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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

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.

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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.

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.

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.

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.

Metabolomics aims to quantitatively measure small-molecule metabolites in biological samples, such as bodily fluids (e.g., urine, blood, and saliva), tissues, and breathe exhalation, which reflects metabolic responses of a living system to pathophysiological stimuli or genetic modification. In the past decade, metabolomics has made notable progresses in providing useful systematic insights into the underlying mechanisms and offering potential biomarkers of many diseases. Metabolomics is a complementary manner of genomics and transcriptomics, and bridges the gap between genotype and phenotype, which reflects the functional output of a biological system interplaying with environmental factors. Recently, the technology of metabolomics study has been developed quickly. This review will discuss the whole pipeline of metabolomics study, including experimental design, sample collection and preparation, sample detection and data analysis, as well as mechanism interpretation, which can help understand metabolic effects and metabolite function for living organism in system level.

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).

Metabolomics aims to quantitatively measure small-molecule metabolites in biological samples, such as bodily fluids (e.g., urine, blood, and saliva), tissues, and breathe exhalation, which reflects metabolic responses of a living system to pathophysiological stimuli or genetic modification. In the past decade, metabolomics has made notable progresses in providing useful systematic insights into the underlying mechanisms and offering potential biomarkers of many diseases. Metabolomics is a complementary manner of genomics and transcriptomics, and bridges the gap between genotype and phenotype, which reflects the functional output of a biological system interplaying with environmental factors. Recently, the technology of metabolomics study has been developed quickly. This review will discuss the whole pipeline of metabolomics study, including experimental design, sample collection and preparation, sample detection and data analysis, as well as mechanism interpretation, which can help understand metabolic effects and metabolite function for living organism in system level.

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.

Although blood plasma has been used to diagnose diseases and to evaluate physiological conditions, it is not easy to establish a global normal concentration range for the targeting components in the plasma due to the inherent metabolic diversity. We show here that NMR spectroscopy coupled with principal component analysis (PCA) may provide a useful method for quantitatively characterizing the metabolic diversity of human blood plasma. We analyzed 70 human blood plasma samples with and without addition of ibuprofen. By defining the PC score values as diversity index (I-div) and the drug-induced PC score value change as interaction index (I-dist), we find that the two indexes are highly correlated (P < 0.0001). Triglycerides, choline-containing phospholipids, lactate, and pyruvate are associated with both indexes (P < 0.0001), respectively. In addition, a significant amount of lactate and pyruvate are in the NMR "invisible" bound forms and can be replaced by ibuprofen. The diffusion and transverse relaxation time weighted NMR approaches gave rise to a better characterization of the diversity and the interaction than that of the one acquired using NOESYPR1D with 100 ms mixing time. These results might be useful for understanding the blood plasma drug interaction and personalized therapy.

As an AAA-ATPase, Vps4 is important for function of multivesicular bodies (MVB) sorting pathway, which involves in cellular phenomena ranging from receptor down-regulation to viral budding to cytokinesis. The activity of Vps4 is stimulated by the interactions between Vta1 N-terminus (named as Vta1NTD) and Did2 fragment (176–204 aa) (termed as Did2176–204) or Vps60 (128–186 aa) (termed as Vps60128–186). The structural basis of how Vta1NTD binds to Did2176–204 is still unclear. To address this, in this report, the structure of Did2176–204 in complex with Vta1NTD was determined by NMR techniques, demonstrating that Did2176–204 interacts with Vta1NTD through its helix α6′ extending over the 2nd and the 3rd α-helices of Vta1NTD microtubule interacting and transport 1 (MIT1) domain. The residues within Did2176–204 helix α6′ in the interface make up of an amino acid sequence as E192′xxL195′xxR198′L199′xxL202′R203′, identical to type 1 MIT-interacting motif (MIM1) (D/E)xxLxxRLxxL(K/R) of CHMP1A180–196 observed in Vps4-CHMP1A complex structure, indicating that Did2 binds to Vta1NTD through canonical MIM1 interactions. Moreover, the Did2 binding does not result in Vta1NTD significant conformational changes, revealing that Did2, similar to Vps60, enhances Vta1 stimulation of Vps4 ATPase activity in an indirect manner.

DNA phosphorothioate (PT) modification, with sulfur replacing a nonbridging phosphate oxygen in a sequence and stereo specific manner, is a novel physiological variation in bacteria. But what effects on DNA properties PT modification has is still unclear. To address this, we prepared three double-stranded (ds) DNA decamers, d(CGPXGCCGCCGA) with its complementary strand d(TCGGCGPXGCCG) (where X = O or S, i.e., PT-free dsDNA, [Sp, Sp]-PT dsDNA or [Rp, Rp]-PT dsDNA) located in gene of Streptomyces lividans. Their melting temperature (Tm) measurement indicates that [Rp, Rp]-PT dsDNA is most unstable. Their electron transfer potential detection presents an order of anti-oxidation properties: Sp-PT DNA > Rp-PT DNA > PT-free DNA. Their NMR structures demonstrate that PT modification doesn’t change their B-form conformation. The sulfur in [Rp, Rp]-PT dsDNA locates in the major groove, with steric effects on protons in the sugar close to modification sites, resulting in its unstability and facilitating its selectively interactions with ScoMcrA. We thought that PT modification was dialectical to the bacteria. It protects the hosting bacteria by working as antioxidant against H2O2 and acts as a marker, directing restriction enzyme observed in other hosts, like ScoMcrA, to correctly cleave the PT modified DNA, so that bacteria cannot spread and survive.

UHRF1 is an important epigenetic regulator for maintenance DNA methylation. UHRF1 recognizes hemi-methylated DNA (hm-DNA) and trimethylation of histone H3K9 (H3K9me3), but the regulatory mechanism remains unknown. Here we show that UHRF1 adopts a closed conformation, in which a C-terminal region (Spacer) binds to the tandem Tudor domain (TTD) and inhibits H3K9me3 recognition, whereas the SET-and-RING-associated (SRA) domain binds to the plant homeodomain (PHD) and inhibits H3R2 recognition. Hm-DNA impairs the intramolecular interactions and promotes H3K9me3 recognition by TTD–PHD. The Spacer also facilitates UHRF1–DNMT1 interaction and enhances hm-DNA-binding affinity of the SRA. When TTD–PHD binds to H3K9me3, SRA-Spacer may exist in a dynamic equilibrium: either recognizes hm-DNA or recruits DNMT1 to chromatin. Our study reveals the mechanism for regulation of H3K9me3 and hm-DNA recognition by URHF1., UHRF1 is involved in the maintenance of DNA methylation, but the regulatory mechanism of this epigenetic regulator is unclear. Here, the authors show that it has a closed conformation and are able to make conclusions about the mechanism of recognition of epigenetic marks.

Vta1 and Vps60 are two ESCRT associated proteins, their direct interaction enhances Vps4 ATPase activity. The N-terminal domain of Vta1 (residues 1–167aa, named as Vta1NTD) contains two tandem MIT domains, which specifically recognize Vps60 and Did2 but not other ESCRT-III subunits. The fragment Vps60 (128–186aa) was reported to display full activity of Vps60, which stimulates Vps4 ATPase in a Vta1-dependent manner. To study the structural basis for the interaction between Vta1 and Vps60, as a first step, here, we report the resonance assignments of the sequential backbone atoms and the side chains of the residues in the two components of Vta1NTD/Vps60128–186 complex at pH 7.0 and 20 °C (BMRB No. 18521).

Structural insights into DndE from Escherichia coli B7A involved in DNA phosphorothioation modification

Structural basis for site-specific reading of unmodified R2 of histone H3 tail by UHRF1 PHD finger

RET protein functions as a receptor-type tyrosine kinase and has been found to be aberrantly expressed in a wide range of human diseases. A highly GC-rich region upstream of the promoter plays an important role in the transcriptional regulation of RET. Here, we report the NMR solution structure of the major intramolecular G-quadruplex formed on the G-rich strand of this region in K(+) solution. The overall G-quadruplex is composed of three stacked G-tetrad and four syn guanines, which shows distinct features for all parallel-stranded folding topology. The core structure contains one G-tetrad with all syn guanines and two other with all anti-guanines. There are three double-chain reversal loops: the first and the third loops are made of 3 nt G-C-G segments, while the second one contains only 1 nt C10. These loops interact with the core G-tetrads in a specific way that defines and stabilizes the overall G-quadruplex structure and their conformations are in accord with the experimental mutations. The distinct RET promoter G-quadruplex structure suggests that it can be specifically involved in gene regulation and can be an attractive target for pathway-specific drug design.

Lung cancer causes serious health problems. Clinical diagnosis of lung cancer relies on histopathological evalution of tissue specimen. However, extensive knowledge of the metabolic biochemistry of tumors can potentially provide important information for accurate diagnosis of lung cancer. High resolution magic-angle spinning NMR spectroscopy has emerged and be widely acknowledged as an excellent tool in investigating tissue metabolism. Moreover, the combination of high resolution magic-angle spinning NMR technique and multivariate data analysis has become an important metabonomics platform for studying the intact biological tissues. This study reported the metabonomic characteristics of 51 lung tissues from 17 patients with lung cancer using the high resolution magic-angle spinning 1H NMR spectroscopy and the multivariate data analysis methods including principal component analysis and orthogonal partial least squares-discriminant analysis. Clear differences among the metabonomic characteristics of lung cancer tissues at various sites were disclosed. Compared with the adjacent noninvolved tissues, the lung cancer tissues had significantly high levels of aspartate, phosphocholine, glycerophosphocholine and lactate but significantly low levels of glucose and valine. Furthermore, significantly positive (or negative) correlations were observed between the levels of some metabolites such as lactate, fatty acids, valine, phosphocholine, and glycerophosphocholine. Magn Reson Med 66:1531-1540, 2011. ( C) 2011 Wiley Periodicals, Inc.

It is demonstrated that combination of diffusion-weighted NMR spectroscopy and principle components analysis (PCA) can be used to discriminate serums of patients with coronary heart disease (CHD) from that of health subjects. Diffusion-weighted H-1 NMR spectroscopy is a powerful approach for enhancing resonances from large molecules and suppressing resonances of low molecular-weight metabolites without disturbing physiological properties of human serum samples. The best diffusion weighting factor is found to be around b = 0.85 x 10(7) s/cm(2), increasing or decreasing the diffusion weighting factor destructs the discrimination efficiency. These preliminary results show that high density and low density lipoproteins (HDL & LDL) are the key components contributed to the discrimination, while very small metabolites (such as lactate etc.) and larger lipoproteins (such as very lower density lipoproteins, very low density lipoproteins (VLDL etc. have less effects.

A large number of studies indicate that oxidative modification of plasma lipoproteins, especially low-density lipoprotein (LDL), is a critical factor in initiation and progression of atherosclerosis. We have previously found that ibuprofen (IBP), a potential antioxidant drug to inhibit LDL oxidation, interacted with lipoproteins in intact human plasma. In the present study, we compare the binding affinities of IBP to LDL and HDL (high-density lipoprotein) by H-1 NMR spectroscopy. When IBP is added into the HDL and LDL samples, the -N+(CH3)(3) moieties of phosphatidylcholine (PC) and sphingomyelin (SM) in lipoprotein particles experience the chemical shift up-field drift. Intermolecular cross-peaks observed in NOESY spectra imply that there are direct interactions between ibuprofen and lipoproteins at both hydrophobic and hydrophilic (ionic) regions. These interactions are likely to be important in the solubility of ibuprofen into lipoprotein particles. Ibuprofen has higher impact on the PC and SM head group (-N+(CH3)(3)) and -(CH2)(n-) group in HDL than that in LDL. This could be explained by either IBP has higher binding affinity to HDL than to LDL, or IBP induces orientation of the phospholipid head group at the surface of the lipoprotein particles. (C) 2007 Elsevier Ireland Ltd. All rights reserved.

Nosiheptide is a parent compound of thiopeptide family that exhibit potent activities against various bacterial pathogens. Its C-terminal amide formation is catalyzed by NosA, which is an unusual strategy for maturating certain thiopeptides by processing their precursor peptides featuring a serine extension. We here report the crystal structure of truncated NosA1-111 variant, revealing three key elements, including basic lysine 49 (K49), acidic glutamic acid 101 (E101) and flexible C-terminal loop NosA112-151, are crucial to the catalytic terminal amide formation in nosiheptide biosynthesis. The side-chain of residue K49 and the C-terminal loop fasten the substrate through hydrogen bonds and hydrophobic interactions. The side-chain of residue E101 enhances nucleophilic attack of H2O to the methyl imine intermediate, leading to Cα-N bond cleavage and nosiheptide maturation. The sequence alignment of NosA and its homologs NocA, PbtH, TpdK and BerI and the enzymatic assay suggest that the mechanistic studies on NosA present an intriguing paradigm about how NosA family members function during thiopeptide biosynthesis.

The catalytic activity of cytochrome c (cyt c) to peroxidize cardiolipin to its oxidized form is required for the release of pro-apoptotic factors from mitochondria, and for execution of the subsequent apoptotic steps. However, the structural basis for this peroxidation reaction remains unclear. In this paper, we determined the three-dimensional NMR solution structure of yeast cyt c Y67H variant with high peroxidase activity, which is almost similar to that of its native form. The structure reveals that the hydrogen bond between Met80 and residue 67 is disrupted. This change destabilizes the sixth coordination bond between heme Fe(3+) ion and Met80 sulfur atom in the Y67H variant, and further makes it more easily be broken at low pH conditions. The steady-state studies indicate that the Y67H variant has the highest peroxidase activities when pH condition is between 4.0 and 5.2. Finally, a mechanism is suggested for the peroxidation of cardiolipin catalyzed by the Y67H variant, where the residue His67 acts as a distal histidine, its protonation facilitates O-O bond cleavage of H2O2 by functioning as an acidic catalyst.

The AAA-ATPase Vps4 is critical for function of the multivesicular body sorting pathway, which impacts cellular phenomena ranging from receptor down-regulation to viral budding to cytokinesis. Vps4 activity is stimulated by the interaction between Vta1 and Vps60, but the structural basis for this interaction is unclear. The fragment Vps60(128–186) was reported to display the full activity of Vps60. Vta1 interacts with Vps60 using its N-terminal domain (Vta1NTD). In this work, the structure of Vps60(128–186) in complex with Vta1NTD was determined using NMR techniques, demonstrating a novel recognition mode of the microtubule-interacting and transport (MIT) domain in which Vps60(128–186) interacts with Vta1NTD through helices α4′ and α5′, extending over Vta1NTD MIT2 domain helices 1–3. The Vps60 binding does not result in Vta1 conformational changes, further revealing the fact that Vps4 ATPase is enhanced by the interaction between Vta1 and Vps60 in an unanticipated manner.

Vta1 and Vps60 are two ESCRT associated proteins, their direct interaction enhances Vps4 ATPase activity. The N-terminal domain of Vta1 (residues 1-167aa, named as Vta1NTD) contains two tandem MIT domains, which specifically recognize Vps60 and Did2 but not other ESCRT-III subunits. The fragment Vps60 (128-186aa) was reported to display full activity of Vps60, which stimulates Vps4 ATPase in a Vta1-dependent manner. To study the structural basis for the interaction between Vta1 and Vps60, as a first step, here, we report the resonance assignments of the sequential backbone atoms and the side chains of the residues in the two components of Vta1NTD/Vps60(128-186) complex at pH 7.0 and 20 °C (BMRB No. 18521).

The neuron-restrictive silencer factor/RE1-silencing transcription factor (NRSF/REST) is regarded as not only a key transcriptional repressor but also an activator in neuron gene expression by specifically interacting with neuron-restrictive silencer element (NRSE/RE1) dsDNA and small NRSE/RE1 dsRNA, respectively. But its exact mechanism remains unclear. One major problem is that it is hard to obtain its functional multiple zinc finger (ZnF) domains in a large quantity for further structural studies. To address this issue, in this study, we for the first time attained soluble NRSF/REST functional domains named as ZnF5-8, ZnF4-8, ZnF3-8 and ZnF2-8 containing four, five, six and seven ZnF motifs in tandem, respectively, by using Circular Dichroism (CD) spectrum and two-dimensional (2D) nucleic magnetic resonance (NMR) (1)H-(1)H NOESY spectrum to monitor the folding of each single ZnF peptide. The data indicated that the residue cysteine 397 (Cys397) plays important roles in the global folding of NRSF/REST multiple ZnFs domain.

To convert cyt c into a peroxidase-like metalloenzyme, the P71H mutant was designed to introduce a distal histidine. Unexpectedly, its peroxidase activity was found even lower than that of the native, and that the axial ligation of heme iron was changed to His71/His18 in the oxidized state, while to Met80/His18 in the reduced state, characterized by UV-visible, circular dichroism, and resonance Raman spectroscopy. To further probe the functional importance of Pro71 in oxidation state dependent conformational changes occurred in cyt c, the solution structures of P71H mutant in both oxidation states were determined. The structures indicate that the half molecule of cyt c (aa 50–102) presents a kind of “zigzag riveting ruler” structure, residues at certain positions of this region such as Pro71, Lys73 can move a big distance by altering the tertiary structure while maintaining the secondary structures. This finding provides a molecular insight into conformational toggling in different oxidation states of cyt c that is principle significance to its biological functions in electron transfer and apoptosis. Structural analysis also reveals that Pro71 functions as a key hydrophobic patch in the folding of the polypeptide of the region (aa 50–102), to prevent heme pocket from the solvent.