhibitor binding sites (maytansine and pironetin binding sites) on tubulin are elucidated, while the typical modulator binding sites (colchicine, taxanes, vinca alkaloids, and laulimalid binding sites) are also characterized. Among all the reported tubulin modulators, several inhibitors that bind to taxanes or vinca alkaloids binding sites have been approved by FDA to treat cancer. Thus, tubulin inhibitors discovery and design is still a hotspot in anticancer drug development. In this review, all the reported tubulin modulators that have co-crystal structures with tubulin in PDB are sorted out and classified according to their binding sites in tubulin. Besides, the ease or complexity of developing tubulin inhibitor on different binding sites on tubulin is also discussed. This work could contribute to discovering and designing new potent and specific modulators of tubulin.

Effects of high pressure processing on the interaction of α-lactalbumin and pelargonidin-3-glucoside

The effects of high pressure processing on the binding interactions of α-lactalbumin and pelargonidin-3-glucoside were studied using the fluorescence quenching, molecular dynamic simulation and molecular docking analysis. The results of fluorescence quenching indicated that the high pressure processing significantly increased the quenching constants of α-lactalbumin at pH 7.4 and pH 8.0. The accessible fraction at pH 8.0 was significantly increased, while the fractions at pH 6.0 and pH 7.4 were increased without significant difference. Molecular dynamic simulation and docking results demonstrated that the coil structures and locations, and the residues structures in the binding site of α-lactalbumin were affected, the binding site was the typical binding site of calcium ion and not changed during the processing. The dissociation constant of histidine residues was in the range of 6.13 to 6.83, the charge on the residues increased when pH value increased, affected the binding interactions and caused the quenching constant difference.

On identifying collective displacements in apo-proteins that reveal eventual binding pathways.

Binding of small molecules to proteins often involves large conformational changes in the latter, which open up pathways to the binding site. Observing and pinpointing these rare events in large scale, all-atom, computations of specific protein-ligand complexes, is expensive and to a great extent serendipitous. Further, relevant collective variables which characterise specific binding or un-binding scenarios are still difficult to identify despite the large body of work on the subject. Here, we show that possible primary and secondary binding pathways can be discovered from short simulations of the apo-protein without waiting for an actual binding event to occur. We use a projection formalism, introduced earlier to study deformation in solids, to analyse local atomic displacements into two mutually orthogonal subspaces—those which are “affine” i.e. expressible as a homogeneous deformation of the native structure, and those which are not. The susceptibility to non-affine displacements among the various residues in the apo- protein is then shown to correlate with typical binding pathways and sites crucial for allosteric modifications. We validate our observation with all-atom computations of three proteins, T4-Lysozyme, Src kinase and Cytochrome P450.

Triptolide attenuates proteinuria and podocyte apoptosis via inhibition of NF-\u03baB/GADD45B

Podocyte injury is a primary contributor to proteinuria. Triptolide is a major active component of Tripterygium wilfordii Hook F that exhibits potent antiproteinuric effects. We used our previously developed in vivo zebrafish model of inducible podocyte-target injury and found that triptolide treatment effectively alleviated oedema, proteinuria and foot process effacement. Triptolide also inhibited podocyte apoptosis in our zebrafish model and in vitro. We also examined the mechanism of triptolide protection of podocyte. Whole-genome expression profiles of cultured podocytes demonstrated that triptolide treatment downregulated apoptosis pathway-related GADD45B expression. Specific overexpression of gadd45b in zebrafish podocytes abolished the protective effects of triptolide. GADD45B is a mediator of podocyte apoptosis that contains typical NF-κB binding sites in the promoter region, and NF-κB p65 primarily transactivates this gene. Triptolide inhibited NF-κB signalling activation and binding of NF-κB to the GADD45B promoter. Taken together, our findings demonstrated that triptolide attenuated proteinuria and podocyte apoptosis via inhibition of NF-κB/GADD45B signalling, which provides a new understanding of the antiproteinuric effects of triptolide in glomerular diseases.

The molecular mechanism of two coreceptor binding site antibodies X5 and 17b neutralizing HIV-1: Insights from molecular dynamics simulation.

The coreceptor binding site of gp120 plays an important role in HIV entry into host cell. X5 and 17b are typical coreceptor binding site antibodies with the ability to broadly neutralize HIV. Thus, here, to study the neutralizing mechanism of two antibodies and identify the source of two antibodies with different neutralizing ability, we performed molecular dynamics simulations for the complexes of X5 and 17b with gp120 and CD4. The simulation results indicate X5 and 17b mainly affects CD4 and coreceptor binding sites. Specifically, for CD4 binding site (CD4bs), the binding of antibodies has different effects on CD4bs with and without CD4. However, for coreceptor binding sites, the binding of the antibodies has consistent influence on the region adjacent to loop V3 despite of the simulated systems with or without CD4. The binding of the antibodies enhances the interactions of gp120 region adjacent to loop V3 with other region of gp120, which are unfavorable for conformational rearrangements of the region adjacent to loop V3 and further binding the coreceptor. Additionally, the interactions of loop V3 and bridging sheet with X5 lead to the close motion of loop V3 in X5 bound form, which further influences the rearrangements in gp120.

Insight into the Molecular Mechanism of the Transcriptional Regulation of amtB Operon in Streptomyces coelicolor.

In Streptomyces coelicolor, amtB transcription is promptly regulated by the global nitrogen regulator GlnR. Although the GlnR binding cis-element has been characterized in amtB promoter, consisting of three GlnR boxes of a3-b3, a1-b1, and a2-b2, its role in GlnR-mediated transcriptional regulation remains unclear. Here, we showed that GlnR had different binding affinity against each pair of GlnR binding sites in amtB promoter (i.e., a3-b3, a1-b1, and a2-b2 sites), and GlnR was able to bind a3-b3 and a1-b1, respectively, but not a2-b2 alone. Consistently, a2 was not a typical GlnR binding site and further experiments showed that a2 was non-essential for GlnR-mediated binding in vitro and transcriptional regulation in vivo. To uncover the physiological role of the three GlnR boxes, we then mutated the wild-type amtB promoter to a typical GlnR-binding motif containing two GlnR boxes (a3-b3–a2-b2), and found although the transcription of the mutated promoter could still be activated by GlnR, its increasing rate was less than that of the wild-type. Based on these findings, one could conclude that the three GlnR boxes assisted GlnR in more promptly activating amtB transcription in response to nitrogen limitation, facilitating bacterial growth under nitrogen stresses.

Multiple Cryptic Binding Sites are Necessary for Robust Fibronectin Assembly

Assembly of elastic, insoluble fibronectin (FN) fibrils from soluble FN monomers is a crucial step in embryonic development and wound healing. However, the mechanism of FN fibrillogenesis is still poorly understood. FN fibril assembly requires cell-generated forces, applied to growing fibrils and exposing cryptic FN-FN binding sites buried in elastic Type III domains. The number and location of cryptic binding sites in Type III domains has been much debated, and there is increasing experimental evidence that multiple Type III domains may contain stretch-dependent FN-FN binding sites. The requirement of cell-dependent forces to generate fibrils limits the ability to recreate fibrils in cell-free experiments, restricting the investigation of the mechanisms underlying assembly. To address this, we use a recently developed biophysical model of FN fibrillogenesis (Weinberg et al, Biophys J, 2017), which allows us to simulate competing hypotheses for the location and properties of the cryptic FN-FN binding sites and quantify the effect of these molecular alterations on the morphology and biomechanical properties of the assembled fibril. Model predictions indicate that a single FN-FN binding site located in a domain N-terminal of the 10th Type III domain (which contains the integrin-binding motif RGD) facilitates negligible fibrillogenesis, while a single FN-FN binding site in a domain located C-terminal of the 10th Type III domain produces FN fibrils that are neither robust nor physiologically similar to actual FN fibrils. In contrast, the inclusion of multiple FN-FN binding sites predicts robust FN fibril assembly, which, importantly, minimally depends on the individual mechanical and chemical binding properties in FN with binding sites in all 15 Type III domains.

THiCweed: fast, sensitive detection of sequence features by clustering big datasets.

We present THiCweed, a new approach to analyzing transcription factor binding data from high-throughput chromatin immunoprecipitation-sequencing (ChIP-seq) experiments. THiCweed clusters bound regions based on sequence similarity using a divisive hierarchical clustering approach based on sequence similarity within sliding windows, while exploring both strands. ThiCweed is specially geared toward data containing mixtures of motifs, which present a challenge to traditional motif-finders. Our implementation is significantly faster than standard motif-finding programs, able to process 30 000 peaks in 1–2 h, on a single CPU core of a desktop computer. On synthetic data containing mixtures of motifs it is as accurate or more accurate than all other tested programs. THiCweed performs best with large ‘window’ sizes (≥50 bp), much longer than typical binding sites (7–15 bp). On real data it successfully recovers literature motifs, but also uncovers complex sequence characteristics in flanking DNA, variant motifs and secondary motifs even when they occur in <5% of the input, all of which appear biologically relevant. We also find recurring sequence patterns across diverse ChIP-seq datasets, possibly related to chromatin architecture and looping. THiCweed thus goes beyond traditional motif finding to give new insights into genomic transcription factor-binding complexity.

Immunogenetic factors driving formation of ultralong VH CDR3 in Bos taurus antibodies.

The antibody repertoire of Bos taurus is characterized by a subset of variable heavy (VH) chain regions with ultralong third complementarity determining regions (CDR3) which, compared to other species, can provide a potent response to challenging antigens like HIV env. These unusual CDR3 can range to over seventy highly diverse amino acids in length and form unique β-ribbon 'stalk' and disulfide bonded 'knob' structures, far from the typical antigen binding site. The genetic components and processes for forming these unusual cattle antibody VH CDR3 are not well understood. Here we analyze sequences of Bos taurus antibody VH domains and find that the subset with ultralong CDR3 exclusively uses a single variable gene, IGHV1-7 (VHBUL) rearranged to the longest diversity gene, IGHD8-2. An eight nucleotide duplication at the 3' end of IGHV1-7 encodes a longer V-region producing an extended F β-strand that contributes to the stalk in a rearranged CDR3. A low amino acid variability was observed in CDR1 and CDR2, suggesting that antigen binding for this subset most likely only depends on the CDR3. Importantly a novel, potentially AID mediated, deletional diversification mechanism of the B. taurus VH ultralong CDR3 knob was discovered, in which interior codons of the IGHD8-2 region are removed while maintaining integral structural components of the knob and descending strand of the stalk in place. These deletions serve to further diversify cysteine positions, and thus disulfide bonded loops. Hence, both germline and somatic genetic factors and processes appear to be involved in diversification of this structurally unusual cattle VH ultralong CDR3 repertoire.

Mutational Analysis at Intersubunit Interfaces of an Anionic Glutamate Receptor Reveals a Key Interaction Important for Channel Gating by Ivermectin.

The broad-spectrum anthelmintic drug ivermectin (IVM) activates and stabilizes an open-channel conformation of invertebrate chloride-selective glutamate receptors (GluClRs), thereby causing a continuous inflow of chloride ions and sustained membrane hyperpolarization. These effects suppress nervous impulses and vital physiological processes in parasitic nematodes. The GluClRs are pentamers. Homopentameric receptors assembled from the Caenorhabditis elegans (C. elegans) GluClα (GLC-1) subunit can inherently respond to IVM but not to glutamate (the neurotransmitter). In contrast, heteromeric GluClα/β (GLC-1/GLC-2) assemblies respond to both ligands, independently of each other. Glutamate and IVM bind at the interface between adjacent subunits, far away from each other; glutamate in the extracellular ligand-binding domain, and IVM in the ion-channel pore periphery. To understand the importance of putative intersubunit contacts located outside the glutamate and IVM binding sites, we introduced mutations at intersubunit interfaces, between these two binding-site types. Then, we determined the effect of these mutations on the activation of the heteromeric mutant receptors by glutamate and IVM. Amongst these mutations, we characterized an α-subunit point mutation located close to the putative IVM-binding pocket, in the extracellular end of the first transmembrane helix (M1). This mutation (αF276A) moderately reduced the sensitivity of the heteromeric GluClαF276A/βWT receptor to glutamate, and slightly decreased the receptor subunits’ cooperativity in response to glutamate. In contrast, the αF276A mutation drastically reduced the sensitivity of the receptor to IVM and significantly increased the receptor subunits’ cooperativity in response to IVM. We suggest that this mutation reduces the efficacy of channel gating, and impairs the integrity of the IVM-binding pocket, likely by disrupting important interactions between the tip of M1 and the M2-M3 loop of an adjacent subunit. We hypothesize that this physical contact between M1 and the M2-M3 loop tunes the relative orientation of the ion-channel transmembrane helices M1, M2 and M3 to optimize pore opening. Interestingly, pre-exposure of the GluClαF276A/βWT mutant receptor to subthreshold IVM concentration recovered the receptor sensitivity to glutamate. We infer that IVM likely retained its positive modulation activity by constraining the transmembrane helices in a preopen orientation sensitive to glutamate, with no need for the aforementioned disrupted interactions between M1 and the M2-M3 loop.

Simulating Force Response at Cellular Junctions: Desmoplakin as a Molecular Force Sensor?

Desmosomes are cell-cell junctions with a key role in mechano-sensation. However, it is currently unknown how the desmosomal complex and its proteins respond to mechanical force, transmitting chemical signals. We study desmoplakin, one of the obligate desmosomal proteins, identifying its possible role in mechanosensing. The most striking feature of this protein is the presence of an SH3 domain embedded in one of its six spectrin repeat domains. Intriguingly, the spectrin repeats hide the typical SH3 binding site, suggesting that either (i) the SH3 domain serves as a stabilizer of the protein through its interaction with one of the spectrin repeats, or (ii) it is activated only under force, initiating a signaling cascade for mechanosensing. To explore these alternatives, we performed equilibrium as well as force-probe Molecular Dynamics simulations on the central region of desmoplakin. To bridge towards physiologically relevant time scales, we repeated our simulations through a wide range of pulling velocities with a total simulation time of several tens of microseconds. We find desmoplakin to feature a clear sawtooth-like force profile, with each peak corresponding to modular spectrin repeat unfolding events followed by the final SH3 unfolding. The SH3 domain not only stabilizes desmoplakin, but also remains intact until the very last part of the trajectory, allowing interactions with downstream partners. Interestingly, we find the same behavior in the homologous plectin protein, suggesting this dual role of the SH3 domain to be conserved throughout the plakin family of proteins. However, plectin unfolds at significantly lower forces, involving an early or forceless loss of the SH3 domain-spectrin repeat interface. We identify specific mutations that promote this unfolding pathway, explaining the evolutionary adaptation of the cytosolic compared to the desmosomal mechano-sensing molecule. Our results suggest a direct role of unique SH3-insertion in spectrin repeat proteins in cellular mechanotransduction, and will be validated by direct comparison to single-molecule force spectroscopy experiments carried out by our collaborators.

Bicyclic Peptide Based Lectinomimic

Peptide based lectin mimetics represent an attractive approach for the development of artificial carbohydrate receptors that might find application in bio-analytical and medicinal fields. Taking into consideration the structure of typical lectin binding site, we have designed a novel artificial receptor molecule possessing a rigid three-dimensional structure, hydrogen-bonding site and lipophilic binding pocket to promote hydrophobic interaction and hydrogen-bonding. A new solid- phase synthetic approach that allows complete synthesis of desired bicyclic peptide 1 on the solid support was developed. CD spectra of peptides 1 and 2 indicate that the structure of 1 is rather rigid and preorganised for the three-dimensional monosaccharide substrates binding. The binding affinities of bicyclic peptide receptor 1 toward various carbohydrate substrates at physiologically relevant conditions were estimated by UV/vis and fluorimetric titration experiments, and the observed values are in the millimolar range. With these results we have demonstrated that the bicyclic peptide 1 represent a promising basis for the design of new and more efficient carbohydrate receptors that may have broader application in bio-analytical or medicinal field.

Cloning of three genes involved in the flavonoid metabolic pathway and their expression during insect resistance in Pinus massoniana Lamb.

Pinus massoniana Lamb. is an important timber and turpentine-producing tree species in China. Dendrolimus punctatus and Dasychira axutha are leaf-eating pests that have harmful effects on P. massoniana production. Few studies have focused on the molecular mechanisms underlying pest resistance in P. massoniana. Based on sequencing analysis of the transcriptomes of insect-resistant P. massoniana, three key genes involved in the flavonoid metabolic pathway were identified in the present study (PmF3H, PmF3'5'H, and PmC4H). Structural domain analysis showed that the PmF3H gene contains typical binding sites for the 2OG-Fe (II) oxygenase superfamily, while PmF3'5'H and PmC4H both contain the cytochrome P450 structural domain, which is specific for P450 enzymes. Phylogenetic analysis showed that each of the three P. massoniana genes, and the homologous genes in gymnosperms, clustered into a group. Expression of these three genes was highest in the stems, and was higher in the insect-resistant P. massoniana varieties than in the controls. The extent of the increased expression in the insect-resistant P. massoniana varieties indicated that these three genes are involved in defense mechanisms against pests in this species. In the insect-resistant varieties, rapid induction of PmF3H increased the levels of PmF3'5'H and PmC4H expression. The enhanced anti-pest capability of the insect-resistant varieties could be related to temperature and humidity. In addition, these results suggest that these three genes maycontribute to the change in flower color during female cone development.