Release of a Group 1 Mycoplasma Virus from Acholeplasma laidlawii After Treatment with Mitomycin C

A rod-shaped group 1 mycoplasma virus was released from Acholeplasma laidlawii strain JA2 after treatment with 2.5 μg of mitomycin C per ml. Similar treatment of A. laidlawii strain Bju failed to stimulate release of any PFU.     

The Ribosome Binding Site of Hepatitis C Virus mRNA

Hepatitis C virus (HCV) infects an estimated 170 million people worldwide, the majority of whom develop a chronic infection which can lead to severe liver disease, and for which no generally effective treatment yet exists. A promising target for treatment is the internal ribosome entry site (IRES) of HCV, a highly conserved domain within a highly variable RNA. Never before have the ribosome binding sites of any IRES domains, cellular or viral, been directly characterized. Here, we reveal that the HCV IRES sequences most closely associated with 80S ribosomes during protein synthesis initiation are a series of discontinuous domains together comprising by far the largest ribosome binding site yet discovered.     

Binding sites of cholinesterases. Alkylation by an aziridinium derivative

1. Alkylation of acetylcholinesterase and butyrylcholinesterase by 2-chloro-N-(chloroethyl)-N-methyl-2-phenylethylamine was observed. This alkylating agent was more potent than related compounds previously described, and less stable (half-life 8.5min. at 23 degrees ). 2. Alkylation had effects on hydrolysis of substrates varying from activation for indophenyl acetate to inhibition for acetylcholine, and intermediate effects with five other substrates. The effects were on V(max.) and not K(m). 3. Alkylation caused a variety of changes in sensitivity to inhibition by five carbamates, five organophosphates and four other inhibitors, varying from total protection against tetraethylammonium to mildly enhanced sensitivity to urea. 4. The findings suggested the existence of three binding sites, one of which was anionic and another hydrophobic, in addition to the esteratic site.     

Anomalous Induction of Mycobacteriophages Mediated by Mitomycin C

Mitomycin C has been found to stimulate the production of long-tailed defective bacteriophages and poly tails in thick cell wall mycobacterial mutants.     

Enhancement of Agrobacterium tumefaciens Infectivity by Mitomycin C

The ability of Agrobacterium tumefaciens to induce pinto leaf tumors may be enhanced two- to threefold after treatment with mitomycin C. The enhancement may be obtained with either lethal or nonlethal concentrations. With 10-min treatments, an optimal response was obtained with 0.005 mug of mitomycin C per ml in the absence of any change in the number of viable cells. Both the tumor induction process and the tumors induced by treated cultures appear qualitatively the same as controls. To account for these results, the antibiotic must increase the proportion of viable cells that will subsequently initiate tumors. One, or at most a few, random lesions in the bacterial chromosome seem to be the necessary requirement for this promotion. At mitomycin concentrations of 1 and 5 mug/ml, the ability of A. tumefaciens to initiate tumors is rapidly lost, indicating that a fairly intact bacterial chromosome is one of the essentials for the tumor induction process.     

The Discovery of a Novel Macrophage Binding Site

1. During the course of studies directed to determine the transport of Angiotensin II AT(2) receptors in the rat brain, we found that stab wounds to the brain revealed a binding site recognized by the AT(2) receptor ligand CGP42112 but not by Angiotensin II. 2. We localized this novel site to macrophages/microglia associated with physical or chemical injuries of the brain. 3. The non-Angiotensin II site was also highly localized to inflammatory lesions of peripheral arteries. 4. In rodent tissues, high binding expression was limited to the spleen and to circulating monocytes. A high-affinity binding site was also characterized in human monocytes. 5. Lack of affinity for many ligands binding to known macrophage receptors indicated the possibility that the non-Angiotensin II CGP42112 binding corresponds to a novel site.6. CGP42112 enhanced cell attachment to fibronectin and collagen and metalloproteinase-9 secretion from human monocytes incubated in serum-free medium but did not promote cytokine secretion. 7. When added in the presence of lipopolysaccharide, CGP42112 reduced the lipopolysaccharide-stimulated secretion of the pro-inflammatory cytokines TNF-alpha, IL-1, IL-1 beta, and IL-6, and increased protein kinase A. 8. Molecular modeling revealed that a CGP42112 derivative was selective for the novel macrophage site and did not recognize the Angiotensin II AT(2) receptor. 9. These results demonstrate that CGP42112, previously considered as a selective Angiotensin II AT(2) ligand, recognizes an additional non-Angiotensin II site different from AT(2) receptors. 10. Our observations indicate that CGP42112 or related molecules could be considered of interest as potential anti-inflammatory compounds.     

Blind Prediction of Charged Ligand Binding Affinities in a Model Binding Site

Predicting absolute protein–ligand binding affinities remains a frontier challenge in ligand discovery and design. This becomes more difficult when ionic interactions are involved because of the large opposing solvation and electrostatic attraction energies. In a blind test, we examined whether alchemical free-energy calculations could predict binding affinities of 14 charged and 5 neutral compounds previously untested as ligands for a cavity binding site in cytochrome c peroxidase. In this simplified site, polar and cationic ligands compete with solvent to interact with a buried aspartate. Predictions were tested by calorimetry, spectroscopy, and crystallography. Of the 15 compounds predicted to bind, 13 were experimentally confirmed, while 4 compounds were false negative predictions. Predictions had a root-mean-square error of 1.95 kcal/mol to the experimental affinities, and predicted poses had an average RMSD of 1.7 Å to the crystallographic poses. This test serves as a benchmark for these thermodynamically rigorous calculations at predicting binding affinities for charged compounds and gives insights into the existing sources of error, which are primarily electrostatic interactions inside proteins. Our experiments also provide a useful set of ionic binding affinities in a simplified system for testing new affinity prediction methods.     

Distortion of the Major Histocompatibility Complex Class I Binding Groove to Accommodate an Insulin-derived 10-Mer Peptide

The non-obese diabetic mouse model of type 1 diabetes continues to be an important tool for delineating the role of T-cell-mediated destruction of pancreatic β-cells. However, little is known about the molecular mechanisms that enable this disease pathway. We show that insulin reactivity by a CD8⁺ T-cell clone, known to induce type 1 diabetes, is characterized by weak T-cell antigen receptor binding to a relatively unstable peptide-MHC. The structure of the native 9- and 10-mer insulin epitopes demonstrated that peptide residues 7 and 8 form a prominent solvent-exposed bulge that could potentially be the main focus of T-cell receptor binding. The C terminus of the peptide governed peptide-MHC stability. Unexpectedly, we further demonstrate a novel mode of flexible peptide presentation in which the MHC peptide-binding groove is able to “open the back door” to accommodate extra C-terminal peptide residues.     

Structure, Properties, and Engineering of the Major Zinc Binding Site on Human Albumin

Most blood plasma zinc is bound to albumin, but the structure of the binding site has not been determined. Zn K-edge extended x-ray absorption fine structure spectroscopy and modeling studies show that the major Zn²⁺ site on albumin is a 5-coordinate site with average Zn-O/N distances of 1.98 Å and a weak sixth O/N bond of 2.48 Å, consistent with coordination to His⁶⁷ and Asn⁹⁹ from domain I, His²⁴⁷ and Asp²⁴⁹ from domain II (residues conserved in all sequenced mammalian albumins), plus a water ligand. The dynamics of the domain I/II interface, thought to be important to biological function, are affected by Zn²⁺ binding, which induces cooperative allosteric effects related to those of the pH-dependent neutral-to-base transition. N99D and N99H mutations enhance Zn²⁺ binding but alter protein stability, whereas mutation of His⁶⁷ to alanine removes an interdomain H-bond and weakens Zn²⁺ binding. Both wild-type and mutant albumins promote the safe management of high micromolar zinc concentrations for cells in cultures.Zinc is not only required for hundreds of essential extra- and intracellular proteins and enzymes but is also recruited by toxins such as anthrax lethal factor (1) and staphylococcal enterotoxin (2). There is a need to understand how zinc transport and distribution is controlled (3).Although considerable progress has been made in the identification and study of membrane-bound zinc transporters, the molecular mechanism of extracellular zinc transport is still obscure. The total concentration of zinc in blood is high, ∼15–20 μm (4), and plasma zinc concentrations are maintained at a relatively constant level, except during periods of dietary zinc depletion and acute responses to stress or inflammation, when they are depressed (5). In humans, ∼98% of so-called “exchangeable” zinc in blood plasma (9–14 μm) is bound to serum albumin (6). Studies on perfused rat intestine have implicated albumin in the transport of newly absorbed zinc in portal blood, from the intestine to the liver (5). Albumin has also been shown to promote zinc uptake by endothelial cells, with receptor-mediated endocytosis as the most likely mechanism (7).Albumin, the most abundant protein in blood plasma (∼40 mg ml⁻¹ and 0.6 mm), is synthesized in the liver and secreted into the blood stream as a 585-residue, single-chain protein after loss of a 24-residue propeptide (8). The protein is largely α-helical and folds into three structurally homologous domains (I, II, and III), each of which contains two subdomains (A and B) (see Fig. 1A) (9). There are 35 Cys residues that form six disulfide bridges in each domain, except for domain I, which contains only five bridges and a free thiol at Cys³⁴.Open in a separate windowFIGURE 1.A, domain structure of human serum albumin (9). Domain I (red), domain II (blue), and domain III (green) are held together solely by linker helices and weak interactions. B, the proposed interdomain zinc site on albumin is formed by two residues from domain I (red) and two residues from domain II (blue).Zinc binding to albumin has also been demonstrated in vitro, and the so-called high-affinity site for zinc on albumin displays a binding constant of K ≈ 10⁷ m⁻¹ (10–13). Although over 50 x-ray structures of albumin have been reported to date (9, 14–16), no experimental structural data are available for the zinc site on albumin. On the basis of ¹¹¹Cd NMR studies, site-directed mutagenesis, and molecular modeling, we have proposed that the major zinc site on albumin, termed site A, is located at the interface of domains I and II and is formed by the side chains of His⁶⁷ and Asn⁹⁹ from domain I and by His²⁴⁷ and Asp²⁴⁹ from domain II (Fig. 1B) (17, 18).Here, we report the first direct structural characterization of a zinc site on albumin, using Zn K-edge x-ray absorption fine structure (EXAFS)² spectroscopy. We also explored the possibility of engineering albumins with increased or decreased zinc-binding affinity by mutating the postulated zinc-binding ligands. NMR methods were used to identify mutated and zinc-binding histidines and to probe the effects of zinc binding on the conformational dynamics of the protein. Finally, we show that albumin at physiological concentrations promotes the culture of hepatocytes at otherwise toxic zinc concentrations.     

Inhibition of HCO(3) Binding to Photosystem II by Atrazine at a Low-Affinity Herbicide Binding Site

In maize chloroplasts, the ratio of HCO₃⁻ (anion) binding sites to high-affinity atrazine binding sites is unity. In the dark, atrazine noncompetitively inhibits the binding of half of the HCO₃⁻ to the photosystem II (PSII) complexes. The inhibition of binding saturates at 5 micromolar atrazine, little inhibition is seen at 0.5 micromolar atrazine, although the high-affinity herbicide binding sites are nearly filled at this concentration. This means that HCO₃⁻ and atrazine interact noncompetitively at a specific low-affinity herbicide binding site that exists on a portion of the PSII complexes. Light abolishes the inhibitory effects of atrazine on HCO₃⁻ binding. Based on the assumption that there is one high-affinity atrazine binding site per PSII complex, we conclude that there is also only one binding site for HCO₃⁻ with a dissociation constant near 80 micromolar. The location of the HCO₃⁻ binding site, and the low-affinity atrazine binding site, is not known.     

Identification of the SSB Binding Site on E. coli RecQ Reveals a Conserved Surface for Binding SSB's C Terminus

RecQ DNA helicases act in conjunction with heterologous partner proteins to catalyze DNA metabolic activities, including recombination initiation and stalled replication fork processing. For the prototypical Escherichia coli RecQ protein, direct interaction with single-stranded DNA-binding protein (SSB) stimulates its DNA unwinding activity. Complex formation between RecQ and SSB is mediated by the RecQ winged-helix domain, which binds the nine C-terminal-most residues of SSB, a highly conserved sequence known as the SSB-Ct element. Using nuclear magnetic resonance and mutational analyses, we identify the SSB-Ct binding pocket on E. coli RecQ. The binding site shares a striking electrostatic similarity with the previously identified SSB-Ct binding site on E. coli exonuclease I, although the SSB binding domains in the two proteins are not otherwise related structurally. Substitutions that alter RecQ residues implicated in SSB-Ct binding impair RecQ binding to SSB and SSB/DNA nucleoprotein complexes. These substitutions also diminish SSB-stimulated DNA helicase activity in the variants, although additional biochemical changes in the RecQ variants indicate a role for the winged-helix domain in helicase activity beyond SSB protein binding. Sequence changes in the SSB-Ct element are sufficient to abolish interaction with RecQ in the absence of DNA and to diminish RecQ binding and helicase activity on SSB/DNA substrates. These results support a model in which RecQ has evolved an SSB-Ct binding site on its winged-helix domain as an adaptation that aids its cellular functions on SSB/DNA nucleoprotein substrates.     

A model for the production of chromosome damage by Mitomycin C

A model is presented, which is based on the idea that the chromosome damage induced by Mitomycin C results directly from repair or misrepair of DNA molecules responsible for the linear continuity of the chromosomes. Testing the model with human cells confirms the prediction that exchanges with complete joining occur between chromosome regions containing homologous, repetitive DNA. Most probably incomplete exchanges involve homologous, but unique DNA sequences. — Prerequisites determining the MC-induced aberration patterns are the distribution of the chemical due to compartmentalization, the somatic pairing of chromosomes, and the occurrence of repeated or unique DNA sequences. — The scoring of different classes of MC-induced chromatid aberrations (attenuation, constriction, gap, break) in alcohol/acetic acid-fixed chromosome has a limited value.     

Validation of an Allosteric Binding Site of Src Kinase Identified by Unbiased Ligand Binding Simulations

Allostery plays a primary role in regulating protein activity, making it an important mechanism in human disease and drug discovery. Identifying allosteric regulatory sites to explore their biological significance and therapeutic potential is invaluable to drug discovery; however, identification remains a challenge. Allosteric sites are often “cryptic” without clear geometric or chemical features. Since allosteric regulatory sites are often less conserved in protein kinases than the orthosteric ATP binding site, allosteric ligands are commonly more specific than ATP competitive inhibitors. We present a generalizable computational protocol to predict allosteric ligand binding sites based on unbiased ligand binding simulation trajectories. We demonstrate the feasibility of this protocol by revisiting our previously published ligand binding simulations using the first identified viral proto-oncogene, Src kinase, as a model system. The binding paths for kinase inhibitor PP1 uncovered three metastable intermediate states before binding the high-affinity ATP-binding pocket, revealing two previously known allosteric sites and one novel site. Herein, we validate the novel site using a combination of virtual screening and experimental assays to identify a V-type allosteric small-molecule inhibitor that targets this novel site with specificity for Src over closely related kinases. This study provides a proof-of-concept for employing unbiased ligand binding simulations to identify cryptic allosteric binding sites and is widely applicable to other protein–ligand systems.     

水稻RNA结合蛋白C3H12与RNA结合位点的鉴定

CCH型锌指蛋白质C3H12是进化上保守的RNA结合蛋白质,它含有5个串联的CCCH锌指结构域ZnF1-5,形成2个紧密的锌指簇ZnF1-3和ZnF4-5。早期的研究发现,C3H12可能通过与mRNA结合的方式在转录后水平调控基因的表达。然而,与C3H12结合的mRNA类型和他们的结合模式,并未通过实验得到证明。本文表达纯化了一系列C3H12截短及全长蛋白质,并合成了一些潜在RNA底物ARE9、ARE19及对照Random21。通过等温滴定量热法 (isothermal titration calorimetry, ITC) 确定了C3H12与富含腺嘌呤尿嘧啶单元 (AU-rich element, ARE) mRNA底物的结合,并揭示了互作核心区域和热力学性质。通过荧光光谱分析和微型热泳动 (microscale thermophoresis, MST)技术对ITC的结果进一步佐证。结果表明：(1) C3H12与ARE底物的相互作用是焓驱动的能量有利的 (ΔG<0) 特异性结合,结合比为1:1。(2) C3H12与ARE19的亲和力较ARE9更高（约2倍）。(3) C3H12中ZnF1-3在与ARE类底物的结合活性中发挥主导作用。(4) C3H12结构中的141个氨基酸残基的接头不直接参与和ARE底物的相互作用。本研究揭示的CCCH型锌指蛋白质C3H12与ARE底物结合模式,将为进一步在分子结构水平阐明C3H12与ARE底物结合的机制奠定基础。     

鳞片状细胞癌抗原Ⅰ与乙型肝炎病毒的结合受反应位点环域疏水性的影响

鳞片状细胞癌抗原Ⅰ (SCCA1)是丝氨酸蛋白酶抑制剂 (serpin)超家族的成员 ,具有多种变异体。有报道其中的两种(BP和AJ515706 )能通过乙型肝炎病毒 (HBV)的前S1抗原促进表达SCCA1的细胞与HBV的结合。本研究从HepG2细胞中扩增出的一株SCCA1(A1)却不具备HBV结合能力。将A1的C末端与BP的C末端互换 ,获得的A1-BP能够结合HBV ,而BP-A1却不能。A1与BP的C末端仅有 3个氨基酸的差异 ,其中 2个位于反应位点环域。一级结构分析发现在该区域内 ,A1的疏水性较弱 ,而BP和AJ515706的疏水性较强。将A1的aa349位的弱疏水性的谷氨酸突变为强疏水性的缬氨酸 ,则可获得HBV结合能力。反之 ,将BP同一位点的缬氨酸突变为谷氨酸 ,则会丧失HBV结合能力。这些结果提示SCCA1与HBV的结合受反应位点环域的疏水性的影响。     

水稻RNA结合蛋白C3H12与RNA结合位点的鉴定

CCH型锌指蛋白质C3H12是进化上保守的RNA结合蛋白质,它含有5个串联的CCCH锌指结构域ZnF1-5,形成2个紧密的锌指簇ZnF1-3和ZnF4-5。早期的研究发现,C3H12可能通过与mRNA结合的方式在转录后水平调控基因的表达。然而,与C3H12结合的mRNA类型和他们的结合模式,并未通过实验得到证明。本文表达纯化了一系列C3H12截短及全长蛋白质,并合成了一些潜在RNA底物ARE9、ARE19及对照Random21。通过等温滴定量热法 (isothermal titration calorimetry, ITC) 确定了C3H12与富含腺嘌呤尿嘧啶单元 (AU-rich element, ARE) mRNA底物的结合,并揭示了互作核心区域和热力学性质。通过荧光光谱分析和微型热泳动 (microscale thermophoresis, MST)技术对ITC的结果进一步佐证。结果表明：(1) C3H12与ARE底物的相互作用是焓驱动的能量有利的 (ΔG<0) 特异性结合,结合比为1:1。(2) C3H12与ARE19的亲和力较ARE9更高（约2倍）。(3) C3H12中ZnF1-3在与ARE类底物的结合活性中发挥主导作用。(4) C3H12结构中的141个氨基酸残基的接头不直接参与和ARE底物的相互作用。本研究揭示的CCCH型锌指蛋白质C3H12与ARE底物结合模式,将为进一步在分子结构水平阐明C3H12与ARE底物结合的机制奠定基础。     

Characteristics of a Membrane-Associated Steroid Binding Site in Rat Liver

Abstract

The glucocorticoid dexamethasone binds a site in microsomes in a saturable manner which by competition studies also binds other classes of steroids. The characteristics of dexamethasone binding to microsomes is distinct from the cytosolic glucocorticoid receptor by virtue of a slower rate of association; a differential competition by the glucocorticoid receptor agonist triamcinolone acetonide and antagonist RU38486; and a lack of sensitivity to the reversible thiol reactive agent arsenite. However, both binding sites have a similar rate constant for complex dissociation; are sensitive to covalent thiol modification by N ethylmaleimide and iodoacetamide; and have a similar concentration-dependent sensitivity to the reversible thiol reactive agent methyl methanethiosulfonate. The binding of dexamethasone by microsomes therefore exhibits distinct properties from the soluble glucocorticoid receptor.     

CD44 Binding to Hyaluronic Acid Is Redox Regulated by a Labile Disulfide Bond in the Hyaluronic Acid Binding Site

CD44 is the primary leukocyte cell surface receptor for hyaluronic acid (HA), a component of the extracellular matrix. Enzymatic post translational cleavage of labile disulfide bonds is a mechanism by which proteins are structurally regulated by imparting an allosteric change and altering activity. We have identified one such disulfide bond in CD44 formed by Cys77 and Cys97 that stabilises the HA binding groove. This bond is labile on the surface of leukocytes treated with chemical and enzymatic reducing agents. Analysis of CD44 crystal structures reveal the disulfide bond to be solvent accessible and in the–LH hook configuration characteristic of labile disulfide bonds. Kinetic trapping and binding experiments on CD44-Fc chimeric proteins show the bond is preferentially reduced over the other disulfide bonds in CD44 and reduction inhibits the CD44-HA interaction. Furthermore cells transfected with CD44 no longer adhere to HA coated surfaces after pre-treatment with reducing agents. The implications of CD44 redox regulation are discussed in the context of immune function, disease and therapeutic strategies.     

Biologic Activity of a Nucleotide Conjugate Between Mitomycin C and Cytarabine Monophosphate

Abstract

A dual prodrug conjugate between the antimetabolite cytarabine monophosphate and the alkylating agent 2,7-diaminomitosene (derived from mitomycin C), cytaramycin, was synthesized and tested for antileukemic activity in sensitive and resistant tumors. The compound was active against parental L-1210, CCRF-CEM, HL-60 and K-562 leukemia cells but did not overcome resistance in sublines developed for (1) multidrug resistance (L-1210/MDR and K-562-R) or (2) for cytarabine resistance (CCRF-CEM/ARA-C and HL-60/ARA-C). Alkaline DNA elution tests demonstrate a predominance of strand breaking activity due to the cytarabine moiety, and a lesser degree of DNA crosslinking, due to the mitosene moiety. The conjugate was active in mice bearing P-388 leukemia (80% increased lifespan), but was not more effective than mitomycin C alone in mice bearing a cytarabine-resistant L-1210 cell line (38% to 31% increased lifespan). These findings suggest that mitomycin nucleotide conjugates do not overcome resistance to the parent antimetabolites.