text:
These authors contributed equally to the work.
Research Article
Structural Basis of the Allosteric Inhibitor Interaction on the HIV-1 Reverse Transcriptase RNase H Domain
Version of Record online: 31 AUG 2012
DOI: 10.1111/cbdd.12010
© 2012 John Wiley & Sons A/S
Chemical Biology & Drug Design
Volume 80, Issue 5, pages 706–716, November 2012
Additional Information
How to Cite
Christen, M. T., Menon, L., Myshakina, N. S., Ahn, J., Parniak, M. A. and Ishima, R. (2012), Structural Basis of the Allosteric Inhibitor Interaction on the HIV-1 Reverse Transcriptase RNase H Domain. Chemical Biology & Drug Design, 80: 706–716. doi: 10.1111/cbdd.12010
Publication History
- Issue online: 5 OCT 2012
- Version of Record online: 31 AUG 2012
- Accepted manuscript online: 31 JUL 2012 02:55AM EST
- Received 8 March 2012, revised 22 June 2012 and accepted for publication 17 July 2012
- acylhydrazone;
- dimer;
- enzyme;
- inhibitor;
- molecular docking;
- protein;
- reverse transcriptase;
- ribonuclease H
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Conclusions
- Acknowledgments
- References
- Supporting Information
HIV-1 reverse transcriptase (RT) has been an attractive target for the development of antiretroviral agents. Although this enzyme is bi-functional, having both DNA polymerase and ribonuclease H (RNH) activities, there is no clinically approved inhibitor of the RNH activity. Here, we characterize the structural basis and molecular interaction of an allosteric site inhibitor, BHMP07, with the wild-type (WT) RNH fragment. Solution NMR experiments for inhibitor titration on WT RNH showed relatively wide chemical shift perturbations, suggesting a long-range conformational effect on the inhibitor interaction. Comparisons of the inhibitor-induced NMR chemical shift changes of RNH with those of RNH dimer, in the presence and absence of Mg2+, were performed to determine and verify the interaction site. The NMR results, with assistance of molecular docking, indicate that BHMP07 preferentially binds to a site that is located between the RNH active site and the region encompassing helices B and D (the 'substrate-handle region'). The interaction site is consistent with the previous proposed site, identified using a chimeric RNH (p15-EC) [Gong et al. (2011) Chem Biol Drug Des 77, 39–47], but with slight differences that reflect the characteristics of the amino acid sequences in p15-EC compared to the WT RNH.
a trihydroxy acylhydrazide compound (24)
- Δδ
chemical shift perturbations (Hz)
sequence-averaged backbone chemical shift perturbation (Hz)
heteronuclear single-quantum correlated spectroscopy
protein – inhibitor equilibrium dissociation constant
- p15-EC
a catalytically active RNH chimeric construct (27)
HIV-1 RT ribonuclease H domain
reverse transcriptase
wild type
HIV-1 reverse transcriptase (RT) is a multifunctional enzyme with both DNA polymerase and ribonuclease H (RNH) activities. Approximately half of the current, clinically approved drugs for the treatment of HIV-1 infection target RT, specifically, the DNA polymerase activity of the enzyme (1–6). Like the DNA polymerase activity, RNH function is essential for viral replication and, thus, plays a crucial role in HIV-1 pathogenesis (7–10). Although effort has been devoted to developing RT-RNH inhibitors (11–19), currently, no clinically approved anti-AIDS drugs based on RNH inhibition are available. In part, this significant absence may be due to the fact that the structural basis of the RNH-inhibitor interaction is not well understood.
Recently, crystal structures of RNH active-site inhibitors in complex with RT or an RNH fragment (20–22) as well as an NMR study of an RNH inhibitor interaction at the active site (23) were published. In contrast to these studies, which were focused on inhibitors chelating with Mg2+ ions at the active site, we recently characterized several acylhydrazone inhibitors that exhibit moderate antiviral activity; one of these, BHMP07, was found to bind the RNH domain at a novel allosteric site (24). This conclusion was based on chemical shift changes of backbone amide NMR signals, in the presence of BHMP07 at various concentrations, demonstrating that BHMP07 primarily recognizes the RNH substrate-handle region encompassing helices B and D (25) (i.e., residues 503–527 in RT). This observation was supported by a recent molecular docking study of hydrazone/hydrazine compounds on RT (26).
The RNH fragment used in our previous NMR study was a chimeric protein, created by replacing a small loop segment of HIV-1 RT-RNH with a 24 residue α-helical substrate-binding loop derived from Escherichia coli RNase H. Although this construct, termed p15-EC, has been widely used to screen RNH inhibitors and characterize protein–inhibitor interactions (27–29), it is unclear whether inhibitor-binding sites obtained for the p15-EC construct are directly applicable to wild-type (WT) RT-RNH. Addressing this issue is of critical importance because little structural information is available about the interaction mechanism of RNH inhibitors with WT RNH.
In this article, we describe the interaction between BHMP07 and the WT RNH fragment, evaluated using NMR spectroscopy. With this method, monitoring of backbone chemical shift perturbations (Δδ) can provide atomic resolution information for weakly interacting protein-ligand complexes, over the course of a titration series, allowing for localization of the ligand-binding site. In general, magnitudes of chemical shift changes qualitatively correlate with the distance from the ligand. To gain insight into the allosteric inhibitor interaction with WT RNH, we monitored the impact of increasing concentrations of BHMP07 on both the monomeric WT RNH and a kinetically trapped dimer form. First, we determined that the dimer interface encompasses the substrate-handle region and does not influence Mg2+ interaction at the active site. Thus, if BHMP07 binds at (or close to) the substrate-handle region, it will not be able to bind the dimer form of RNH. Our data indicate that this is the case: BHMP07 interacted with monomeric WT RNH but not with the dimeric form. Next, we demonstrated that Mg2+ prevents binding to both the monomeric and dimeric forms. Based on these results, we conclude that BHMP07 interacts with the monomeric RNH in a way that involves residues near both the substrate-handle region and the active site. Thus, while involvement of the substrate-handle region is consistent with our previous study of p15-EC, overlap of the BHMP07 interaction site with areas affected by Mg2+ binding is more pronounced in the WT RNH compared to p15-EC (24). This shift of the binding site is not surprising, as it is concomitant with a smaller number of hydrophobic amino acid side chains in the substrate-handle region of WT RNH compared to the chimeric construct. Finally, computational docking of BHMP07 to an RNH domain structure revealed three potential binding sites, one of which is located between the substrate-handle region and the active site and is consistent with the NMR data.
Materials and Methods
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Conclusions
- Acknowledgments
- References
- Supporting Information
Sample preparation
We generated the isolated RNH domain (RT residues 427–560, with an additional N-terminal-peptide S–E–L) by expressing the domain in E. coli. In brief, the coding sequence was inserted into pET32a plasmid (EMD Chemicals Inc., San Diego, CA, USA) as a thioredoxin fusion construct that included a tobacco etch virus (TEV) protease recognition site at its C-terminus (30). For NMR experiments, the cells were cultured in modified M9 minimal media containing 15NH4Cl (1 g/L) as the sole nitrogen source in the absence or presence of [u-13C]-glucose as the sole carbon source (30). The protein was isolated from the cell lysate using a Hi-Trap SP ion-exchange column (GE Healthcare, Piscataway, NJ, USA) at pH 7.5, using a 0–1 m NaCl gradient, followed by further purification by size exclusion chromatography on a Superdex 200 26/60 column (GE Healthcare), using buffer containing 25 mm sodium phosphate buffer (pH 7.0), 0.02% sodium azide, 100 mm NaCl, and 1 mm DTT. The RNH protein was eluted at two positions (Fraction #1 and Fraction #2); both peaks were pooled and separately subjected to TEV digestion. Subsequently, each digested fraction was re-suspended in 20× volume of 25 mm Tris buffer at pH 8.5 containing 2 mm DTT and subjected to a HiTrap TMQ SP column. Purity of the two protein species were confirmed by running 15% acrylamide gels in both SDS denatured and non-denaturing (native) conditions (PhastGel system; GE Healthcare). The final eluted protein fractions (∼2–3 mg/mL) were stored separately in aliquots at −80 °C. To prevent confusion owing to the extra three residues in the N-terminus, in what follows, we report both the residue numbers reflecting the experimental construct used and, in parentheses, the RT numbering (see Figure S1).
Multi-angle static light scattering
Multi-angle static light scattering experiments were performed on a HELEOS instrument (Wyatt Technology, Santa Barbara, CA, USA) equipped with refractive index detectors (Optilab rEX; Wyatt Technology) and an analytical Superdex 200 column (1 × 30 cm; GE Healthcare), pre-equilibrated at room temperature with 25 mm sodium phosphate, 100 mm NaCl, 2 mm DTT, and 0.02% (w/v) sodium azide at pH 7.0. One hundred microlitres of thawed protein Fractions #1 and #2 (∼3 mg/mL) were separately injected at a flow rate of 0.5 mL/min and eluted with the same buffer.
NMR experiments
Thawed aliquots of WT RNH Fraction #1 and Fraction #2 were dialyzed into 25 mm sodium phosphate containing 100 mm sodium chloride and 2 mm DTT, pH 7.0, and the proteins were concentrated, typically to ∼200 μm, using Amicon Vivaspin concentrators (Millipore, Billerica, MA, USA). NMR samples were prepared by combining ∼380 μL protein solution with 20 μL D2O (to allow for deuterium locking) and stored in 5-mm NMR tubes susceptibility-matched to D2O (BMS-005B; Shigemi, Inc., Allison Park, PA, USA).
All NMR experiments were recorded at 20 °C on a Bruker Avance 600 spectrometer (Bruker BioSpin, Billerica, MA, USA) equipped with a 5-mm triple-resonance z-gradient cryogenic probe. Backbone 1H/15N signals were assigned via essentially the same approach used for the p15-EC RNH study (24) by recording HNCA, CBCACONH, and HNCACB experiments using 13C/15N-labeled protein at ∼500 μm concentration (31). To facilitate backbone assignment, a separate set of experiments was collected on a sample of 13C/15N-labeled protein at ∼500 μm concentration in the presence of 40 mm MgCl2. NMR spectra were processed and analyzed using NMRPipe, NMRview, and ccpnmr analysis 2.1.5 (32–34). After manual assignment of ∼75% of the sequence, existing spin systems were frozen, and further sequential links were identified with the aid of predictions generated independently by the PINE server 1.0 (35), and the Nexus automated assignment protocol that is included in the ccpnmr analysis software. A series of 1H-15N heteronuclear single-quantum correlated spectroscopy (HSQC) experiments were recorded at different Mg2+ concentrations (0, 5, 10, 15, and 20 mm) to identify the Mg2+ interaction sites. Similarly, a series of 1H-15N HSQC experiments were recorded at different concentrations of BHMP07 (inhibitor/protein ratios ranging from 0 to ∼1.5:1) to identify the inhibitor-interaction site in the presence and absence of 20 mm Mg2+. All titration series were conducted under the same conditions and each completed within a half day.
Combined chemical shift changes for each step i of the titration series are calculated as the quadratically weighed amplitudes of the 1H and 15N chemical shift differences:
- (1)
End-points for the titrations, although falling short of complete ligand saturation, were chosen because higher concentrations of the inhibitor solution (in DMSO) resulted in significant precipitation. Hence, the maximal amplitude of chemical shift changes is shown at the 1.5:1 ratio, to facilitate comparison between monomer and dimer and to minimize the contribution of unspecific-binding effects.
Determination of dissociation constants
Dissociation constants (KD) were derived from the chemical shift changes logged for discrete resonances by fitting the obtained titration curves individually via nonlinear regression analysis. Assuming single-site binding for a system in fast exchange, at each titration step i, the theoretical is given based on the total protein concentration [P]total and the current ligand concentration [L]i as (36):
- (2)
The unknown parameters, that is, KD and the extent of chemical shift change at saturation, Δδ∞ = δbound − δfree, were optimized by minimizing the difference between the experimentally measured Δδobs and the Δδcal for each titration curve, that is, minimizing the chi-squared function
- (3)
Uncertainties of the optimized parameters were estimated via Monte Carlo resampling, by generating 50 sets of synthetic data of Δδ with a Gaussian distribution whose standard deviation (σ) is given by an experimental chemical shift uncertainty, δnoise (37).
Computational docking experiments
Molecular docking calculations were carried out with the Schrödinger Suite 2011 programs. The structure of the wild-type RNH fragment of HIV-1 RT, previously determined by X-Ray crystallography (PDB access code 3KP2), was downloaded from the Protein Data Bank. To prepare structures for further docking calculations, hydrogen atom and hydrogen bond assignment, side chain ionization, and energy minimization were carried out with the Protein Preparation Wizard Protocol (Schrödinger Suite 2011 Protein Preparation Wizard; epik version 2.2, Schrödinger, LLC, New York, NY, 2011; impact version 5.7, Schrödinger, LLC, New York, NY, 2011; prime version 3.0, Schrödinger, LLC, New York, NY, 2011).
The geometry of BHMP07 was minimized with MacroModel (macromodel, version 9.9, Schrödinger, LLC, New York, NY, 2011), and then the ligand was prepared for docking by force field assignment and ionization states prediction (at target pH = 7.5 ± 1.0) by LigPrep application (ligprep, version 2.5, Schrödinger, LLC, New York, NY, 2011). Prepared structures were then used to generate conformers with confgen (version 2.3, Schrödinger, LLC, New York, NY, 2011) and conformers with Boltzmann populations >5% were taken into further docking. The receptor grid box was centered on the residues that showed the largest chemical shifts upon NMR binding (see below) and made to accommodate a ligand within 20 Å of length (i.e., the approximate length of the most extended BHMP07 conformer). The potential of nonpolar parts of the receptor was softened by applying a 0.8 scaling factor to van der Waals radii of atoms with partial charges <0.15 a.u. Docking computations were carried out using Glide XP in extra precision mode (glide, version 5.7, Schrödinger, LLC, New York, NY, 2011).
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Conclusions
- Acknowledgments
- References
- Supporting Information
Isolation of dimer and monomer fractions of RNH in solution
We previously synthesized and characterized acylhydrazones that inhibit RT RNH activity and exhibit moderate antiviral activity (24,38). To further characterize the binding mechanism of acylhydrazones to the RT RNH fragment, we prepared WT RNH by expressing the domain in E. coli. Interestingly, the domain was eluted in two different fractions from gel filtration column chromatography, henceforth referred to as Fraction #1 and #2. The protein fractions exhibited similar migration on SDS PAGE and the same molecular masses as determined by mass spectroscopy (15094.8 and 15094.7 Da for non-isotope labeled Fractions #1 and #2, respectively; data not shown), confirming that both fractions contain the same polypeptide. However, the gel filtration profiles of the two fractions and the associated molecular masses, determined by multi-angle light scattering equipped with a refractive index detector, revealed that Fraction #1 is predominantly a dimer (Figure 1). Consistent with this observation, native gel profiles showed different migration patterns for Fraction #1 and Fraction #2 (Figure 1, inset). 1H-15N HSQC spectra of the two fractions, despite differing in ∼20 distinct cross-peaks, exhibit overall similar resonance patterns, suggesting that the basic secondary and tertiary fold of the dimer is almost identical to that of the monomer (Figure 2A). No interconversion between the two forms was seen at 20 or 40 °C over 3 days. However, upon unfolding by adding concentrated urea and then re-folding, the biophysical characteristics of the protein in Fraction #1 became consistent with those of Fraction #2 (data not shown). Taken together, the data suggest that the RNH domain in Fraction #1 represents a kinetically trapped dimer. To our knowledge, no dimer of RNH in solution has been heretofore reported in the literature.
Assignment of NMR spectra
Using spectra recorded in the absence of metal ions or in the presence of 40 mm MgCl2, we were able to assign amide resonances for 83.5% of the RNH residues in Fraction #2 (total 111 residues among 133 backbone amide. Note that there are four Pro residues). This represents a significant increase in completeness over published assignments (39) and, notably, includes most residues in the C-terminal helix. Thus, we were able to assign the amide resonances of 118 residues in Fraction #1 and directly compare 109 chemical shifts between Fractions #1 and #2 (Figure 2).
Characterization of the dimer interface in solution
Based on the NMR signal assignments, significant backbone chemical shift differences (, i.e., greater than one standard deviation from the average response) in the dimer compared to the monomer were found to be restricted to a relatively few residues, including Glu55(478) [Δδ ∼ 83 Hz], Gln77(500) [Δδ ∼ 138 Hz], Ala79(502) [Δδ ∼ 49 Hz], Leu80(503) [Δδ ∼ 75 Hz], Ile82(505) [Δδ ∼ 113 Hz], Ile83(506) [Δδ ∼ 86 Hz], Ala85(508) [Δδ ∼ 116 Hz], Asp88(511) [Δδ ∼ 106 Hz], and Ala111(534) [Δδ ∼ 46 Hz] (Figure 2B). Thus, the interface helix is located at the substrate-handle region (residues 80–104 in our NMR sequence, see Figure S1 and Figure 3A). Crucially, this interface area is largely distinct from the active site (Figure 3A); this is confirmed by the fact that Mg2+ interacts with both, resulting in congruous effects in the monomer and dimer forms (Figure S2). Indeed, both amplitude (Figure S2, compare panels A and B) and spatial localization (Figure S2, compare panels C and D) of the chemical shift perturbation upon Mg2+ addition were almost identical for both protein forms.
Interestingly, the interface mapped by NMR spectroscopy coincides with one identified in a previously reported crystallographic dimer structure [PDB = 3K2P, (21)]. Indeed, mapping of the chemical shift differences between the monomer and dimer in solution presents a high degree of overlap with the interface in the previously reported crystallographic dimer (Figure 3B). It must, however, be emphasized that the kinetically trapped RNH dimer form in solution, discussed here, is distinct from the crystallographic dimer which solely reflects packing effects. While further structural studies would be needed for an in-depth characterization, we propose that this stable dimer may involve domain swapping at the interfacial α-helix in the substrate-handle region. Because such a domain swap would retain most of the secondary and tertiary structure of the monomer, it would explain why the pattern of NMR chemical shifts of the dimer was similar to that of monomer, with only 24 resonances significantly altered between the two structures (Figure 2A).
BHMP07 binds to the RNH monomer in the absence of Mg2+
Upon addition of BHMP07 to the RNH monomer, in absence of metal, a number of 1H-15N HSQC cross-peaks shifted monotonically with increasing ligand concentration, implying a fast exchange régime for the protein/ligand interaction (Figure S3A). The most strongly affected polypeptide backbone amide NH resonances were from residues located at the dimer interface, notably Glu55(478) [Δδ ∼ 47 Hz], Ser76(499) [Δδ ∼ 46 Hz] and Ala79(502) [Δδ ∼ 87 Hz]; or in the vicinity of the active site, such as Ala23(446) [Δδ ∼ 58 Hz], Asn24(447) [Δδ ∼ 33 Hz], Arg25(448) [Δδ ∼ 43 Hz], Gly30(453) [Δδ ∼ 47 Hz], Ala32(455) [Δδ ∼ 40 Hz] and Gly118(541) [Δδ ∼ 67 Hz]. Additional strong perturbations were observed for C-terminal residues, including Ile133(556) [Δδ ∼ 42 Hz], Lys135(558) [Δδ ∼ 48 Hz] and Val136(559) [Δδ ∼ 50 Hz] (Figure 4A). Overall, the sequence-averaged perturbation was ∼ 17.3 ± 15.4 Hz, the large σ reflecting the site-specificity of the binding interaction. The equilibrium dissociation constant (KD) between BHMP07 and RNH monomer was ∼ 49 ± 25 μm in the absence of Mg2+, determined by nonlinearly fitting the ligand titration profiles for 26 individual residues that exhibited significant changes compared to the average perturbation (i.e., , Table S1). It must be noted that although complete saturation could not be observed experimentally (vide supra), the measured titration profiles are clearly hyperbolic (Table S1 and Figure S3B). Here, the relatively small uncertainty of the KD, given as a standard deviation, indicates that the binding is specific and cooperative. Although this in vitro KD for the RNH fragment is rather large, it nevertheless compares favorably to the KD ∼ 142 ± 36 μm that was previously derived for the p15-EC RNH construct (24).
Mg2+ negatively impacts BHMP07 binding in the RNH monomer
Pre-addition of 20 mm MgCl2 to the RNH monomer resulted in significantly reduced perturbation effects during titration with BHMP07 (Figure S4A), with a sequence-averaged perturbation of ∼ 7.8 ± 5.8 Hz (Figure 4B) and with no residue exhibiting Δδ > 30 Hz. The minute amplitude of the observed chemical shift changes forms compelling evidence that, in essence, BHMP07 does not bind to RNH monomer in the presence of Mg2+. Furthermore, the observed small shifts are clearly independent of any conformational changes caused by the Mg2+ interaction: indeed, the Δδ for the latter interaction do not correlate with the perturbations induced by BHMP07 (Figure S2). Further inspection of chemical shift changes of the individual 1H (Figure S5) and 15N (Figure S6), chemical shift differences support these results. This result is in agreement with our previous study on p15-EC. However, it is rather idiosyncratic that the present distribution of small chemical shift perturbations closely mirrors the one recorded without Mg2+. Qualitatively, these tiny Δδs yield KD ∼ 53 ± 129 μm from nonlinear fitting of 15 curves (Figure S4B), a value that is similar to the one obtained in the absence of Mg2+. As interaction of Mg2+ with WT RNH is known to be weak (39), an appreciable population (∼5%) of metal-free RNH is expected to exist in the current condition. Hence, our tentative interpretation is that the observed BHMP07-induced Δδ in the presence of 20 mm Mg2+ (Figure 4B) were attributed to inhibitor interaction with the Mg2+-free form of RNH in solution.
BHMP07 does not specifically interact with the RNH dimer
As discussed above, the RNH dimer interface is located at the substrate-handle region (Figure 3). Indeed, chemical shift differences between the monomer and the dimer (Figure 2B) overlap with the shift perturbations of the monomer caused by BHMP07 interaction (Figure 4A). Thus, any interaction with BHMP07 involving the substrate-handle region would likely be impeded in the dimer form of RNH because of its reduced accessibility upon dimerization. To test this hypothesis, analogous titrations with BHMP07 were performed on the RNH dimer.
Clearly, the addition of BHMP07 to the dimer resulted in weaker chemical shift changes ( ∼ 10.2 ± 7.5 Hz) when compared to the monomer (Figure 4, compare panels A and C). In particular, most titration curves did not show a consistent hyperbolic profile but were rather erratic (not shown), implying that the corresponding Δδs were not indicative of a specific binding interaction. The significant reduction in chemical shift changes in the dimeric RNH demonstrates that the substrate-handle region is involved in the BHMP07 interaction. Pre-addition of Mg2+ did not lead to an increased BHMP07 perturbation for most residues either (Figure 4D), with the sequence-averaged perturbations being ∼9.8 ± 8.0 Hz. Thus, the above titration results using the RNH dimer verify that the substrate-handle region is involved in the BHMP07 interaction.
Molecular docking
The NMR data above indicate that BHMP07 interacts with the monomer RNH at a region near the active site and the substrate-handle region. Using this information, we carried out theoretical molecular docking studies. NMR chemical shift perturbations not only map direct binding effects but are also sensitive to indirect, long-range effects that may result from conformational changes. Thus, for our docking calculations, we designed a receptor grid to encompass residues with strongly perturbed chemical shifts upon BHMP07 interaction, namely Ala23(446), Asp24(447), Asn25(448), Gly30(453), Ala32(455), Ser76(499), and Ala79(502) (see Figure 4A). We did not include the residues at the loop or hinge regions that may undergo significant chemical shift changes when the torsion angles vary or slight conformational adjustments occur; for the same reason, we also did not include the residues at the C-terminus, which are dynamic in solution (39,41).
The docking results revealed three potential interaction sites: Site I involves predominantly the substrate-handle region; Site II is located in a region that bridges the substrate-handle and the active site regions; and Site III is located at the active site region (Figure 5A). Residues that interact with BHMP07 are explicitly indicated in Figure S7. Based on calculated docking scores BHMP07 shows strong affinity to Site I and II, while binding into Site III is less favorable. It can be argued that an interaction involving exclusively Site I would not fully satisfy the experimental NMR results as it cannot explain the observed perturbations near the active site. On the other hand, an interaction similarly dependent on Site III exclusively would not cause significant chemical shift changes in the substrate-handle region. By contrast, Site II is located proximal to the substrate-handle region and involves active site residues. Furthermore, inclusion of manganese ions at the active site prevents in silico docking of BHMP07 at Site II as a result of steric clash (data not shown), consistent with our NMR results that showed suppression of BHMP07 binding in the presence of Mg2+ (Figure 4).
The Site II position suggests that BHMP07 is partially sandwiched between two secondary structural units, β-strand 1 and the α-helix B. In this conformation, residues near BHMP07 include Asp20(443), Gly21(444), Ala24(445) in the β-strand 1; and Ser76(499), Gln77(500), Tyr(501), Ser76(499) in the α-helix B (Figures S7 and S8). These residues overlap with the residues included in the molecular docking [Ala23(446), Ala24(447), Asn25(448) in the β-strand 1; and Asp76(499), Ala79(502) in the α-helix B]. There is a slight difference between the NMR-observed Δδs and the BHMP07 interacting residues, presumably because chemical shift perturbations can also reflect indirect effects such as conformational shift induced by ligand interaction with residue side chains. In addition, since docking was performed into a rigid receptor, possible slight conformational changes of protein backbone and side chain orientations, which might be caused by ligand binding, were not taken into account. Overall, while the binding configurations of Site I and Site III may not be discarded out of hand, a BHMP07 interaction mechanism that predominantly involves Site II correlates the best with our NMR results.
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Conclusions
- Acknowledgments
- References
- Supporting Information
In the protein energy landscape, slight changes in the balance of energetics can result in significant effects in protein folding and molecular interaction (42–44). Thus, protein crystals may show inhibitors trapped at interaction sites in a manner different from that predicted by biochemical experiments, because the energetics in a crystal are different from those in solution. For example, in the crystal structure of RT with an N-acylhydrazone analog, the inhibitor DHBNH, while specifically affecting RNH function, was found to bind to the non-nucleoside RT inhibitor binding pocket in the polymerase domain and not to the RNH site (38). In another crystallographic study, the non-nucleoside inhibitor nevirapine was soaked into RT crystals to block the polymerase site and to allow an RNH active-site inhibitor to bind at the RNH site rather than the polymerase (22). Dynamic structural changes of RT in solution have been observed by computational and experimental studies (45–47). Inspection of inhibitor interactions using RNH in solution, as a complement strategy of the RT crystal, is, therefore, of paramount importance (24). However, in cases where the binding is weak, particularly if the inhibitor is not highly soluble in aqueous solution, structure determination of protein-inhibitor complexes by NMR becomes challenging. Accordingly, the present study focuses on a hybrid approach, combining NMR-binding site identification with computational prediction of the binding site. Because BHMP07 has moderate antiviral activity (24), understanding the mechanism of inhibition at an atomic level is critically important for future design of RT RNH inhibitors.
Our identification of a previously unreported dimeric form of RNH in solution is not surprising; indeed, proteins are not static entities and undergo various conformational fluctuations that can result in various quaternary structures, including oligomerization and domain swapping (48–55). For RNase H, although dimers have been detected during purification for some RNHs (56,57), so far, only crystallographic dimers have been observed. The dimer that we observed in solution most likely results from domain swapping at the helix region (Figure 3). Previously, the helix region was observed to be unstable in the folding intermediate of the RNH fragment (58), suggesting that the helix can be swapped during the folding process. Indeed, unfolding of the WT RNH monomer in 6 m urea followed by re-folding at very high-protein concentration in NMR buffer (>10 mm) yielded almost exclusively Fraction #1 (data not shown), in line with earlier observations for other domain swapped dimers (53).
Here, we demonstrate that BHMP07 binds to the monomer RNH but has little effect on the dimer (Figure 4), strongly supporting our earlier hypothesis that the substrate-handle region is involved in the inhibitor interaction (24). From combined in silico molecular docking results and in vitro NMR data, we predict that for WT RNH, the preferred BHMP07-binding site is located at the juncture between the active site and the substrate-handle region, that is, Site II (Figure 5B). This result, thus, refines our earlier predictions using p15-EC, in which BHMP07 was seen to bind to the substrate-handle region itself (24). Indeed, compared to the WT RNH, the p15-EC construct contains 24 additional residues, including three additional Trp, in the substrate-handle region. We speculate that these surplus hydrophobic residues distort the interaction site of p15-EC for BHMP07 and lead it to bind at the elongated substrate-binding handle. This hypothesis is supported by the fact that BHMP07 binds to WT RNH with a KD approximately threefold lower than the value obtained for p15-EC, demonstrating that the binding mode discussed in the present study is the more favorable of the two.
In striking contrast to RNH inhibitors that exhibit NMR chemical shift changes in the presence of Mg2+, we find that BHMP07 causes significant perturbation of RNH signals only in the absence of Mg2+. Consequently, the BHMP07-binding mode that we propose in this and our previous study is clearly different from that of RNH active-site inhibitors, which requires metal coordination (20–23). As revealed by the observed chemical shift changes (Figure 4), BHMP07 also structurally affects residues in areas remote from the likely binding site (Figure 5), clearly demonstrating the allosteric modulation effect on the structure. Combined with the fact that BHMP07 was previously shown to present moderate anti-HIV activity (24), knowing this allosteric mechanism is critically important to future design of RT RNH inhibitors.
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Conclusions
- Acknowledgments
- References
- Supporting Information
Although extensive screening has identified a variety of small molecular RNH inhibitors, little information about the structural basis of their interaction with RNH is known. Here, we report on a kinetically trapped dimer form of RNH in which the substrate-handle region provides the dimer interface. We also demonstrate that the substrate-handle region and the active site of a wild-type RNH fragment are conjointly involved in the interaction with an acylhydrazone inhibitor, BHMP07. Our results support the presence of an inhibitor-binding site between the substrate-handle region and the active site of RNH. This binding mechanism is significantly different from active site metal-mediated inhibitors, promoting an alternative line of attack for future RNH inhibitor design.
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Conclusions
- Acknowledgments
- References
- Supporting Information
This study was supported by grants from the National Institutes of Health (AI077424 to R.I. and M.A.P., and AI073975 to M.A.P.), the National Science Foundation (MCB 0814905 to R.I.), and funds from the University of Pittsburgh. We thank Wazo Myint and Maria DeLucia for technical assistance in this work and Teresa Brosenitsch for critical reading of the manuscript.
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Conclusions
- Acknowledgments
- References
- Supporting Information
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Supporting Information
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Conclusions
- Acknowledgments
- References
- Supporting Information
Figure S1. Explicit presentation of the amino acid sequence of the RT RNH fragment used in this study, i.e. residues 427–560, with an additional N-terminal peptide, S–E–L (underlined).
Figure S2. Chemical shift perturbations induced by Mg2+ on (A) monomer and (B) dimer RNH fractions, and their highlight on the ribbon structure presentations (C and D, respectively).
Figure S3. (A) Overlaid 1H-15N HSQC spectra of the RNH monomer recorded in absence (black) or presence of 0.2, 0.4, 0.8 and 1.6× BHMP07 (orange, blue, pink, and magenta, respectively). (B) Normalized chemical shift changes as a function of BHMP07 concentration for some representative residues, E55 (filled circle), A79 (filled triangle), L80 (filled squares), A85 (filled diamond), S92 (open circle), and S76 (open triangle).
Figure S4. (A) Overlaid 1H-15N HSQC spectra of the RNH monomer after pre-addition of 20 mM MgCl2 recorded in absence (black) or presence of 0.25, 0.5 and 1.5× BHMP07 (orange, light blue and magenta, respectively). (B) Normalized chemical shift changes as a function of BHMP07 concentration (same residues and representation as in Figure S3B).
Figure S5. Histogram of backbone amide proton resonance shifts induced in RNH monomer by (A) BHMP07 in absence of Mg2+, (B) BHMP07 in 20 mM MgCl2 and (C) 20 mM MgCl2 only.
Figure S6. Histogram of backbone amide nitrogen resonance shifts induced in RNH monomer by (A) BHMP07 in absence of Mg2+, (B) BHMP07 in 20 mM MgCl2 and (C) 20 mM MgCl2 only.
Figure S7. Ligand interaction diagrams for BHMP07 docked into RNH shown for each predicted binding site.
Figure S8. Per-residue interaction scores for BHMP07 bound to Site II in its best pose (highest docking score).
Table S1. Individual KD for the interaction of monomeric RNH with BHMP07.
|Supporting info item
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.


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Egypt's Press Review July 26: USD exchange rate reaches 13.25 EGP, CBE head to be blamed for $ crisis; Security agencies discover Sisi's attempted assassination
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Wrap-up of Egypt's top news July 25: 2 Thanaweya Amma students suicide after failure in exam; Assassination plot cancels Sisi's visit to Mauritania for Arab Summit; women 'negatively portrayed' in Ramadan drama
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Egypt's business digest July 25: EGX closes on collective rise Monday; EGP weakens on black market amid devaluation talk
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Egypt's sport digest July 25: Tunisian footballer Ali Maaloul arrives in Cairo to join al-Ahly; Algy officially signs deal to transfer Ramadan Sobhi to Stoke City
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Egypt's business digest July 25: Egypt to receive $1 bln from Saudi Arabia in few days; Egypt's 50% of investing companies see political instability causes slowdown in economic growth- global report
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Egypt's political digest July 25: Assassination plot cancels Sisi's visit to Mauritania; Pope Tawadros II says 37 attacks carried out on churches over three years
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Egypt's local news digest July 25: Thanaweya Amma student shot herself dead, another set himself on fire over failure in final exams
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Egypt's press review July 25: MP Emad Mahrous calls for granting Turkish cleric Fethullah Gulen asylum in Egypt
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Wrap-up of Egypt's top news July 24: Education minister approves Thanaweya Amma exam results
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SANTA CLARA – Just in case the 49ers defenders needed a reminder that their next opponent was going to provide perhaps the toughest test of the season, they got it on Monday night.
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American English: /ˈkɔrp(ə)rət/ British English: /ˈkɔːp(ə)rət/
Translation of corporate in Spanish:
- 1 1.1 (of a company)(headquarters/lawyer)corporate membership
de la empresa
de la compañíasuscripción (feminine) de empresathe corporate imagela imagen corporativahe is still at the bottom of the corporate ladder
la imagen de la empresa or compañíatodavía está en el primer peldaño del escalafón de la empresacorporate name corporate tax (in US)(impuesto a la renta pagado por sociedades)Example sentences1.2 (of big business)
- The previous year, the three men hired a corporate finance house to find a buyer for the company.
- Using debt to finance a corporate takeover is precisely the same as taking out a mortgage.
- It is understood that almost all major corporate finance houses have expressed an interest.
- 2 (joint, collective)(action/decision/responsibility)
Definition of corporate in:
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Media Newsletter
Tips, Trips, and Happenings from
Friends of Travel Classics Writers Conferences
American and Swiss Artists Featured at Fondation Beyeler in Basel
From 29 May through 4 September 2016, the Fondation Beyeler is presenting a comprehensive exhibition devoted to American artist Alexander Calder and Swiss artists Peter Fischli and David Weiss, who worked as partners under the name of Fischli/Weiss. See
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New Toboggan Track in Bern
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Special exhibition NRLA at Swiss Museum of Transport
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. Media contact:
Sibylle Gerardi
, +41 41 227 17 33.
New Rooms, Beach and BBQ at Tschuggen's Lakefront Hotel Eden Roc
Lakefront Hotel Eden Roc in Ascona will unveil 15 redesigned rooms this April, as well as a brand new white sand beach and barbecue station later this spring. Hotel Eden Roc's pergola, which is situated on Lago Maggiore, will feature fine-grained white sand, new lounge chairs, a fire pit grill station, and more. Creating a beach-chic vibe, guests can throw their own barbecue get-togethers and grill fresh ingredients, if they so choose. This will be coupled with complimentary Swiss BBQ workshops once per week courtesy of an Eden Roc Chef. Visit
. Media contact:
Gabriele Sappok
, 212-922 1961.
Free beer on the "Quöllfrisch Express" in Switzerland
A refreshing late summer season surprise awaits Appenzell Railways passengers in 2016. On four Sundays in September the Quöllfrisch Express will operate from Gossau to Wasserauen in eastern Switzerland, with passengers being offered a free first beer. But beer or no beer -- beautiful Appenzell is always worth a vacation visit, with its many scenic splendors between Lake Constance and Mount Säntis. Route included in the Swiss Travel Pass! Visit
. Media contact:
Ladina Wunderli
, 0041795939550.
Lake Como Four Ways
A boat excursion is a must during your visit to Grand Hotel Villa Serbelloni on Lake Como. For a different view, take the
Funicolare Como-Brunate
to the tiny village of Brunate for spectacular views and sightseeing. Looking for a more active experience? Take a kayak tour with
Bellagio Water Sports Kayak Club
and see historic architecture and gardens from a unique vantage point. For the truly adventurous, choose from canyoneering tours of varying lengths and levels of challenge with experienced guide Claudio Pozzi of
via jumps, slides, zip lines and rappelling. The Concierge team at Grand Hotel Villa Serbelloni can help you choose activities and arrange transportation. Visit
. Media contact:
Noel M. Carroll
, 914-961-0334.
The Hottest Digs at Sea: Family Travel Goes Next-Level
Highly-acclaimed CruiseCompete provides the latest about family suites and planning a great family cruise vacation. Comprehensive information about family accommodations & suites' provides families assistance in their cruise vacation planning. Lines featured include Princess, Holland America, Celebrity and Disney and overviews include sizes and such available features as sofa beds, butler service and Wi-Fi service. Visit
. Media contact:
Heidi Allison
, 248-651-4491.
Four New Hotels Join Small Luxury Hotels of the World
Small Luxury Hotels of the World (SLH) Small Luxury Hotels of the World is pleased to introduce four new hotels to join the luxury collection this March. From a glamorous retreat in the south of France and an art deco villa in Germany to a congregation of exclusive townhouses in Japan's northern Hokkaido Island, these hotels all have something unique and indulgent to offer the independently-minded traveler. Muse St Tropez, Kasara Niseko Village Townhouse, RIVA - Das Hotel Am Bodensee & Villa Franca. Visit
. Media contact:
Erin Woodward
, 917-864-57171.
Andaz Scottsdale Resort & Spa to Debut
Prepare to be swept away by Scottsdale's newest resort addition, Andaz Scottsdale Resort & Spa, slated to open in September 2016 near the base of Camelback Mountain. The midcentury modern property will include 201 bungalow-style guest rooms, a full service spa and salon, and more than 20,000 square feet of meeting space on 22 acres of land. Most excitingly, the property has announced a partnership with nearby artist compound Cattletrack, the longtime home and studio of some of the Southwest's most notable artists such as Philip Curtis and Fritz Scholder. For additional details, see
. Visit
. Media contact:
Laura McMurchie
, 480-429-2253.
Great Weather, Great Rodeos in Arizona
Summer in northern Arizona means great weather and great rodeos. From Payson's 132nd Annual Oldest Continuous Rodeo to the 129th Annual Prescott Frontier Days & World's Oldest Rodeo and from the Annual Show Low Deuces Wild Rodeo to the 104th Annual Apache County Round Valley Rodeo, professional cowboys descend upon Arizona each summer to compete to be the best. More at
. Visit
. Media contact:
Tony Alba
, 602-364-3715.
Fashion Week Comes to Tucson
Tucson Fashion Week will take place October 13-14, 2016. This year's exciting lineup includes a rock-and-roll fashion show curated by Cesar Padilla, owner of Cherry Vintage and author of "Ripped: T-shirts From the Underground" on Friday evening. Saturday evening's event will feature Richie Rich, iconic designer and creator of the fashion brand and cosmetic line Heatherette along with Shahida Parides, who returns to TFW as a featured runway designer. Visit
. Media contact:
Mary Rittmann
, 520-770-2127.
Snag a Spanish Summer Escape at Omni Scottsdale Resort
For travelers looking ahead to summer, guests are invited to take time to reconnect without reaching for their passport. Whether basking poolside in a private cabana, unwinding at Joya Spa, indulging in wine tasting and savory tapas at award-winning Prado or joining in on any one of the specialty weekend events, Omni Scottsdale Resort provides the perfect Spanish escape. The luxury resort's Summer Escapes Package includes two nights in luxurious accommodations with a third night free, in addition to exclusive offers throughout their stay. Visit
. Media contact:
James Bardwell
, 212-683-2442.
New Tennis, Golf and Spa Programs at The Boulders
The Boulders introduces new wellness programs that include "Tennis Yoga Mind & Moves," the "Golf & Fitness Combo" package and new autumn-inspired spa treatments that employ fresh products harvested from the Organic Garden, focusing on renewing skin damaged by the summer sun. Guests can also enjoy the new multi-million dollar renovations, the new waterfall rappelling and autumn desert romance packages. Visit
. Media contact:
Debi Bridges
, 480-306-4242.
Foodie-Cyclist Event in Whistler
The Bici Gusti Gourmet Ride was created for the insatiable love for gourmet food and cycling. Join an intimate group of 70 riders for an early season 70 km spin through the Whistler and Callaghan Valleys -- peppered with moderate climbs and garnished with flowing descents. Post-ride is an exquisite dinner created with ingredients within 70 km, this is the ride for you and your taste buds. See
. Visit
. Media contact:
Mary Zinck
, 604-938-2702.
Three New Arts Venues in Phoenix, Arizona
Phoenix adds three new properties to its portfolio channeling arts. The Hilton Garden Inn Phoenix Downtown opened in a historic architectural gem constructed in 1939 with art deco embellishments. The Founde:Re (opening Summer 2016) will reflect modern and local art just steps from Roosevelt Row Arts District. The Camby boasts "playful fun" with 5 C's of Arizona (Climate, Citrus, Cotton, Cattle & Copper). Visit
. Media contact:
Mariah Hurst
, 480-390-4213.
Victoria, B.C.'s Eagle Wing Tours Partners with First Nation on Marine Tour
Discover incredible local wildlife and learn about Aboriginal culture in Victoria, BC with Eagle Wing Tours on their new tour beginning in summer 2016. Developed in partnership with Songhees First Nation, this experience will give visitors the unique opportunity to learn from a Songhees Nation guide as they cruise the waters around their traditional territory while looking for local marine life. Half of the tour cost will go directly to the Songhees Nation to help revitalize the area and promote long-term sustainability for the people and ecosystems in Tl'chés. Visit
. Media contact:
Amanda Eyolfson
, 250-414-6998.
Babymoon Package Pampers Parents at Four Seasons Scottsdale
Make magical memories together with the new Hello Baby babymoon package at Four Seasons Resort Scottsdale at Troon North. The package includes a monogrammed baby onesie, decadent chocolate welcome amenity, and complimentary ice cream sundae in Proof. Guests also receive $100 credit for every two paid consecutive nights. While on property, enjoy complimentary guided hikes and stargazing to complement the experience. Visit
. Media contact:
Kim Cole
, 480-513-5049.
Italian-Inspired Indulgence Celebrated at Four Seasons Hualalai
La Dolce Vita, the annual Resort-wide celebration of the sweet life, returns to Four Seasons Resort Hualalai with a series of celebratory events, June 2-4, 2016. Co-hosted by Executive Chef Massimo Falsini and winemakers Steve and Chrystal Clifton (Palmina, La Voix), La Dolce Vita offers a variety of spa, retail and lifestyle experiences add to the weekend of Italian-inspired indulgence. Visit
. Media contact:
Brad Packer
, 310-634-2452.
New All-Inclusive Luxury Experience at Four Seasons Bora Bora
Four Seasons Resort Bora Bora announces the new, inclusive experience for guests of its seven beachfront villa estates. These accommodations range from one to three bedrooms, encompassing between 2,722 and 5,380 square feet. Each includes its own private plunge pool, expansive beachfront lawn, white sand beach and spectacular views. All Beachfront Villa Estate guests receive: airport transfers, all meals, a villa personal assistant, private beach set-up, private bar stocking and more. Visit
. Media contact:
Brad Packer
, 310-634-2452.
Wales Welcomes Roald Dahl 100 Celebrations Programme
A programme of events to celebrate the centenary of Welsh born author and the world's number one story teller, Roald Dahl in 2016. The Roald Dahl 100 celebrations Programme will include City of the Unexpected, a city-wide performance celebrating all things Dahl, produced by National Theatre Wales and Wales Millennium Centre. Visit
. Media contact:
Rob Jones
, +44 (0)300 061 6087.
New Victorinox Swiss Army Brand Store Opening in NYC's Rockefeller Center
This summer Victorinox Swiss Army will host a launch celebration for the company's new brand store in the heart of New York City's Rockefeller Center located at 620 Fifth Avenue. A one-stop-shop for everything Victorinox, the store will feature a wide selection of Swiss Army Knives, including new and U.S. exclusive designs, along with the company's most popular watch styles, apparel and travel accessories. Media contact:
Rachael Lyon
, 203-944-2348
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Child of Eden
Child of Eden is the sort of game that brings out the worst in reviewers. It's a stunning game - innovative, smart and genuinely beautiful, with dazzling abstract graphics and screen-filling blooms of neon light and colour. Both the visuals and the gameplay are ingeniously tied to a fabulous electronic soundtrack, and there's a wonder to the way each level unfolds, combining audio, video and motion control into a powerful experience of music, light and colour. This is all too much for us.
Suddenly, people who spend most of their working lives comparing games in which you shoot people in the face start waxing lyrical as if they actually spend their evenings hanging out in galleries and reading poetry before wandering down to their local repertory for an evening of early Russian non-narrative film.
Now, when someone starts waffling on about "synaesthesia" before describing Child of Eden as a life-changing work of art, we'd take it with a pinch of salt. That doesn't mean, however, that it is not an extraordinary game (or a fascinating work of art). More importantly for some of us, it is the best reason in a good 6 months to own an Xbox 360 with Kinect.
We won't go too far into the premise or the plot, for the simple reason that the game doesn't really need it. All you need to know is that, at some point in the dim and distant future, whatever's left of humanity has recreated a girl inside some super future version of the Internet - Eden - with the intention of restoring the lost memories of the human race. All looks good, as evinced by video clips of the aforementioned lovely wandering around a garden in diaphanous gown, but then some viruses pop up and spoil the party. It's up to you to destroy the viruses by means of flying on-rails through the different parts of Eden, blasting away at the abusive chunks of code. Some of you may find this inspirational and deeply moving, while others will chuckle and make sarcastic comments about haircare adverts. What matters is how it all plays out.
To be brutally honest, playing Child of Eden involves standing in front of the screen, waving your right hand to move a targeting cursor. When the cursor passes over viruses it selects them for targeting, at which point thrusting your palm forwards will release energy beams to blow them up. Of course, the viruses won't take this lying down. Some of them will even fire back. Luckily, by thrusting your left hand forwards you can stream out rapid-fire shots, destroying missiles and any orange-coloured critters in your sights. Shoot blue spheres and you regain some energy. Shoot orange spheres and you can build up euphoria - a screen-clearing smart bomb you can fire by waving both arms aloft (increasing the sense that you're out at an early-90s rave). That's pretty much it.
Well, maybe not. Like its spiritual ancestor, Rez, Child of Eden is as much a rhythm action game as a shooter. Sure, you can get through the levels just by blasting everything that moves, but the real trick is in timing your waves of target and release so that the sounds you make blasting tie in with the pulse of the soundtrack. Get it right and the big scores beckon and your star ratings improve. As stars are the only way to unlock the next level, this is a fairly big deal.
Now, all of this might sound simple and maybe a bit pretentious, but play Child of Eden on a decent-sized screen with the sound whacked up and the lights turned down, and it really can be amazing. The Kinect controls are - with some caveats - responsive, and there's something about your direct physical interaction with the game that takes the experience to a whole new level. Not wishing to labour a point, the graphics and music are astonishing. Beyond the first levels geometric forms and neon glows you'll find weird worlds of organic, plant-like shapes, hulking whales and swimming manta-rays, butterflies, flowers and soaring birds. Strange, mechanical cityscapes emerge and collapse, and rockets blast through tunnels on their way to some unknown destination. As the music surges, chills, pumps and soothes, each level reaches a climax of furious sound and colour. It's the kind of sensory overload that leaves you feeling good.
As a game, though, Child of Eden isn't quite perfect. Despite having only five worlds and an unlockable challenge mode, length isn't actually a deal-breaker. We'd hate to see the power of the game diluted by stretching out the running time, and there's plenty of replay value, with multiple rewards, visual effects, difficulty levels and achievements to unlock as you come back. However, there are moments where the Kinect controls struggle to keep up with the pace, particularly when you need to switch between normal attacks and rapid fire to stave off missiles. A secondary control system where you clap to switch modes can help, but limits your efficiency in later levels. What's more, there are sections with sudden changes of view, or where you need to change the view yourself by dragging the cursor to the edge of the screen. At these points the action feels a little stiff and out of control, leading to frustration.
The answer, of course, is to try harder difficulty levels with the conventional Xbox 360 controller, and effectively play Child of Eden like a score attack arcade game. This works extremely well, with the beat of the music replicated in the rumble of the controller, and will give Rez fans the trip they've been looking for for the last decade.
Child of Eden might not have the staying power of some triple A blockbusters, but it is an extraordinary game that anyone with Kinect ought to play. We'd take some of the adulation with a pinch of salt, but when the spectacular visuals, powerful soundtrack and immersive, motion-controlled gameplay all kick in at once, it's a dazzling, trippy experience you really need to sample for yourself.
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Starting off 2016 right!
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Look into my eyes and tell me what you see. Does it burn when I look at you like that? Does it? DOES IT?
Undocumented code is the root of all evil. That hardly counts. I mean you even have to multiply it with itself to get all evil. How bad can it be?
Testing is this thing that breaks your code. Don't be mean. Don't break code. Don't test things.
Don't write documentation. Write a program instead that implants the required knowledge into someone's brain instead. Don't write documentation for this program either, but make it run on every human and android you encounter.
Want to contact me about something? If you have an account on Wikipedia and verify your email-address, you can send me an email via Wikipedia.
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.... beautiful and unexpected stories
- About Us [+]Rockethouse is a small production company that makes radio, film and other delights. The company was founded by Matt Thompson and takes its name from the old building on the Scottish coast where he works. Below is the view from the office of the Bass Rock.
- News... [+]May 2016The Jungle Book an audiobook directed by Cherry Cookson for Amazon/Audible has won a prestigious prize in the USA. I did the sound design for it. http://www.audiofilemagazine.com/audies/?category=&winners=trueMay 2016Two lovely radio programmes made by Frances Byrnes at The Rockethouse have gone out recently on Radio 4.
The Sprung Floor
Dancer and choreographer Dane Hurst has bought a former Rambert Company vinyl dance floor to take back to his home in South Africa, for under-privileged children to dance on.
An Excellent Dumb Discourse: Shakespeare in Silence
Isn't silent Shakespeare an oxymoron? All that's nuanced, beautiful, meaningful in the poetry - silenced. Yet between 1899 and 1927, when the first commercial sound film was released, nearly three hundred Shakespeare films were made. Presented by Professor Judith Buchanan. Produced by Frances Byrnes
http://www.bbc.co.uk/programmes/b0773ld6March 2016Our first film 'No Hot Ashes' has been accepted for the Nashville Film Festival. https://nashvillefilmfestival.org/February 2016
Les Liaisons Dangereues. The Donmar Warehouse Production now downloadable as an audio play made possible by Audible Originals and Wireless Theatre. Rockethouse edited, designed sound and mastered the audio. We kept it minimal. Starring Dominic West, Janet McTeer and the original cast. You can get a copy for free HERE.January 2016
Film 2 in post production
Sinister starring Thom Tuck and Ishbel McFarlane is now being edited by Sue Moles. It will be less than 15' long and in B&W. Camera is Leo Bruges. 1st AD Sandy Harris. Sound Paul Chan. More credits and details to come.January 2016
'An Excellent Dumb Discourse': Shakespeare in Silence - BBC Radio 4 - Frances Byrnes in production with presenter Professor Judith Buchanan, University of York. 300 Shakespeare films were made in the silent era! Why? Isn't Shakespeare's power in his blank verse and big ideas? TX 17 April 2016
2015 - 2013
The Jungle Book - Audible/Amazon - Sound design for Cherry Cookson directed production by Wireless Theatre Company.
A Cold War dance - World Service andRadio 4 30 July 2015 - Martha Graham's dance company made a propaganda tour of SE Asia at the height of the Vietnam war in 1974. Presented and produced by Frances Byrnes
The Scrapbook: Memoirs of a Forensic Investigator -'Between The Ears' about a retied US Forensic Investigator Jack Sturiano - TX 30 May 2015 Radio 3 21.30
Skylarking and Portable Heroes of a Thousand Nights shortlisted for Prix Maurlic 2015
'Dialling Amok' by Sebastian Fitzek - Post production for a Cherry Cookson directed (Wireless Theatre) Audible audiobook.
Every BBC Radio 4 and World Service documentary produced by The Rockethouse in the last 15 months has been selected for BBC Radio 4's Pick of the Week.
Snow White and the Seven Signs of Ageing Radio 4 13 March 2015 About the Evil Queen and alternative ways to deal with ageing. Cathy FitzGerald presents/produces.
Woods Beyond a Cornfield Radio 4 23 Nov 2014 About a girl who goes missing. Poem by Stanley Cook. Frances Byrnes production.
Night Fishing Radio 4 02 Nov 2014 About the poet Tom Rawling and how he fished at night for sea trout. With Grevel Lindop and Finlay Wilson.
Paul is Dead Radio 4 14 Oct 2014 About the rumour that Paul McCartney had been killed in a car accident and replaced by an impostor.
How to Dig a Grave Radio 4 08 Oct 2014 About how to dig a grave. Cathy FitzGerald as presenter and producer.
Skylarking Radio 3 05 July 2014 About day dreaming of the sky when you are in it or looking at it. Cathy FitzGerald as presenter and producer. Music by Joe Acheson. Nominated for Prix Europa 2014.
Radio 3 Between the Ears about a retired US forensic investigator is in production.
No Hot Ashes - short film now in post production. Cinematography by Holger Mohaupt. Madeline Brolly and Sandy Grierson star.
Strawberry and Vanilla - sound mastering for film documentary about an ice cream seller. Co-directed by Tracey Fearnehough and Holger Mohaupt.
Semi - Recent
British Library Enduring War exhibition - sound design. (5.1 surround installation)
North Berwick Coastal Communities Museum - sound design
Immigrant cabbies in NYC for BBC World Service. Excellent reviews. Can be heard here: Yellow Cab Blues
Two intervals for BBC Radio 3. Stalking The Hun and Gramophones on the Front.
A Profile of Norwegian writer Per Petterson recorded in Oslo for a 45' Radio 3 with presenter David Szalay.
The First Action Movie with Penny Woolcock and produced by Frances Byrnes. The first ever action movie may well have been made in Sheffield in 1903.
Heart and Soul - A Place To Dwell BBC World Service Tx's 28/12/13 with Cathy FitzGerald. A Sikh Doctor in Harlem is attacked and forgives.
Sound restoration on BBC The Lost Tapes of Orson Welles. John Sugar production.
Nov 2013 Radio Productions Awards Indie of Year and Sound Designer nominations.
Oct 2013 Third Coast Award (Honourable mention) for No Greater Love.
Oct 2013 Prix Europa, Berlin Documentary 5th prize White Stiletto Dreams.
BBC Radio 4. Noise - A Human History - 30 parts - Written and presented by Professor David Hendy.
Complete series now available via
(NB AudioGo has closed)
Blog Here
Book Here
No Greater Love wins prize at Prix Maurilic 2013. Listen here.
Staying Bright - BBC Radio 3 tx's 21/04/2013 A history of Stainless Steel presented by poet Simon Armitage. Produced by Frances Byrnes
- A Scrapbook [+]
…. of images and quotes which I found inspiring.
Jean Renoir
Consistency requires you to be as ignorant today as you were a year ago. - Bernard Berenson
My movie is born first in my head, dies on paper; is resuscitated by the living persons and real objects I use, which are killed on film but, placed in a certain order and projected on to a screen, come to life again like flowers in water. - Robert Bresson
1930's photo of a Japanese street storyteller.


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© WBT - Thomas Blairon
- Address:
Avenue des Robiniers
1020 Brussels (BRUXELLES)
- MAPS: Google Maps
The area is not a playground as such, but it has been set up in a playful way which the children appreciate.
There is a labyrinth, designer outdoor furniture, greenhouses... anything can turn into a game.
These gardens are laid out in order to enhance the contemporary look of the garden's arts.
The park is also completely accessible to persons with reduced mobility and to prams!
This information was provided in collaboration with Bruxelles Environnement - IBGE
Visit our partners' webpages
- Galler Chocolates Grand-Place
- The Wellington Museum in Waterloo
- Musée Oldmasters Museum, Royal Museums of Fine Arts
- La Fonderie - Brussels Museum of Industry and Labour
- The Atomium - Monument and Symbol Built for Expo 58


text:
July 2, 2012
The End of the Affair, Graham Greene
Tactile pleasure of pint to paper.
Long time resident of Florida, and now a proud resident of North carolina. Involved in marketing and internet sales for businesses in screen printing supplies.
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The Michael and Son Sportsplex at Rockville is proud to offer sports surfaces and activities all year around for all ages. Check this page for our special weekly offers!
Click Here for field space available.
New ID's REQUIRED after 4/1. Click here for details.
Hoopstars Camp: Sign up for the full week of camp and receive 15% off. This promotion can not be combine with any other offers. Expires July 30, 2016.
What is the Rockville A&A Tax?


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Schultz Gareau Insurance Agency offers a wide range of insurance services to tackle all of your insurance needs. Some of the insurance services we offer are;More
$1 mil. - $5 mil.in Revenue
10 - 20EmployeesContact people at this company
1135 Lakewood Court NReginaSaskatchewanS4X 3S3Canadaview map
(306) 924-2212x24(306) 924-2465Fax
Enterprise_Iteration-NT.1545[05] SEIDXWK-31


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DEFINITION of 'Capital Tax'
A tax on a corporation's taxable capital, comprising capital stock, surpluses, indebtedness and reserves. Capital tax is applicable to capital owned by a company, not its spending. Capital taxes, in contrast to income taxes, are charged regardless of the profitability of the firm.
Also known as "corporation capital tax".
Next Up
BREAKING DOWN 'Capital Tax'
In British Columbia, the corporation capital tax (CCT) is a tax levied on financial corporations with a permanent establishment in British Columbia and net paid-up capital in excess of a minimum threshold amount. For the purposes of the CCT, a financial corporation is a bank, trust company, credit union or loan corporation.
Capital Gains TaxA type of tax levied on capital gains incurred by individuals ...
Tax RateThe percentage at which an individual or corporation is taxed. ...
Capital Gains TreatmentThe specific taxes assessed on investment capital gains as determined ...
Effective Tax RateThe average rate at which an individual or corporation is taxed. ...
Corporate TaxA levy placed on the profit of a firm, with different rates used ...
TaxesAn involuntary fee levied on corporations or individuals that ...
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