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Microphthalmia transcription factor (MITF) regulates melanocyte development and is the "lineage-specific survival" oncogene of melanoma. MITF is essential for melanoma initiation, progression, and relapse and has been considered an important therapeutic target; however, direct inhibition of MITF through small molecules is considered impossible, due to the absence of a ligand-binding pocket for drug design. Here, our structural analyses show that the structure of MITF is hyperdynamic because of its out-of-register leucine zipper with a 3-residue insertion. The dynamic MITF is highly vulnerable to dimer-disrupting mutations, as we observed that MITF loss-of-function mutations in human Waardenburg syndrome type 2 A are frequently located on the dimer interface and disrupt the dimer forming ability accordingly. These observations suggest a unique opportunity to inhibit MITF with small molecules capable of disrupting the MITF dimer. From a high throughput screening against 654,650 compounds, we discovered compound TT-012, which specifically binds to dynamic MITF and destroys the latter's dimer formation and DNA-binding ability. Using chromatin immunoprecipitation assay and RNA sequencing, we showed that TT-012 inhibits the transcriptional activity of MITF in B16F10 melanoma cells. In addition, TT-012 inhibits the growth of high-MITF melanoma cells, and inhibits the tumor growth and metastasis with tolerable toxicity to liver and immune cells in animal models. Together, this study demonstrates a unique hyperdynamic dimer interface in melanoma oncoprotein MITF, and reveals a novel approach to therapeutically suppress MITF activity.

(+)-Valencene is a bioactive sesquiterpene that can be used for flavoring and fragrances, and microbial production provides an alternative sustainable access. However, the complexity of cellular metabolism makes it challenging for its high-level production. Here, we report the global rewiring cellular metabolism for

FtmOx1 is an -ketoglutarate (-KG) dependent mononuclear non-haem iron enzyme, which catalyzes endoperoxidation in fu-mitremorgin B to form verruculogen. The main products of the reaction are extremely complicated dependent on...

APOBEC3A (A3A) deaminates deoxycytidine in target motif TC in a single-stranded DNA (we termed it as TC DNA), which mortally mutates viral pathogens and immunoglobulins, and leads to the diversification and lethality of cancers. The crystal structure of A3A-DNA revealed a unique U-shaped recognition mode of target base dC0 . However, when TC DNA was titrated into 15 N-labeled A3A solution, we observed two sets of 1 H-15 N cross-peaks of A3A in HSQC spectra, and two sets of 1 H-1 H cross-peaks of DNA in two-dimensional 13 C,15 N- filtered TOCSY spectra, indicating two different kinds of conformers of either A3A or TC DNA existing in solution. Here, mainly by NMR, we demonstrated that one DNA conformer interacted with one A3A conformer, forming a specific complex A3AS -DNAS in a way almost similar to that observed in the reported crystal A3A-DNA structure, where dC0 inserted into zinc ion binding center. While the other DNA conformer bound with another A3A conformer, but dC0 did not extend into the zinc-binding pocket, forming a non-specific A3ANS -DNANS complex. The NMR solution structure implied three sites Asn61 , His182 and Arg189 were necessary to DNA recognition. These observations indicate a distinctive way from that reported in X-ray crystal structure, suggesting an unexpected mode of deaminase APOBEC3A to identify target motif TC in DNA in solution. This article is protected by copyright. All rights reserved.

Epilepsy is the results from the imbalance between inhibition and excitation in neural circuits, which is mainly treated by some chemical drugs with side effects. Gain-of-function of BK channels or knockout of its β4 subunit associates with spontaneous epilepsy. Currently, few reports were published about the efficacy of BK(α + β4) channel modulators in epilepsy prevention. Charybdotoxin is a non-specific inhibitor of BK and other K

APOBEC3G (A3G) is a member of cytosine deaminase family with a variety of innate immune functions. It displays activities against retrovirus and retrotransposon by inhibition of virus infectivity factor (Vif)-deficient HIV-1 replication. The interaction between A3G N-terminal domain and Vif directs the cellular Cullin 5 E3-ubiquitin ligase complex to ubiquitinate A3G, and leads to A3G proteasomal degradation, which is a potential target for anti-HIV drug. Currently, there are very few reports about stable small molecules targeting the interaction between A3G and Vif. In this study, we screened two series of small molecules containing carbamyl sulfamide bond or disulfide bond as bridges of two different aromatic rings. Five asymmetrical disulfides were successfully identified against interaction between A3G and Vif with the IC

Cyclina sinensis is an edible clam widely distributed along the coastal waters of Asia. In the present study, a polysaccharide (CSP-1) isolated from C. sinensis was purified by a DEAE-Sepharose Fast Flow column, and it had an average molecular weight of 3.8 × 105 Da and a prevalent component monosaccharide of Glc. The results of methylation analysis and 1D/2D NMR indicated that CSP-1 was a glycogen constructed with α-1,4-Glc and branched at C-6 every 9 Glc residues. In addition, Cong red test suggests CSP-1 was not a helical conformation, and irregular and spherical lumps were observed by AFM. Moreover, CSP-1 was found to possess potent immunostimulatory activity on the basis of its significant abilities to enhance NO production and cytokines (TNF-α, IL-1β and IL-6) secretion in RAW 264.7 macrophages.

Current yeast metabolic engineering in isoprenoids production mainly focuses on rewiring of cytosolic metabolic pathway. However, the precursors, cofactors and the enzymes are distributed in various sub-cellular compartments, which may hamper isoprenoid biosynthesis. On the other side, pathway compartmentalization provides several advantages for improving metabolic flux toward target products. We here summarize the recent advances on harnessing sub-organelle for isoprenoids biosynthesis in yeast, and analyze the knowledge about the localization of enzymes, cofactors and metabolites for guiding the rewiring of the sub-organelle metabolism. This review may provide some insights for constructing efficient yeast cell factories for production of isoprenoids and even other natural products.

The DNA phosphorothioate modification is a novel physiological variation in bacteria. DndE controls this modification by binding to dsDNA via a mechanism that remains unclear. Structural analysis of the wild-type DndE tetramer suggests that a positively charged region in its center is important for DNA binding. In the present study, we replaced residues G21 and G24 in this region with lysines, which increases the DNA binding affinity but does not affect the DNA degradation phenotype. Structural analysis of the mutant indicates that it forms a new tetrameric conformation and that DndE interacts with DNA as a monomer rather than as a tetramer. A structural model of the DndE-DNA complex, based on its structural homolog P22 Arc repressor, indicates that two flexible loops in DndE are determinants of DNA binding.

Human APOBEC3G (A3G) inhibits the replication of human immunodeficiency virus-1 by deaminating cytidine at the 3'-end in the target motif 5'-CCC-3' in viral cDNA during reverse transcription. It in vitro deaminates two consecutive cytidines in a 3'->5' order. Although a crystal structure of the A3G catalytic domain (A3G-CD2) with DNA was reported, it is unknown why residues involved in enzymatic reaction are distributed widely. Here, we introduced an iodine atom into the C-5 position of cytidine (dC6 I ) in DNA 5'-ATTC4 C5 C6 I A7 ATT-3' (TCCC6 I ). It switches the deamination sequence preference from CCC to TCC, although small dC6 I deamination was observed. Solution structures of A3G-CD2 in complexes with products DNA TCUC6 I and TCUU6 I indicate that the substrate DNA binds A3G-CD2 in TCC and CCC modes. The dC6 deamination correlates with the 4th base type. The CCC mode favours dC6 deamination, while the TCC mode results in dC5 deamination. These studies present an extensive basis to design inhibitors to impede viral evolvability.

Nucleic acid mimics of fluorescent proteins can be valuable tools to locate and image functional biomolecules in cells. Stacking between the internal G-quartet, formed in the mimics, and the exogenous fluorophore probes constitutes the basis for fluorescence emission. The precision of recognition depends upon probes selectively targeting the specific G-quadruplex in the mimics. However, the design of probes recognizing a G-quadruplex with high selectivity in vitro and in vivo remains a challenge. Through structure-based screening and optimization, we identified a light-up fluorescent probe, 9CI that selectively recognizes c-MYC Pu22 G-quadruplex both in vitro and ex vivo. Upon binding, the biocompatible probe emits both blue and green fluorescence with the excitation at 405 nm. With 9CI and c-MYC Pu22 G-quadruplex complex as the fluorescent response core, a DNA mimic of fluorescent proteins was constructed, which succeeded in locating a functional aptamer on the cellular periphery. The recognition mechanism analysis suggested the high selectivity and strong fluorescence response was attributed to the entire recognition process consisting of the kinetic match, dynamic interaction, and the final stacking. This study implies both the single stacking state and the dynamic recognition process are crucial for designing fluorescent probes or ligands with high selectivity for a specific G-quadruplex structure.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

G-quadruplexes (G4s) are frequently formed in the promoter regions of oncogenes, considered as promising drug targets for anticancer therapy. Due to high structure similarity of G4s, discovering ligands selectively interacting with only one G4 is extremely difficult. Here, mainly by NMR, we report that colchicine selectively binds to oncogene RET G4-DNA.

Acidic polysaccharides are attractive functional ingredients in shellfish which are consumed as delicious and nutritious foods world widely. In the present study, acidic polysaccharides from 21 species of edible shellfish were characterized and quantified by analyzing their repeated disaccharides using the multiple reaction monitoring (MRM) mode of triple quadrupole mass spectrometer upon acid degradation and 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization. A total of 6 glycosaminoglycans (GAGs) and 8 non-GAGs with repeated disaccharide units of a hexuronic acid linked to a hexosamine or a hexose were detected. Among them, chondroitin sulfate, heparin, →4)-β-GlcA-(1 → 2)-α-Man-(1 → and →3)- β-GlcA-(1 → 3)-α-Gal-(1 → were identified unambiguously by comparing with the references. The quantification results revealed that the contents of these polysaccharide varied greatly among shellfish species with a maximum over 100 mg/100 g. Furthermore, the dendrogram of hierarchical clustering analysis indicated that the composition of acidic polysaccharides in some shellfish species was related with the genetic relationship. Thus, the present study provides a more comprehensive knowledge about the distribution of acidic polysaccharides in various shellfish species.

DNA can form diverse structures, which predefine their physiological functions. Besides duplexes that carry the genetic information, quadruplexes are the most well-studied DNA structures. In addition to their important roles in recombination, replication, transcription and translation, DNA quadruplexes have also been applied as diagnostic aptamers and antidisease therapeutics. Herein we further expand the sequence and structure complexity of DNA quadruplex by presenting a high-resolution crystal structure of DNA1 (5′-AGAGAGATGGGTGCGTT-3′). This is the first quadruplex structure that contains all the internal A-, G-, C-, T-tetrads, A:T:A:T tetrads and bulged nucleotides in one single structure; as revealed by site-specific mutagenesis and biophysical studies, the central ATGGG motif plays important role in the quadruplex formation. Interestingly, our structure also provides great new insights into cation recognition, including the first-time reported Pb2+, by tetrad structures.

Because the glycosidic linkage of uronic acid is most resistant to acid, oligosaccharides may be formed during the acid hydrolysis of acidic polysaccharides. To take chondroitin sulfate (CS) as an example of acidic polysaccharides, the present study characterized the oligosaccharides released through acid hydrolysis and demonstrated their usefulness for structural confirmation. Acid hydrolysates of commercial standard CSs from shark cartilage and porcine bone were elucidated using HPLC-MSn after 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization, and altogether 11 di-, tri- and tetra-saccharides with or without sulfate/acetyl groups were identified by their multi-stage mass spectra. Meanwhile the trends of reaction yields of these oligosaccharides alone with trifluoroacetic acid (TFA) concentrations (0.1-2.0 M) were investigated, and 0.2 M TFA was recommended. Then three real samples, sturgeon backbone, porcine trachea and sea cucumber were analyzed, and their CSs were identified by detection of characteristic oligosaccharide fragments. The present study indicated that acid hydrolysis could provide information for acetyl substitution, sulfation and glycosidic linkages, and was helpful for the structural analysis of acidic polysaccharides.

Gain-of-function of BK channels or knockout of their β4 subunit is associated with spontaneous epilepsy. Currently, efficacy of BK (α + β4) channel modulators in preventing epilepsy was never reported. Here, we show that martentoxin selectively inhibits BK (α + β4) channels by interaction with the extracellular loop of the BK β4 subunit (hβ4-loop) at a molar ratio 4:1 (hβ4-loop vs martentoxin). Residues Glu104, Glu122, Gln124, Lys125, and Glu128 of the hβ4-loop form hydrogen bonds with residues Asp5, Glu13, Lys20, Ser24, Gln26, Lys28, and Arg35 of martentoxin, by which martentoxin reduces the neuronal spiking frequency and increases interspike intervals. Intrahippocampal infusion of martentoxin significantly increases the latency time of seizure, reduces seizure duration and seizure numbers on pentylenetetrazole-induced presensitized rats, inhibits hippocampal hyperexcitability and c-Fos expression, and displays neuroprotective effects on hippocampal neurons. These results suggest that the BK (α + β4) channel is a novel therapeutic target of intractable epilepsy and martentoxin contributes to the rational drug design for epilepsy treatment.

Elliptical accretion disk models for tidal disruption events (TDEs) have been recently proposed and independently developed by two groups. Although these two models are characterized by a similar geometry, their physical properties differ considerably. In this paper, we further investigate the properties of the elliptical accretion disk of the nearly uniform distribution of eccentricity within the disk plane. Our results show that the elliptical accretion disks have distinctive hydrodynamic structures and spectral energy distributions, associated with TDEs. The soft X-ray photons generated at pericenter and nearby are trapped in the disk and advected around the ellipse because of large electron scattering opacity. They are absorbed and reprocessed into emission lines and low-frequency continuum via recombination and bremsstrahlung emission. Because of the rapid increase of bound-free and free-free opacities with radius, the low-frequency continuum photons become trapped in the disk at large radius and are advected through apocenter and back to the photon-trapping radius. Elliptical accretion disks predict sub-Eddington luminosities and emit mainly at the photon-trapping radius of thousands of Schwarzschild radii with a blackbody spectrum of nearly single temperature of typically about 3X10^4 K. Because of the self-regulation, the photon-trapping radius expands and contracts following the rise and fall of accretion rate. The radiation temperature is nearly independent of BH mass and accretion rate and varies weakly with the stellar mass and the viscosity parameter. Our results are well consistent with the observations of optical/UV TDEs., 54 pages, 7 figures; to match the published version

Spectral overlap makes it difficult to use NMR for mapping the conformational profile of heterogeneous conformational ensembles of macromolecules. Here, we apply a 1H-14N HSQC experiment to monitor the alkaline conformational transitions of yeast iso-1 cytochrome c (ycyt c) at natural isotopic abundance. Trimethylated Lys72 of ycyt c is selectively detected by a 1H-14N HSQC experiment, and used as a probe to trace conformational transitions of ycyt c under alkaline conditions. It was found that at least four different conformers of ycyt c coexisted under alkaline conditions. Besides the native structure, Lys73 or Lys79 coordinated conformers and a partially unfolded state with exposed heme were observed. These results indicate that the method is powerful at simplifying spectra of a trimethylated protein, which makes it possible to study complex conformational transitions of naturally extracted or chemically modified trimethylated protein at natural isotopic abundance.

A family of DEDDh 3′→5′ exonucleases known as Small RNA Degrading Nucleases (SDNs) initiates the turnover of ARGONAUTE1 (AGO1)-bound microRNAs in Arabidopsis by trimming their 3′ ends. Here, we report the crystal structure of Arabidopsis SDN1 (residues 2-300) in complex with a 9 nucleotide single-stranded RNA substrate, revealing that the DEDDh domain forms rigid interactions with the N-terminal domain and binds 4 nucleotides from the 3′ end of the RNA via its catalytic pocket. Structural and biochemical results suggest that the SDN1 C-terminal domain adopts an RNA Recognition Motif (RRM) fold and is critical for substrate binding and enzymatic processivity of SDN1. In addition, SDN1 interacts with the AGO1 PAZ domain in an RNA-independent manner in vitro, enabling it to act on AGO1-bound microRNAs. These extensive structural and biochemical studies may shed light on a common 3′ end trimming mechanism for 3′→5′ exonucleases in the metabolism of small non-coding RNAs., Small RNA degrading nucleases (SDNs) can degrade short RNAs. Here the authors report the crystal structure of Arabidopsis SDN1 in complex with a single-stranded RNA, and provide new insight into 3′ end trimming mechanism of 3′ to 5′ riboexonucleases in the metabolism of various species of small RNAs.

Tumor angiogenesis is mainly regulated by vascular endothelial growth factor (VEGF) produced by cancer cells. It is active on the endothelium via VEGF receptor 2 (VEGFR-2). G-quadruplexes are DNA secondary structures formed by guanine-rich sequences, for example, within gene promoters where they may contribute to transcriptional activity. The proximal promoter of VEGFR-2 contains a G-quadruplex, which has been suggested to interact with small molecules that inhibit VEGFR-2 expression and thereby tumor angiogenesis. However, its structure is not known. Here, we determined its NMR solution structure, which is composed of three stacked G-tetrads containing three syn guanines. The first guanine (G(1)) is positioned within the central G-tetrad. We also observed that a noncanonical, V-shaped loop spans three G-tetrad planes, including no bridging nucleotides. A long and diagonal loop, which includes six nucleotides, connects reversal double chains. With a melting temperature of 54.51 °C, the scaffold of this quadruplex is stabilized by one G-tetrad plane stacking with one nonstandard bp, G(3)–C(8), whose bases interact with each other through only one hydrogen bond. In summary, the NMR solution structure of the G-quadruplex in the proximal promoter region of the VEGFR-2 gene reported here has uncovered its key features as a potential anticancer drug target.

Large-conductance Ca2+- and voltage-dependent K+ (BK) channels display diverse biological functions while their pore-forming α subunit is coded by a single Slo1 gene. The variety of BK channels is correlated with the effects of BKα coexpression with auxiliary β (β1-β4) subunits, as well as newly defined γ subunits. Charybdotoxin (ChTX) blocks BK channel through physically occluding the K+-conduction pore. Human brain enriched β4 subunit (hβ4) alters the conductance-voltage curve, slows activation and deactivation time courses of BK channels. Its extracellular loop (hβ4-loop) specifically impedes ChTX to bind BK channel pore. However, the structure of β4 subunit’s extracellular loop and the molecular mechanism for gating kinetics, toxin sensitivity of BK channels regulated by β4 are still unclear. To address them, here, we first identified four disulfide bonds in hβ4-loop by mass spectroscopy and NMR techniques. Then we determined its three-dimensional solution structure, performed NMR titration and electrophysiological analysis, and found that residue Asn123 of β4 subunit regulated the gating and pharmacological characteristics of BK channel. Finally, by constructing structure models of BKα/β4 and thermodynamic double-mutant cycle analysis, we proposed that BKα subunit might interact with β4 subunit through the conserved residue Glu264(BKα) coupling with residue Asn123(β4).

Autolysis is not only a major reason for postharvest quality deterioration of sea cucumber, but also a promising alternative for exogenous protease to produce peptides or polysaccharides. However, little has been known about the effects of autolysis on bioactive polysaccharides of sea cucumber. Concerning the quality and safety of sea cucumber products involved autolysis reaction, the present study focused on the chemical composition of sulfated polysaccharides (SPs) released by autolysis reaction. Chemical analysis indicated that after 3-day autolysis 63% of sulfated polysaccharides were liberated but with protein chains at their reducing ends. Then the composition of SP obtained by autolysis (A-SP) was compared with that of total SPs (T-SP) via a series of analysis techniques, including FTIR, 1H NMR, HPLC and mass spectroscopy. As indicated by the results, fucan to fucosylated chondroitin sulfate ratio was found high in A-SP compared to T-SP, fucan with a lower molecular weight was the major fraction in A-SP, and the di-sulfated Fuc residue observed in T-SP was absent in A-SP. To conclude, A-SP differed greatly from T-SP in the chemical composition, suggesting possible changes on their bioactivities.

Large-conductance Ca2+-activated K+ (BK) channels are composed of a pore-forming α and a variable number of auxiliary β subunits and play important roles in regulating excitability, action potential waveforms and firing patterns, particularly in neurons and endocrine and cardiovascular cells. The β2 subunits increase the diversity of gating and pharmacological properties. Its extracellular loop contains eight cysteine residues, which can pair to form a high-order structure, underlying the stability of the extracellular loop of β2 subunits and the functional effects on BK channels. However, how these cysteines form disulfide bonds still remains unclear. To address this, based on the fact that the rectification and association of BK α to β2 subunits are highly sensitive to disruption of the disulfide bonds in the extracellular loop of β2, we developed a rectification ratio based assay by combining the site-directed mutagenesis, electrophysiology and enzymatic cleavage. Three disulfide bonds: C1(C84)-C5(C113), C3(C101)-C7(C148) and C6(C142)-C8C(174) are successfully deduced in β2 subunit in complex with a BK α subunit, which are helpful to predict structural model of β2 subunits through computational simulation and to understand the interface between the extracellular domain of the β subunits and the pore-forming α subunit.

Nosiheptide is a member of the thiopeptide family of antibiotics which demonstrates potent activities against various bacterial pathogens. The formation of its C-terminal amide is catalysed by NosA in an unusual strategy for maturating certain thiopeptides by processing precursor peptides featuring a serine extension. Here, a recombinant C-terminally truncated selenomethionine-derivatized NosA1–111variant fromStreptomyces actuosusconsisting of residues 1–111, named SeMet NosA1–111, was crystallized using the sitting-drop vapour-diffusion method. Diffraction data were collected to 2.40 Å resolution using synchrotron radiation. The crystals belonged to the primitive cubic space groupP4132, with unit-cell parametersa=b=c= 143.3 Å. Assuming the presence of three molecules in the asymmetric unit, the calculated Matthews coefficient was 3.94 Å3 Da−1and the corresponding solvent content was 40.3%.

APOBEC3G (A3G) is a DNA cytidine deaminase (CD) that demonstrates antiviral activity against human immunodeficiency virus 1 (HIV-1) and other pathogenic virus. It has an inactive N-terminal CD1 virus infectivity factor (Vif) protein binding domain (A3G-CD1) and an actively catalytic C-terminal CD2 deamination domain (A3G-CD2). Although many studies on the structure of A3G-CD2 and enzymatic properties of full-length A3G have been reported, the mechanism of how A3G interacts with HIV-1 single-stranded DNA (ssDNA) is still not well characterized. Here, we reported a crystal structure of a novel A3G-CD2 head-to-tail dimer (in which the N terminus of the monomer H (head) interacts with the C terminus of monomer T (tail)), where a continuous DNA binding groove was observed. By constructing the A3G-CD1 structural model, we found that its overall fold was almost identical to that of A3G-CD2. We mutated the residues located in or along the groove in monomer H and the residues in A3G-CD1 that correspond to those seated in or along the groove in monomer T. Then, by performing enzymatic assays, we confirmed the reported key elements and the residues in A3G necessary to the catalytic deamination. Moreover, we identified more than 10 residues in A3G essential to DNA binding and deamination reaction. Therefore, this dimer structure may represent a structural model of full-length A3G, which indicates a possible binding mode of A3G to HIV-1 ssDNA.

It is still a challenge to identify mixed polysaccharides isolated from biological tissues. The present study aimed to develop a protocol that allowed for quick characterization of mixed polysaccharides by selecting Cyclina sinensis polysaccharides as test beds. Disaccharides were gradually released by graded acid hydrolysis, separated with 3 kDa molecular weight cut-off membrane, and analyzed by HPLC-ESI-MSn. Furthermore, the monosaccharide composition of the filtrate during the gradient acid hydrolysis process and the final retentate were also determined to provide composition information of these disaccharides. Thus, 6 disaccharides were detected and identified, indicating the existence of polysaccharides constructed of corresponding repeated disaccharide fragments in C. sinensis. The present study demonstrated a potential strategy to characterize mixed polysaccharides without separation and purification.