Influence of ground-state structure and Mg2+ binding on folding kinetics of the guanine-sensing riboswitch aptamer domain

Riboswitch RNAs fold into complex tertiary structures upon binding to their cognate ligand. Ligand recognition is accomplished by key residues in the binding pocket. In addition, it often crucially depends on the stability of peripheral structural elements. The ligand-bound complex of the guanine-sensing riboswitch from Bacillus subtilis, for example, is stabilized by extensive interactions between apical loop regions of the aptamer domain. Previously, we have shown that destabilization of this tertiary loop–loop interaction abrogates ligand binding of the G37A/C61U-mutant aptamer domain (Gswloop) in the absence of Mg2+. However, if Mg2+ is available, ligand-binding capability is restored by a population shift of the ground-state RNA ensemble toward RNA conformations with pre-formed loop–loop interactions. Here, we characterize the striking influence of long-range tertiary structure on RNA folding kinetics and on ligand-bound complex structure, both by X-ray crystallography and time-resolved NMR. The X-ray structure of the ligand-bound complex reveals that the global architecture is almost identical to the wild-type aptamer domain. The population of ligand-binding competent conformations in the ground-state ensemble of Gswloop is tunable through variation of the Mg2+ concentration. We quantitatively describe the influence of distinct Mg2+ concentrations on ligand-induced folding trajectories both by equilibrium and time-resolved NMR spectroscopy at single-residue resolution

Основні підходи до розроблення дизайну упаковки

Упаковка – останній призов, який бачить покупець, і останній шанс переконати його купити товар [1], тому над розробленням цікавого, оригінального дизайну упаковки працює ціла армія професіоналів. Дизайн упаковки включає гармонічну сукупність таких елементів, як: форма, матеріал, розміри, якість виготовлення, вид друку, кольори

Comprehensive Fragment Screening of the SARS-CoV-2 Proteome Explores Novel Chemical Space for Drug Development

12 pags., 4 figs., 3 tabs.SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti-virals. Within the international Covid19-NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR-detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure-based drug design against the SCoV2 proteome.Work at BMRZ is supported by the state of Hesse. Work in Covid19-NMR was supported by the Goethe Corona Funds, by the IWBEFRE-program 20007375 of state of Hesse, the DFG through CRC902: “Molecular Principles of RNA-based regulation.” and through infrastructure funds (project numbers: 277478796, 277479031, 392682309, 452632086, 70653611) and by European Union’s Horizon 2020 research and innovation program iNEXT-discovery under grant agreement No 871037. BY-COVID receives funding from the European Union’s Horizon Europe Research and Innovation Programme under grant agreement number 101046203. “INSPIRED” (MIS 5002550) project, implemented under the Action “Reinforcement of the Research and Innovation Infrastructure,” funded by the Operational Program “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and co-financed by Greece and the EU (European Regional Development Fund) and the FP7 REGPOT CT-2011-285950—“SEE-DRUG” project (purchase of UPAT’s 700 MHz NMR equipment). The support of the CERM/CIRMMP center of Instruct-ERIC is gratefully acknowledged. This work has been funded in part by a grant of the Italian Ministry of University and Research (FISR2020IP_02112, ID-COVID) and by Fondazione CR Firenze. A.S. is supported by the Deutsche Forschungsgemeinschaft [SFB902/B16, SCHL2062/2-1] and the Johanna Quandt Young Academy at Goethe [2019/AS01]. M.H. and C.F. thank SFB902 and the Stiftung Polytechnische Gesellschaft for the Scholarship. L.L. work was supported by the French National Research Agency (ANR, NMR-SCoV2-ORF8), the Fondation de la Recherche Médicale (FRM, NMR-SCoV2-ORF8), FINOVI and the IR-RMN-THC Fr3050 CNRS. Work at UConn Health was supported by grants from the US National Institutes of Health (R01 GM135592 to B.H., P41 GM111135 and R01 GM123249 to J.C.H.) and the US National Science Foundation (DBI 2030601 to J.C.H.). Latvian Council of Science Grant No. VPP-COVID-2020/1-0014. National Science Foundation EAGER MCB-2031269. This work was supported by the grant Krebsliga KFS-4903-08-2019 and SNF-311030_192646 to J.O. P.G. (ITMP) The EOSC Future project is co-funded by the European Union Horizon Programme call INFRAEOSC-03-2020—Grant Agreement Number 101017536. Open Access funding enabled and organized by Projekt DEALPeer reviewe

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form

Magnetization transfer to enhance NOE cross-peaks among labile protons: applications to imino–imino sequential walks in SARS-CoV-2-derived RNAs

2D NOESY plays a central role in structural NMR spectroscopy. We have recently discussed methods that rely on solvent-driven exchanges to enhance NOE correlations between exchangeable and non-exchangeable protons in nucleic acids. Such methods, however, fail when trying to establish connectivities within pools of labile protons. This study introduces an alternative that also enhances NOEs between such labile sites, based on encoding a priori selected peaks by selective saturations. The resulting selective magnetization transfer (SMT) experiment proves particularly useful for enhancing the imino–imino cross-peaks in RNAs, which is a first step in the NMR resolution of these structures. The origins of these enhancements are discussed, and their potential is demonstrated on RNA fragments derived from the genome of SARS-CoV-2, recorded with better sensitivity and an order of magnitude faster than conventional 2D counterparts

Synthesis and in vitro evaluation of novel 5-nitroindole derivatives as c-Myc G-quadruplex binders with anticancer activity

Lead-optimization strategies for compounds targeting c-Myc G-quadruplex (G4) DNA are being pursued to develop anticancer drugs. Here, we investigate the structure-activity- relationship (SAR) of a newly synthesized series of molecules based on the pyrrolidine-substituted 5-nitro indole scaffold to target G4 DNA. Our synthesized series allows modulation of flexible elements with a structurally preserved scaffold. Biological and biophysical analyses illustrate that substituted 5-nitroindole scaffolds bind to the c-Myc promoter G-quadruplex. These compounds downregulate c-Myc expression and induce cell-cycle arrest in the sub-G1/G1 phase in cancer cells. They further increase the concentration of intracellular reactive oxygen species. NMR spectra show that three of the newly synthesized compounds interact with the terminal G-quartets (5′- and 3′-ends) in a 2 : 1 stoichiometry

Disentangling the Coil: Modulation of Conformational and Dynamic Properties by Site-Directed Mutation in the Non-Native State of Hen Egg White Lysozyme

The conformational analysis of non-native states of proteins remains one of the most difficult problems in structural biology, because such states are represented by a superimposition of several states that are rapidly interconverting. Hence, model building of the conformational ensemble remains challenging, although many different biophysical observables can be determined in non-native states of proteins. Here, we present a comprehensive analysis of non-native states of wild-type and mutant forms of the model protein lysozyme by nuclear magnetic resonance spectroscopy. Relaxation rates, chemical shifts, backbone and side chain coupling constants, residual dipolar couplings, diffusion rate constants, and small-angle scattering data merged with computational approaches, such as <i>flexible meccano</i> and ASTEROIDS, allow the description of the non-native state of hen egg white lysozyme in unprecedented detail