The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5′ end, the ribosomal frameshift segment and the 3′-untranslated region (3′-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.
Channelrhodopsin-2 from Chlamydomonas reinhardtii is a lightgated ion channel. Over recent years, this ion channel has attracted considerable interest because of its unparalleled role in optogenetic applications. However, despite considerable efforts, an understanding of how molecular events during the photocycle, including the retinal trans-cis isomerization and the deprotonation/reprotonation of the Schiff base, are coupled to the channel-opening mechanism remains elusive. To elucidate this question, changes of conformation and configuration of several photocycle and conducting/nonconducting states need to be determined at atomic resolution. Here, we show that such data can be obtained by solid-state NMR enhanced by dynamic nuclear polarization applied to 15 N-labeled channelrhodopsin-2 carrying 14,15-13 C 2 retinal reconstituted into lipid bilayers. In its dark state, a pure all-trans retinal conformation with a stretched C14-C15 bond and a significant out-of-plane twist of the H-C14-C15-H dihedral angle could be observed. Using a combination of illumination, freezing, and thermal relaxation procedures, a number of intermediate states was generated and analyzed by DNP-enhanced solid-state NMR. Three distinct intermediates could be analyzed with high structural resolution: the early P 500 1 K-like state, the slowly decaying late intermediate P 480 4 , and a third intermediate populated only under continuous illumination conditions. Our data provide novel insight into the photoactive site of channelrhodopsin-2 during the photocycle. They further show that DNP-enhanced solid-state NMR fills the gap for challenging membrane proteins between functional studies and X-ray-based structure analysis, which is required for resolving molecular mechanisms.ince their discovery (1), channelrhodopsins (ChRs) have generated enormous interest because of the rapid development of their applications in optogenetics (2-7). Commonly, ChR2 from Chlamydomonas reinhardtii (8) and its variants are used thanks to their favorable expression levels. They are the only proteins known today functioning as light-gated ion channels (Fig. 1A). Like other microbial retinal proteins, they undergo a periodic photocycle. In ChRs, this photocycle is coupled to channel opening and closing as revealed in electrophysiological recordings (8). A chimera of ChR1 and ChR2 has been crystallized to yield a structure at 2.3-Å resolution (9). However, little is known on how this coupling functions on a molecular level, and a large number of studies based on visible (10-13), IR (11,[14][15][16][17][18][19], resonance Raman spectroscopy (20, 21), and EPR spectroscopy (22, 23) has been performed to address this question.The photocycles of microbial rhodopsins are usually compared with bacteriorhodopsin, the first discovered and most studied lightdriven proton pump (24). Without any illumination, microbial retinal proteins thermally equilibrate into a dark state (25). In the case of bacteriorhodopsin, for example, this state contains a mixture of two species terme...
Protein phosphatases have very recently emerged as important targets for chemical biology and medicinal chemistry research, and new phosphatase inhibitor classes are in high demand. The underlying frameworks of natural products represent the evolutionarily selected fractions of chemical space explored by nature so far and meet the criteria of relevance to nature and biological prevalidation most crucial to inhibitor development. We refer to synthesis efforts and compound collection development based on these criteria as biology-oriented synthesis. For the discovery of phosphatase inhibitor classes by means of this approach, four natural product-derived or -inspired medium-sized compound collections were synthesized and investigated for inhibition of the tyrosine phosphatases VE-PTP, Shp-2, PTP1B, MptpA, and MptpB and the dual-specificity phosphatases Cdc25A and VHR. The screen yielded four unprecedented and selective phosphatase inhibitor classes for four phosphatases with high hit rates. For VE-PTP and MptpB the first inhibitors were discovered. These results demonstrate that biology-oriented synthesis is an efficient approach to the discovery of new compound classes for medicinal chemistry and chemical biology research that opens up new opportunities for the study of phosphatases, which may lead to the development of new drug candidates.chemical biology ͉ medicinal chemistry ͉ phosphatase inhibition P rotein phosphatases are key regulators of innumerable biological processes (1, 2). Small-molecule modulators of phosphatase activity have proven to be powerful tools for the study of the chemical biology of these enzymes (3), and, in particular, protein tyrosine phosphatases (PTPs) (4, 5) and dual-specificity phosphatases (6) have recently moved into the focus of a growing number of drug discovery programs, for instance in diabetes and anticancer research. However, although important progress has been made, the development of potent and selective phosphatase inhibitors is still in its early stages, and structurally new phosphatase inhibitor classes are in high demand.Relevance to nature is one of the most important criteria to be met by compound classes for chemical biology and medicinal chemistry research. The underlying frameworks of natural products (NPs) provide evolutionarily selected chemical structures encoding the properties required for binding to proteins, and their structural scaffolds represent the biologically relevant and prevalidated fractions of chemical space explored by nature so far (7-9). Consequently, it is to be expected that compound collections designed on the basis of NP structure will be enriched in biochemical and biological activity. Based on this reasoning, we have introduced a structural classification of natural products (SCONP) in a tree-like arrangement as an idea-and hypothesis-generating tool for the design and synthesis of compound collections (8). It permits the selection of library scaffolds based on relevance to and prevalidation by nature. We refer to synthesis efforts base...
Pin1 is a peptidyl-prolyl cis/trans isomerase (PPIase) essential for cell cycle regulation. Pin1-catalyzed peptidyl-prolyl isomerization provides a key conformational switch to activate phosphorylation sites with the common phospho-Ser/Thr-Pro sequence motif. This motif is ubiquitously exploited in cellular response to a variety of signals. Pin1 is able to bind phospho-Ser/Thr-Procontaining sequences at two different sites that compete for the same substrate. One binding site is located within the N-terminal WW domain, which is essential for protein targeting and localization. The other binding site is located in the C-terminal catalytic domain, which is structural homologous to the FK506-binding protein (FKBP) class of PPIases. A flexible linker of 12 residues connects the WW and catalytic domain. To characterize the structure and dynamics of full-length Pin1 in solution, high resolution NMR methods have been used to map the nature of interactions between the two domains of Pin1. In addition, the influence of target peptides on domain interactions has been investigated. The studies reveal a dynamic picture of the domain interactions.15 N spin relaxation data, differential chemical shift mapping, and residual dipolar coupling data indicate that Pin1 can either behave as two independent domains connected by the flexible linker or as a single intact domain with some amount of hinge bending motion depending on the sequence of the bound peptide. The functional importance of the modulation of relative domain flexibility in light of the multitude of interaction partners of Pin1 is discussed. Most peptidyl-prolyl cis/trans isomerases (PPIase)1 play an important role during protein folding by catalyzing the cis/ trans isomerization of peptidyl-prolyl imide bonds (1-4). Recently, with the discovery of the parvulin class of PPIases, peptidyl-prolyl isomerization has been identified as a key step in cell cycle regulation, oncogenesis, signal transduction, and a multitude of other cellular processes. Pin1 is the best-characterized PPIase of the parvulins and selectively catalyzes cis/ trans isomerization of peptide sequences containing the phospho-Ser/Pro or phospho-Thr/Pro motif (pS/T-P) (5-7). Lu et al.(5) have proposed that, in the presence of certain kinases, Pin1 participates in a "tag and twist" mechanism to activate its substrate: the kinase phosphorylates the serine or threonine side chain (tagging), and Pin1 subsequently cis/trans isomerizes (twists) the peptide bond preceding proline in a di-peptide pS/T-P, thus introducing a marked kink in the backbone of the peptide chain.Substrates of Pin1 include the mitotic regulators (Cdc25 phosphatase (8) and NIMA (9), PLK I, Wee, and Myt1 kinases); several transcription factors like -catenin, c-Jun, and the tumor suppressor protein p53 (10 -12); and some specific proteins like the RNA polymerase II, the cytoskeleton protein tau, and the G 1 /S protein cyclin D1 (13, 14).Pin1 is a two-domain protein of 18.4 kDa consisting of an N-terminal WW domain (Pin1 WW ) important for...
A molecule with an anisotropic magnetic susceptibility is spontaneously aligned in a static magnetic field. Alignment of such a molecule yields residual dipolar couplings and pseudocontact shifts. Lanthanide ions have recently been successfully used to provide an anisotropic magnetic susceptibility in target molecules either by replacing a calcium ion with a lanthanide ion in calcium-binding proteins or by attaching an EDTA derivative to a cysteine residue via a disulfide bond. Here we describe a novel enantiomerically pure EDTA derived tag that aligns stronger due to its shorter linker and does not suffer from stereochemical diversity upon lanthanide complexation. We observed residual (15)N,(1)H-dipolar couplings of up to 8 Hz at 800 MHz induced by a single alignment tensor from this tag.
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