Structural Insights into the Substrate Specificity of a 6-Phospho-β-glucosidase BglA-2 from Streptococcus pneumoniae TIGR4

The 6-phospho-β-glucosidase BglA-2 (EC 3.2.1.86) from glycoside hydrolase family 1 (GH-1) catalyzes the hydrolysis of β-1,4-linked cellobiose 6-phosphate (cellobiose-6′P) to yield glucose and glucose 6-phosphate. Both reaction products are further metabolized by the energy-generating glycolytic pathway. Here, we present the first crystal structures of the apo and complex forms of BglA-2 with thiocellobiose-6′P (a non-metabolizable analog of cellobiose-6′P) at 2.0 and 2.4 Å resolution, respectively. Similar to other GH-1 enzymes, the overall structure of BglA-2 from Streptococcus pneumoniae adopts a typical (β/α)₈ TIM-barrel, with the active site located at the center of the convex surface of the β-barrel. Structural analyses, in combination with enzymatic data obtained from site-directed mutant proteins, suggest that three aromatic residues, Tyr¹²⁶, Tyr³⁰³, and Trp³³⁸, at subsite +1 of BglA-2 determine substrate specificity with respect to 1,4-linked 6-phospho-β-glucosides. Moreover, three additional residues, Ser⁴²⁴, Lys⁴³⁰, and Tyr⁴³² of BglA-2, were found to play important roles in the hydrolytic selectivity toward phosphorylated rather than non-phosphorylated compounds. Comparative structural analysis suggests that a tryptophan versus a methionine/alanine residue at subsite −1 may contribute to the catalytic and substrate selectivity with respect to structurally similar 6-phospho-β-galactosidases and 6-phospho-β-glucosidases assigned to the GH-1 family.     

Structure of RNA 3′-phosphate cyclase bound to substrate RNA

RNA 3′-phosphate cyclase (RtcA) catalyzes the ATP-dependent cyclization of a 3′-phosphate to form a 2′,3′-cyclic phosphate at RNA termini. Cyclization proceeds through RtcA–AMP and RNA(3′)pp(5′)A covalent intermediates, which are analogous to intermediates formed during catalysis by the tRNA ligase RtcB. Here we present a crystal structure of Pyrococcus horikoshii RtcA in complex with a 3′-phosphate terminated RNA and adenosine in the AMP-binding pocket. Our data reveal that RtcA recognizes substrate RNA by ensuring that the terminal 3′-phosphate makes a large contribution to RNA binding. Furthermore, the RNA 3′-phosphate is poised for in-line attack on the P–N bond that links the phosphorous atom of AMP to N^ε of His307. Thus, we provide the first insights into RNA 3′-phosphate termini recognition and the mechanism of 3′-phosphate activation by an Rtc enzyme.     

2′-Phosphate cyclase activity of RtcA: a potential rationale for the operon organization of RtcA with an RNA repair ligase RtcB in Escherichia coli and other bacterial taxa

RNA terminal phosphate cyclase catalyzes the ATP-dependent conversion of a 3′-phosphate RNA end to a 2′,3′-cyclic phosphate via covalent enzyme-(histidinyl-Nϵ)-AMP and RNA(3′)pp(5′)A intermediates. Here, we report that Escherichia coli RtcA (and its human homolog Rtc1) are capable of cyclizing a 2′-phosphate RNA end in high yield. The rate of 2′-phosphate cyclization by RtcA is five orders of magnitude slower than 3′-phosphate cyclization, notwithstanding that RtcA binds with similar affinity to RNA_3′p and RNA_2′p substrates. These findings expand the functional repertoire of RNA cyclase and suggest that phosphate geometry during adenylate transfer to RNA is a major factor in the kinetics of cyclization. RtcA is coregulated in an operon with an RNA ligase, RtcB, that splices RNA 5′-OH ends to either 3′-phosphate or 2′,3′-cyclic phosphate ends. Our results suggest that RtcA might serve an end healing function in an RNA repair pathway, by converting RNA 2′-phosphates, which cannot be spliced by RtcB, to 2′,3′-cyclic phosphates that can be sealed. The rtcBA operon is controlled by the σ⁵⁴ coactivator RtcR encoded by an adjacent gene. This operon arrangement is conserved in diverse bacterial taxa, many of which have also incorporated the RNA-binding protein Ro (which is implicated in RNA quality control under stress conditions) as a coregulated component of the operon.     

RNase L releases a small RNA from HCV RNA that refolds into a potent PAMP

Triggering and propagating an intracellular innate immune response is essential for control of viral infections. RNase L is a host endoribonuclease and a pivotal component of innate immunity that cleaves viral and cellular RNA within single-stranded loops releasing small structured RNAs with 5′-hydroxyl (5′-OH) and 3′-monophosphoryl (3′-p) groups. In 2007, we reported that RNase L cleaves self RNA to produce small RNAs that function as pathogen-associated molecular patterns (PAMPs). However, the precise sequence and structure of PAMP RNAs produced by RNase L is unknown. Here we used hepatitis C virus RNA as substrate to characterize RNase L mediated cleavage products [named suppressor of virus RNA (svRNA)] for their ability to activate RIG-I like receptors (RLR). The NS5B region of HCV RNA was cleaved by RNase L to release an svRNA that bound to RIG-I, displacing its repressor domain and stimulating its ATPase activity while signaling to the IFN-β gene in intact cells. All three of these RIG-I functions were dependent on the presence in svRNA of the 3′-p. Furthermore, svRNA suppressed HCV replication in vitro through a mechanism involving IFN production and triggered a RIG-I-dependent hepatic innate immune response in mice. RNase L and OAS (required for its activation) were both expressed in hepatocytes from HCV-infected patients, raising the possibility that the OAS/RNase L pathway might suppress HCV replication in vivo. It is proposed that RNase L mediated cleavage of HCV RNA generates svRNA that activates RIG-I, thus propagating innate immune signaling to the IFN-β gene.     

Structure and mechanism of E. coli RNA 2′,3′-cyclic phosphodiesterase

2H (two-histidine) phosphoesterase enzymes are distributed widely in all domains of life and are implicated in diverse RNA and nucleotide transactions, including the transesterification and hydrolysis of cyclic phosphates. Here we report a biochemical and structural characterization of the Escherichia coli 2H protein YapD, which was identified originally as a reversible transesterifying “nuclease/ligase” at RNA 2′,5′-phosphodiesters. We find that YapD is an “end healing” cyclic phosphodiesterase (CPDase) enzyme that hydrolyzes an _HORNA>p substrate with a 2′,3′-cyclic phosphodiester to a _HORNAp product with a 2′-phosphomonoester terminus, without concomitant end joining. Thus we rename this enzyme ThpR (two-histidine 2′,3′-cyclic phosphodiesterase acting on RNA). The 2.0 Å crystal structure of ThpR in a product complex with 2′-AMP highlights the roles of extended histidine-containing motifs ⁴³HxTxxF⁴⁸ and ¹²⁵HxTxxR¹³⁰ in the CPDase reaction. His43-Nε makes a hydrogen bond with the ribose O3′ leaving group, thereby implicating His43 as a general acid catalyst. His125-Nε coordinates the O1P oxygen of the AMP 2′-phosphate (inferred from geometry to derive from the attacking water nucleophile), pointing to His125 as a general base catalyst. Arg130 makes bidentate contact with the AMP 2′-phosphate, suggesting a role in transition-state stabilization. Consistent with these inferences, changing His43, His125, or Arg130 to alanine effaced the CPDase activity of ThpR. Phe48 makes a π–π stack on the adenine nucleobase. Mutating Phe28 to alanine slowed the CPDase by an order of magnitude. The tertiary structure and extended active site motifs of ThpR are conserved in a subfamily of bacterial and archaeal 2H enzymes.     

Two Carotenoid Oxygenases Contribute to Mammalian Provitamin A Metabolism

Mammalian genomes encode two provitamin A-converting enzymes as follows: the β-carotene-15,15′-oxygenase (BCO1) and the β-carotene-9′,10′-oxygenase (BCO2). Symmetric cleavage by BCO1 yields retinoids (β-15′-apocarotenoids, C₂₀), whereas eccentric cleavage by BCO2 produces long-chain (>C₂₀) apocarotenoids. Here, we used genetic and biochemical approaches to clarify the contribution of these enzymes to provitamin A metabolism. We subjected wild type, Bco1^−/−, Bco2^−/−, and Bco1^−/−Bco2^−/− double knock-out mice to a controlled diet providing β-carotene as the sole source for apocarotenoid production. This study revealed that BCO1 is critical for retinoid homeostasis. Genetic disruption of BCO1 resulted in β-carotene accumulation and vitamin A deficiency accompanied by a BCO2-dependent production of minor amounts of β-apo-10′-carotenol (APO10ol). We found that APO10ol can be esterified and transported by the same proteins as vitamin A but with a lower affinity and slower reaction kinetics. In wild type mice, APO10ol was converted to retinoids by BCO1. We also show that a stepwise cleavage by BCO2 and BCO1 with APO10ol as an intermediate could provide a mechanism to tailor asymmetric carotenoids such as β-cryptoxanthin for vitamin A production. In conclusion, our study provides evidence that mammals employ both carotenoid oxygenases to synthesize retinoids from provitamin A carotenoids.     

A 3-hydroxy β-end group in xanthophylls is preferentially oxidized to a 3-oxo ε-end group in mammals

We previously found that mice fed lutein accumulated its oxidative metabolites (3′-hydroxy-ε,ε-caroten-3-one and ε,ε-carotene-3,3′-dione) as major carotenoids, suggesting that mammals can convert xanthophylls to keto-carotenoids by the oxidation of hydroxyl groups. Here we elucidated the metabolic activities of mouse liver for several xanthophylls. When lutein was incubated with liver postmitochondrial fraction in the presence of NAD⁺, (3′R,6′R)-3′-hydroxy-β,ε-caroten-3-one and (6RS,3′R,6′R)-3′-hydroxy-ε,ε-caroten-3-one were produced as major oxidation products. The former accumulated only at the early stage and was assumed to be an intermediate, followed by isomerization to the latter. The configuration at the C3′ and C6′ of the ε-end group in lutein was retained in the two oxidation products. These results indicate that the 3-hydroxy β-end group in lutein was preferentially oxidized to a 3-oxo ε-end group via a 3-oxo β-end group. Other xanthophylls such as β-cryptoxanthin and zeaxanthin, which have a 3-hydroxy β-end group, were also oxidized in the same manner as lutein. These keto-carotenoids, derived from dietary xanthophylls, were confirmed to be present in plasma of normal human subjects, and β,ε-caroten-3′-one was significantly increased by the ingestion of β-cryptoxanthin. Thus, humans as well as mice have oxidative activity to convert the 3-hydroxy β-end group of xanthophylls to a 3-oxo ε-end group.     

Regulation of 5-phosphoribosyl-1-pyrophosphate synthesis in human fibroblasts by the concentration of inorganic phosphate

During the growth cycle of normal fibroblasts and of fibroblasts deficient in glucose-6-phosphate dehydrogenase activity, the concentration of 5-phosphoribosyl-1-pyrophosphate and of P_i, as well as the activity of 5-phosphoribosyl-1-pyrophosphate synthetase, decreased to stable values in confluent cultures. A high degree of correlation (0.89 and 0.91 for two normal and 0.69 for one glucose-6-phosphate dehydrogenase-deficient cell strain, respectively) was shown between intracellular P_i and 5-phosphoribosyl-1-pyrophosphate concentrations under varying culture and incubation conditions. 5-phosphoribosyl-1-pyrophosphate concentrations were elevated in normal fibroblasts incubated with methylene blue only if intracellular P_i levels were high. Neither methylene blue nor 6-aminonicotinamide, singly, affected intracellular P_i concentrations. However, when normal cells were pretreated with 6-aminonicotinamide and then with methylene blue, intracellular P_i decreased, 5-phosphoribosyl-1-pyrophosphate was depleted, and its rate of generation decreased. Under similar conditions, glucose-6-phosphate dehydrogenase-deficient fibroblasts maintained unaltered P_i levels, and 5-phosphoribosyl-1-pyrophosphate concentration and generation were slightly increased. The decrease in intracellular P_i in normal cells after the combined treatment was commensurate with an accumulation of 6-phosphogluconate, which did not take place in mutant cells. The changes in 5-phosphoribosyl-1-pyrophosphate synthesis, whether due to the stage of growth or various experimental manipulations, were always concordant with changes in intracellular P_i level. The regulatory role of P_i is consistent with the known enzymic properties of 5-phosphoribosyl-1-pyrophosphate synthetase.     

Group VIA Phospholipase A2 (iPLA2β) Modulates Bcl-x 5′-Splice Site Selection and Suppresses Anti-apoptotic Bcl-x(L) in β-Cells

Diabetes is a consequence of reduced β-cell function and mass, due to β-cell apoptosis. Endoplasmic reticulum (ER) stress is induced during β-cell apoptosis due to various stimuli, and our work indicates that group VIA phospholipase A₂β (iPLA₂β) participates in this process. Delineation of underlying mechanism(s) reveals that ER stress reduces the anti-apoptotic Bcl-x(L) protein in INS-1 cells. The Bcl-x pre-mRNA undergoes alternative pre-mRNA splicing to generate Bcl-x(L) or Bcl-x(S) mature mRNA. We show that both thapsigargin-induced and spontaneous ER stress are associated with reductions in the ratio of Bcl-x(L)/Bcl-x(S) mRNA in INS-1 and islet β-cells. However, chemical inactivation or knockdown of iPLA₂β augments the Bcl-x(L)/Bcl-x(S) ratio. Furthermore, the ratio is lower in islets from islet-specific RIP-iPLA₂β transgenic mice, whereas islets from global iPLA₂β^−/− mice exhibit the opposite phenotype. In view of our earlier reports that iPLA₂β induces ceramide accumulation through neutral sphingomyelinase 2 and that ceramides shift the Bcl-x 5′-splice site (5′SS) selection in favor of Bcl-x(S), we investigated the potential link between Bcl-x splicing and the iPLA₂β/ceramide axis. Exogenous C₆-ceramide did not alter Bcl-x 5′SS selection in INS-1 cells, and neutral sphingomyelinase 2 inactivation only partially prevented the ER stress-induced shift in Bcl-x splicing. In contrast, 5(S)-hydroxytetraenoic acid augmented the ratio of Bcl-x(L)/Bcl-x(S) by 15.5-fold. Taken together, these data indicate that β-cell apoptosis is, in part, attributable to the modulation of 5′SS selection in the Bcl-x pre-mRNA by bioactive lipids modulated by iPLA₂β.     

Euryarchaeal β-CASP Proteins with Homology to Bacterial RNase J Have 5′- to 3′-Exoribonuclease Activity

In the Archaea only a handful of ribonucleases involved in RNA processing and degradation have been characterized. One potential group of archaeal ribonucleases are homologues of the bacterial RNase J family, which have a β-CASP metallo-β-lactamase fold. Here we show that β-CASP proteins encoded in the genomes of the hyperthermophilic Euryarchaeota Pyrococcus abyssi and Thermococcus kodakaraensis are processive exoribonucleases with a 5′ end dependence and a 5′ to 3′ directionality. We named these enzymes Pab-RNase J and Tk-RNase J, respectively. RNAs with 5′-monophosphate or 5′-hydroxyl ends are preferred substrates of Pab-RNase J, whereas circularized RNA is resistant to Pab-RNase J activity. Degradation of a 3′ end-labeled synthetic RNA in which an internal nucleoside is substituted by three ethylene glycol units generates intermediates demonstrating 5′ to 3′ directionality. The substitution of conserved residues in Pab-RNase J predicted to be involved in the coordination of metal ions demonstrates their importance for ribonuclease activity, although the detailed geometry of the catalytic site is likely to differ from bacterial RNase J. This is the first identification of a 5′-exoribonuclease encoded in the genomes of the Archaea. Phylogenetic analysis shows that euryarchaeal RNase J has been inherited vertically, suggesting an ancient origin predating the separation of the Bacteria and the Archaea.     

Design and green synthesis of novel quinolinone derivatives of potential anti-breast cancer activity against MCF-7 cell line targeting multi-receptor tyrosine kinases

A new set of 4,6,7,8-tetrahydroquinolin-5(1H)-ones were designed as cytotoxic agents against breast cancer cell line (MCF-7) and synthesised under ultrasonic irradiation using chitosan decorated copper nanoparticles (CS/CuNPs) catalyst. The new compounds 4b, 4j, 4k, and 4e exhibited the most potent cytotoxic activity of IC₅₀ values (0.002 − 0.004 µM) comparing to Staurosporine of IC₅₀; 0.005 μM. The latter derivatives exhibited a promising safety profile against the normal human WI38 cells of IC₅₀ range 0.0149 − 0.048 µM. Furthermore, the most promising cytotoxic compounds 4b, 4j were evaluated as multi-targeting agents against the RTK protein kinases; EGFR, HER-2, PDGFR-β, and VEGFR-2. Compound 4j showed promising inhibitory activity against HER-2 and PDGFR-β of IC₅₀ values 0.17 × 10⁻³, 0.07 × 10⁻³ µM in comparison with the reference drug sorafenib of IC₅₀; 0.28 × 10⁻³, 0.13 × 10⁻³ µM, respectively. In addition, 4j induced apoptotic effect and cell cycle arrest at G2/M phase preventing the mitotic cycle in MCF-7 cells.     

Inhibition of purine phosphoribosyltransferases of Ehrlich ascites-tumour cells by 6-mercaptopurine

1. The formation of adenosine 5′-phosphate, guanosine 5′-phosphate and inosine 5′-phosphate from [8-¹⁴C]adenine, [8-¹⁴C]guanine and [8-¹⁴C]hypoxanthine respectively in the presence of 5-phosphoribosyl pyrophosphate and an extract from Ehrlich ascites-tumour cells was assayed by a method involving liquid-scintillation counting of the radioactive nucleotides on diethylaminoethylcellulose paper. The results obtained with guanine were confirmed by a spectrophotometric assay which was also used to assay the conversion of 6-mercaptopurine and 5-phosphoribosyl pyrophosphate into 6-thioinosine 5′-phosphate in the presence of 6-mercaptopurine phosphoribosyltransferase from these cells. 2. At pH 7·8 and 25° the Michaelis constants for adenine, guanine and hypoxanthine were 0·9 μm, 2·9 μm and 11·0 μm in the assay with radioactive purines; the Michaelis constant for guanine in the spectrophotometric assay was 2·6 μm. At pH 7·9 the Michaelis constant for 6-mercaptopurine was 10·9 μm. 3. 25 μm-6-Mercaptopurine did not inhibit adenine phosphoribosyltransferase. 6-Mercaptopurine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 4·7 μm) and hypoxanthine phosphoribosyltransferase (K_i 8·3 μm). Hypoxanthine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 3·4 μm). 4. Differences in kinetic parameters and in the distribution of phosphoribosyltransferase activities after electrophoresis in starch gel indicate that different enzymes are involved in the conversion of adenine, guanine and hypoxanthine into their nucleotides. 5. From the low values of K_i for 6-mercaptopurine, and from published evidence that ascites-tumour cells require supplies of purines from the host tissues, it is likely that inhibition of hypoxanthine and guanine phosphoribosyltransferases by free 6-mercaptopurine is involved in the biological activity of this drug.     

Sphingosine Kinase 1 Is Regulated by Peroxisome Proliferator-activated Receptor α in Response to Free Fatty Acids and Is Essential for Skeletal Muscle Interleukin-6 Production and Signaling in Diet-induced Obesity

We previously demonstrated that sphingosine kinase 1 (Sphk1) expression and activity are up-regulated by exogenous palmitate (PAL) in a skeletal muscle model system and in diet-induced obesity in mice; however, potential functions and in vivo relevance of this have not been addressed. Here, we aimed to determine the mechanism by which PAL regulates SphK1 in muscle, and to determine potential roles for its product, sphingosine-1-phosphate (S1P), in muscle biology in the context of obesity. Cloning and analysis of the mouse Sphk1 promoter revealed a peroxisome proliferator-activated receptor (PPAR) α cis-element that mediated activation of a reporter under control of the Sphk1 promoter; direct interaction of PPARα was demonstrated by chromatin immunoprecipitation. PAL treatment induced the proinflammatory cytokine interleukin (IL)-6 in a manner dependent on SphK1, and this was attenuated by inhibition of the sphingosine-1-phosphate receptor 3 (S1PR3)_. Diet-induced obesity in mice demonstrated that IL-6 expression in muscle, but not adipose tissue, increased in obesity, but this was attenuated in Sphk1^−/− mice. Moreover, plasma IL-6 levels were significantly decreased in obese Sphk1^−/− mice relative to obese wild type mice, and muscle, but not adipose tissue IL-6 signaling was activated. These data indicate that PPARα regulates Sphk1 expression in the context of fatty acid oversupply and links PAL to muscle IL-6 production. Moreover, this function of SphK1 in diet-induced obesity suggests a potential role for SphK1 in obesity-associated pathological outcomes.     

Detection of Reaction Intermediates during Human Cystathionine β-Synthase-monitored Turnover and H2S Production

Human cystathionine β-synthase (CBS), a novel heme-containing pyridoxal 5′-phosphate enzyme, catalyzes the condensation of homocysteine and serine or cysteine to produce cystathionine and H₂O or H₂S, respectively. The presence of heme in CBS has limited spectrophotometric characterization of reaction intermediates by masking the absorption of the pyridoxal 5′-phosphate cofactor. In this study, we employed difference stopped-flow spectroscopy to characterize reaction intermediates formed under catalytic turnover conditions. The reactions of l-serine and l-cysteine with CBS resulted in the formation of a common aminoacrylate intermediate (k_obs = 0.96 ± 0.02 and 0.38 ± 0.01 mm⁻¹ s⁻¹, respectively, at 24 °C) with concomitant loss of H₂O and H₂S and without detectable accumulation of the external aldimine or other intermediates. Homocysteine reacted with the aminoacrylate intermediate with k_obs = 40.6 ± 3.8 s⁻¹ and re-formed the internal aldimine. In the reverse direction, CBS reacted with cystathionine, forming the aminoacrylate intermediate with k_obs = 0.38 ± 0.01 mm⁻¹ s⁻¹. This study provides the first insights into the pre-steady-state kinetic mechanism of human CBS and indicates that the reaction is likely to be limited by a conformational change leading to product release.     

Pyrrole-imidazole hairpin polyamides with high affinity at 5'-CGCG-3' DNA sequence; influence of cytosine methylation on binding

To investigate the binding of 5′–CpG–3′ sequences by small molecules, two pyrrole (Py)–imidazole (Im) hairpin polyamides, PyImPyIm–γ–PyImPyIm–β–Dp (1) and PyIm–β–Im–γ–PyIm–β–Im–β–Dp (2), which recognize the sequence 5′–CGCG–3′, were synthesized. The binding affinities of the 5′–CGCG–3′ sequence to the Py–Im hairpin polyamides were measured by surface plasmon resonance (SPR) analysis. SPR data revealed that dissociation equilibrium constants (K_d) of polyamides 1 and 2 were 1.1 (± 0.3) × 10^–6 M and 1.7 (± 0.4) × 10^–8 M, respectively. Polyamide 2 possesses great binding affinity for this sequence, 65-fold higher than polyamide 1. Moreover, when all cytosines in 5′–CpGpCpG–3′ were replaced with 5-methylcytosines (^mCs), the K_d value of polyamide 2 increased to 5.8 (± 0.7) × 10^–9 (M), which indicated about 3-fold higher binding than the unmethylated 5′–CGCG–3′ sequence. These results suggest that polyamide 2 would be suitable to target CpG-rich sequences in the genome.     

Structure–activity relationships in human RNA 3′-phosphate cyclase

RNA 3′-phosphate cyclase (Rtc) enzymes are a widely distributed family that catalyze the synthesis of RNA 2′,3′ cyclic phosphate ends via an ATP-dependent pathway comprising three nucleotidyl transfer steps: reaction of Rtc with ATP to form a covalent Rtc-(histidinyl-N)-AMP intermediate and release PP_i; transfer of AMP from Rtc1 to an RNA 3′-phosphate to form an RNA(3′)pp(5′)A intermediate; and attack by the terminal nucleoside O2′ on the 3′-phosphate to form an RNA 2′,3′ cyclic phosphate product and release AMP. Here we used the crystal structure of Escherichia coli RtcA to guide a mutational analysis of the human RNA cyclase Rtc1. An alanine scan defined seven conserved residues as essential for the Rtc1 RNA cyclization and autoadenylylation reactions. Structure–activity relationships were clarified by conservative substitutions. Our results are consistent with a mechanism of adenylate transfer in which attack of the Rtc1 His320 nucleophile on the ATP α phosphorus is facilitated by proper orientation of the PP_i leaving group via contacts to Arg21, Arg40, and Arg43. We invoke roles for Tyr294 in binding the adenine base and Glu14 in binding the divalent cation cofactor. We find that Rtc1 forms a stable binary complex with a 3′-phosphate terminated RNA, but not with an otherwise identical 3′-OH terminated RNA. Mutation of His320 had little impact on RNA 3′-phosphate binding, signifying that covalent adenylylation of Rtc1 is not a prerequisite for end recognition.     

Positive and Negative Allosteric Modulation of an α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor by Binding to a Site in the Transmembrane Domain at the γ+-β? Interface

In the process of developing safer general anesthetics, isomers of anesthetic ethers and barbiturates have been discovered that act as convulsants and inhibitors of γ-aminobutyric acid type A receptors (GABA_ARs) rather than potentiators. It is unknown whether these convulsants act as negative allosteric modulators by binding to the intersubunit anesthetic-binding sites in the GABA_AR transmembrane domain (Chiara, D. C., Jayakar, S. S., Zhou, X., Zhang, X., Savechenkov, P. Y., Bruzik, K. S., Miller, K. W., and Cohen, J. B. (2013) J. Biol. Chem. 288, 19343–19357) or to known convulsant sites in the ion channel or extracellular domains. Here, we show that S-1-methyl-5-propyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (S-mTFD-MPPB), a photoreactive analog of the convulsant barbiturate S-MPPB, inhibits α1β3γ2 but potentiates α1β3 GABA_AR responses. In the α1β3γ2 GABA_AR, S-mTFD-MPPB binds in the transmembrane domain with high affinity to the γ⁺-β⁻ subunit interface site with negative energetic coupling to GABA binding in the extracellular domain at the β⁺-α⁻ subunit interfaces. GABA inhibits S-[³H]mTFD-MPPB photolabeling of γ2Ser-280 (γM2–15′) in this site. In contrast, within the same site GABA enhances photolabeling of β3Met-227 in βM1 by an anesthetic barbiturate, R-[³H]methyl-5-allyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), which differs from S-mTFD-MPPB in structure only by chirality and two hydrogens (propyl versus allyl). S-mTFD-MPPB and R-mTFD-MPAB are predicted to bind in different orientations at the γ⁺-β⁻ site, based upon the distance in GABA_AR homology models between γ2Ser-280 and β3Met-227. These results provide an explanation for S-mTFD-MPPB inhibition of α1β3γ2 GABA_AR function and provide a first demonstration that an intersubunit-binding site in the GABA_AR transmembrane domain binds negative and positive allosteric modulators.     

Adenosine 5'-sulphatophosphate kinase activity in spinach leaf tissue

1. An F⁻-insensitive 3′-nucleotidase was purified from spinach leaf tissue; the enzyme hydrolysed 3′-AMP, 3′-CMP and adenosine 3′-phosphate 5′-sulphatophosphate but not adenosine 5′-nucleotides nor PP_i. The pH optimum of the enzyme was 7.5; K_m (3′-AMP) was approx. 0.8mm and K_m (3′-CMP) was approx. 3.3mm. 3′-Nucleotidase activity was not associated with chloroplasts. Purified Mg²⁺-dependent pyrophosphatase, free from F⁻-insensitive 3′-nucleotidase, catalysed some hydrolysis of 3′-AMP; this activity was F⁻-sensitive. 2. Adenosine 5′-sulphatophosphate kinase activity was demonstrated in crude spinach extracts supplied with 3′-AMP by the synthesis of the sulphate ester of 2-naphthol in the presence of purified phenol sulphotransferase; purified ATP sulphurylase and pyrophosphatase were also added to synthesize adenosine 5′-sulphatophosphate. Adenosine 5′-sulphatophosphate kinase activity was associated with chloroplasts and was released by sonication. 3. Isolated chloroplasts synthesized adenosine 3′-phosphate 5′-sulphatophosphate from sulphate and ATP in the presence of a 3′-nucleotide; the formation of adenosine 5′-sulphatophosphate was negligible. In the absence of a 3′-nucleotide the synthesis of adenosine 3′-phosphate 5′-sulphatophosphate was negligible, but the formation of adenosine 5′-sulphatophosphate was readily detected. Some properties of the synthesis of adenosine 3′-phosphate 5′-sulphatophosphate by isolated chloroplasts are described. 4. Adenosine 3′-phosphate 5′-sulphatophosphate, synthesized by isolated chloroplasts, was characterized by specific enzyme methods, electrophoresis and i.r. spectrophotometry. 5. Isolated chloroplasts catalysed the incorporation of sulphur from sulphate into cystine/cysteine; the incorporation was enhanced by 3′-AMP and l-serine. It was concluded that adenosine 3′-phosphate 5′-sulphatophosphate is an intermediate in the incorporation of sulphur from sulphate into cystine/cysteine.     

Resolving Two-dimensional Kinetics of the Integrin αIIbβ3-Fibrinogen Interactions Using Binding-Unbinding Correlation Spectroscopy

Using a combined experimental and theoretical approach named binding-unbinding correlation spectroscopy (BUCS), we describe the two-dimensional kinetics of interactions between fibrinogen and the integrin αIIbβ3, the ligand-receptor pair essential for platelet function during hemostasis and thrombosis. The methodology uses the optical trap to probe force-free association of individual surface-attached fibrinogen and αIIbβ3 molecules and forced dissociation of an αIIbβ3-fibrinogen complex. This novel approach combines force clamp measurements of bond lifetimes with the binding mode to quantify the dependence of the binding probability on the interaction time. We found that fibrinogen-reactive αIIbβ3 pre-exists in at least two states that differ in their zero force on-rates (k_on1 = 1.4 × 10⁻⁴ and k_on2 = 2.3 × 10⁻⁴ μm²/s), off-rates (k_off1 = 2.42 and k_off2 = 0.60 s⁻¹), and dissociation constants (K_d1 = 1.7 × 10⁴ and K_d2 = 2.6 × 10³ μm⁻²). The integrin activator Mn²⁺ changed the on-rates and affinities (K_d1 = 5 × 10⁴ and K_d2 = 0.3 × 10³ μm⁻²) but did not affect the off-rates. The strength of αIIbβ3-fibrinogen interactions was time-dependent due to a progressive increase in the fraction of the high affinity state of the αIIbβ3-fibrinogen complex characterized by a faster on-rate. Upon Mn²⁺-induced integrin activation, the force-dependent off-rates decrease while the complex undergoes a conformational transition from a lower to higher affinity state. The results obtained provide quantitative estimates of the two-dimensional kinetic rates for the low and high affinity αIIbβ3 and fibrinogen interactions at the single molecule level and offer direct evidence for the time- and force-dependent changes in αIIbβ3 conformation and ligand binding activity, underlying the dynamics of fibrinogen-mediated platelet adhesion and aggregation.