Fragment-based identification of determinants of conformational and spectroscopic change at the ricin active site

<p>Abstract</p> <p>Background</p> <p>Ricin is a potent toxin and known bioterrorism threat with no available antidote. The ricin A-chain (RTA) acts enzymatically to cleave a specific adenine base from ribosomal RNA, thereby blocking translation. To understand better the relationship between ligand binding and RTA active site conformational change, we used a fragment-based approach to find a minimal set of bonding interactions able to induce rearrangements in critical side-chain positions.</p> <p>Results</p> <p>We found that the smallest ligand stabilizing an open conformer of the RTA active site pocket was an amide group, bound weakly by only a few hydrogen bonds to the protein. Complexes with small amide-containing molecules also revealed a switch in geometry from a parallel towards a splayed arrangement of an arginine-tryptophan cation-pi interaction that was associated with an increase and red-shift in tryptophan fluorescence upon ligand binding. Using the observed fluorescence signal, we determined the thermodynamic changes of adenine binding to the RTA active site, as well as the site-specific binding of urea. Urea binding had a favorable enthalpy change and unfavorable entropy change, with a ΔH of -13 ± 2 kJ/mol and a ΔS of -0.04 ± 0.01 kJ/(K*mol). The side-chain position of residue Tyr80 in a complex with adenine was found not to involve as large an overlap of rings with the purine as previously considered, suggesting a smaller role for aromatic stacking at the RTA active site.</p> <p>Conclusion</p> <p>We found that amide ligands can bind weakly but specifically to the ricin active site, producing significant shifts in positions of the critical active site residues Arg180 and Tyr80. These results indicate that fragment-based drug discovery methods are capable of identifying minimal bonding determinants of active-site side-chain rearrangements and the mechanistic origins of spectroscopic shifts. Our results suggest that tryptophan fluorescence provides a sensitive probe for the geometric relationship of arginine-tryptophan pairs, which often have significant roles in protein function. Using the unusual characteristics of the RTA system, we measured the still controversial thermodynamic changes of site-specific urea binding to a protein, results that are relevant to understanding the physical mechanisms of protein denaturation.</p

Structure of RiVax: a recombinant ricin vaccine

The X-ray crystal structure (at 2.1 Å resolution) of an immunogen under development as part of a ricin vaccine for humans is presented and structure-based analysis of the results was conducted with respect to related proteins and the known determinants for inducing or suppressing the protective immune response

Observation of an Excited Bc+ State

Using pp collision data corresponding to an integrated luminosity of 8.5 fb-1 recorded by the LHCb experiment at center-of-mass energies of s=7, 8, and 13 TeV, the observation of an excited Bc+ state in the Bc+π+π- invariant-mass spectrum is reported. The observed peak has a mass of 6841.2±0.6(stat)±0.1(syst)±0.8(Bc+) MeV/c2, where the last uncertainty is due to the limited knowledge of the Bc+ mass. It is consistent with expectations of the Bc∗(2S31)+ state reconstructed without the low-energy photon from the Bc∗(1S31)+→Bc+γ decay following Bc∗(2S31)+→Bc∗(1S31)+π+π-. A second state is seen with a global (local) statistical significance of 2.2σ (3.2σ) and a mass of 6872.1±1.3(stat)±0.1(syst)±0.8(Bc+) MeV/c2, and is consistent with the Bc(2S10)+ state. These mass measurements are the most precise to date

Embodied viewing and Degas’s Little Dancer Aged Fourteen: a multi-disciplinary experiment in eye-tracking and motion capture

This paper presents a cross-disciplinary project based on an experiment in eye-tracking and motion capture (Sainsbury’s Centre for Visual Arts), which aimed to study viewers’ movements around an iconic sculpture: Edgar Degas’s Little Dancer Aged Fourteen. The experiment studies how viewers respond to this three-dimensional artwork not only by looking at it but also through their own bodily reactions to it, such as by unconsciously mimicking a represented attitude or gesture. We compared two groups of viewers: classically trained dancers and non-dancers. Our hypothesis was that the skills and embodied experiences of the dancers would alter the ways in which they engage bodily with the work compared to the non-dancers. Our underlying research question was: how are vision and the body interlinked in esthetic and kinesthetic experience? This paper does not give results, which are forthcoming. It focuses on methodology and provides a commentary on the design and development of the interdisciplinary collaboration behind the project. It explores an interdisciplinary collaboration that bridges the humanities and experimental sciences and asks how being confronted with unfamiliar methodologies forces researchers in a given field to critically self-examine the limits and presuppositions of their practices

Bose-Einstein correlations of same-sign charged pions in the forward region in pp collisions at √s=7 TeV

Bose-Einstein correlations of same-sign charged pions, produced in protonproton collisions at a 7 TeV centre-of-mass energy, are studied using a data sample collected by the LHCb experiment. The signature for Bose-Einstein correlations is observed in the form of an enhancement of pairs of like-sign charged pions with small four-momentum difference squared. The charged-particle multiplicity dependence of the Bose-Einstein correlation parameters describing the correlation strength and the size of the emitting source is investigated, determining both the correlation radius and the chaoticity parameter. The measured correlation radius is found to increase as a function of increasing charged-particle multiplicity, while the chaoticity parameter is seen to decreas

Measurement of the inelastic pp cross-section at a centre-of-mass energy of 13TeV

The cross-section for inelastic proton-proton collisions at a centre-of-mass energy of 13TeV is measured with the LHCb detector. The fiducial cross-section for inelastic interactions producing at least one prompt long-lived charged particle with momentum p &gt; 2 GeV/c in the pseudorapidity range 2 &lt; η &lt; 5 is determined to be ϭ acc = 62:2 ± 0:2 ± 2:5mb. The first uncertainty is the intrinsic systematic uncertainty of the measurement, the second is due to the uncertainty on the integrated luminosity. The statistical uncertainty is negligible. Extrapolation to full phase space yields the total inelastic proton-proton cross-section ϭ inel = 75:4 ± 3:0 ± 4:5mb, where the first uncertainty is experimental and the second due to the extrapolation. An updated value of the inelastic cross-section at a centre-of-mass energy of 7TeV is also reported

Computer-Aided Lead Optimization: Improved Small-Molecule Inhibitor of the Zinc Endopeptidase of Botulinum Neurotoxin Serotype A

Optimization of a serotype-selective, small-molecule inhibitor of botulinum neurotoxin serotype A (BoNTA) endopeptidase is a formidable challenge because the enzyme-substrate interface is unusually large and the endopeptidase itself is a large, zinc-binding protein with a complex fold that is difficult to simulate computationally. We conducted multiple molecular dynamics simulations of the endopeptidase in complex with a previously described inhibitor (Kiapp of 7±2.4 µM) using the cationic dummy atom approach. Based on our computational results, we hypothesized that introducing a hydroxyl group to the inhibitor could improve its potency. Synthesis and testing of the hydroxyl-containing analog as a BoNTA endopeptidase inhibitor showed a twofold improvement in inhibitory potency (Kiapp of 3.8±0.8 µM) with a relatively small increase in molecular weight (16 Da). The results offer an improved template for further optimization of BoNTA endopeptidase inhibitors and demonstrate the effectiveness of the cationic dummy atom approach in the design and optimization of zinc protease inhibitors

Potent New Small-Molecule Inhibitor of Botulinum Neurotoxin Serotype A Endopeptidase Developed by Synthesis-Based Computer-Aided Molecular Design

Botulinum neurotoxin serotype A (BoNTA) causes a life-threatening neuroparalytic disease known as botulism. Current treatment for post exposure of BoNTA uses antibodies that are effective in neutralizing the extracellular toxin to prevent further intoxication but generally cannot rescue already intoxicated neurons. Effective small-molecule inhibitors of BoNTA endopeptidase (BoNTAe) are desirable because such inhibitors potentially can neutralize the intracellular BoNTA and offer complementary treatment for botulism. Previously we reported a serotype-selective, small-molecule BoNTAe inhibitor with a Kiapp value of 3.8±0.8 µM. This inhibitor was developed by lead identification using virtual screening followed by computer-aided optimization of a lead with an IC50 value of 100 µM. However, it was difficult to further improve the lead from micromolar to even high nanomolar potency due to the unusually large enzyme-substrate interface of BoNTAe. The enzyme-substrate interface area of 4,840 Å2 for BoNTAe is about four times larger than the typical protein-protein interface area of 750–1,500 Å2. Inhibitors must carry several functional groups to block the unusually large interface of BoNTAe, and syntheses of such inhibitors are therefore time-consuming and expensive. Herein we report the development of a serotype-selective, small-molecule, and competitive inhibitor of BoNTAe with a Ki value of 760±170 nM using synthesis-based computer-aided molecular design (SBCAMD). This new approach accounts the practicality and efficiency of inhibitor synthesis in addition to binding affinity and selectivity. We also report a three-dimensional model of BoNTAe in complex with the new inhibitor and the dynamics of the complex predicted by multiple molecular dynamics simulations, and discuss further structural optimization to achieve better in vivo efficacy in neutralizing BoNTA than those of our early micromolar leads. This work provides new insight into structural modification of known small-molecule BoNTAe inhibitors. It also demonstrates that SBCAMD is capable of improving potency of an inhibitor lead by nearly one order of magnitude, even for BoNTAe as one of the most challenging protein targets. The results are insightful for developing effective small-molecule inhibitors of protein targets with large active sites

Updated Determination of D⁰–D¯⁰Mixing and CP Violation Parameters with D⁰→K⁺π⁻ Decays

We report measurements of charm-mixing parameters based on the decay-time-dependent ratio of D⁰→K⁺π⁻ to D⁰→K⁻π⁺ rates. The analysis uses a data sample of proton-proton collisions corresponding to an integrated luminosity of 5.0  fb⁻¹ recorded by the LHCb experiment from 2011 through 2016. Assuming charge-parity (CP) symmetry, the mixing parameters are determined to be x′²=(3.9±2.7)×10⁻⁵, y′=(5.28±0.52)×10⁻³, and R[subscript D]=(3.454±0.031)×10⁻³. Without this assumption, the measurement is performed separately for D⁰ and D[over ¯]⁰ mesons, yielding a direct CP-violating asymmetry A[subscript D]=(-0.1±9.1)×10⁻³, and magnitude of the ratio of mixing parameters 1.00<|q/p|<1.35 at the 68.3% confidence level. All results include statistical and systematic uncertainties and improve significantly upon previous single-measurement determinations. No evidence for CP violation in charm mixing is observed

Observation of D⁰ Meson Decays to Π⁺π⁻μ⁺μ⁻ and K⁺K⁻μ⁺μ⁻ Final States

The first observation of the D⁰→π⁺π⁻μ⁺μ⁻ and D⁰→K⁺K⁻μ⁺μ⁻ decays is reported using a sample of proton-proton collisions collected by LHCb at a center-of-mass energy of 8 TeV, and corresponding to 2  fb⁻¹ of integrated luminosity. The corresponding branching fractions are measured using as normalization the decay D⁰→K⁻π⁺[μ⁺μ⁻][subscript ρ⁰/ω], where the two muons are consistent with coming from the decay of a ρ⁰ or ω meson. The results are B(D⁰→π⁺π⁻μ⁺μ⁻)=(9.64±0.48±0.51±0.97)×10⁻⁷ and B(D⁰→K⁺K⁻μ⁺μ⁻)=(1.54±0.27±0.09±0.16)×10⁻⁷, where the uncertainties are statistical, systematic, and due to the limited knowledge of the normalization branching fraction. The dependence of the branching fraction on the dimuon mass is also investigated