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A plethora of modified nucleotides extends the chemical and conformational space for natural occurring RNAs. tRNAs constitute the class of RNAs with the highest modification rate. The extensive modification modulates their overall stability, the fidelity and efficiency of translation. However, the impact of nucleotide modifications on the local structural dynamics is not well characterized. Here we show that the incorporation of the modified nucleotides in tRNAfMet from Escherichia coli leads to an increase in the local conformational dynamics, ultimately resulting in the stabilization of the overall tertiary structure. Through analysis of the local dynamics by NMR spectroscopic methods we find that, although the overall thermal stability of the tRNA is higher for the modified molecule, the conformational fluctuations on the local level are increased in comparison to an unmodified tRNA. In consequence, the melting of individual base pairs in the unmodified tRNA is determined by high entropic penalties compared to the modified. Further, we find that the modifications lead to a stabilization of long-range interactions harmonizing the stability of the tRNA’s secondary and tertiary structure. Our results demonstrate that the increase in chemical space through introduction of modifications enables the population of otherwise inaccessible conformational substates.
Nuclear magnetic resonance (NMR) spectroscopy is a powerful and popular technique for probing the molecular structures, dynamics and chemical properties. However the conventional NMR spectroscopy is bottlenecked by its low sensitivity. Dynamic nuclear polarization (DNP) boosts NMR sensitivity by orders of magnitude and resolves this limitation. In liquid-state this revolutionizing technique has been restricted to a few specific non-biological model molecules in organic solvents. Here we show that the carbon polarization in small biological molecules, including carbohydrates and amino acids, can be enhanced sizably by in situ Overhauser DNP (ODNP) in water at room temperature and at high magnetic field. An observed connection between ODNP 13C enhancement factor and paramagnetic 13C NMR shift has led to the exploration of biologically relevant heterocyclic compound indole. The QM/MM MD simulation underscores the dynamics of intermolecular hydrogen bonds as the driving force for the scalar ODNP in a long-living radical-substrate complex. Our work reconciles results obtained by DNP spectroscopy, paramagnetic NMR and computational chemistry and provides new mechanistic insights into the high-field scalar ODNP.
Translational riboswitches are cis-acting RNA regulators that modulate the expression of genes during translation initiation. Their mechanism is considered as an RNA-only gene-regulatory system inducing a ligand-dependent shift of the population of functional ON- and OFF-states. The interaction of riboswitches with the translation machinery remained unexplored. For the adenine-sensing riboswitch from Vibrio vulnificus we show that ligand binding alone is not sufficient for switching to a translational ON-state but the interaction of the riboswitch with the 30S ribosome is indispensable. Only the synergy of binding of adenine and of 30S ribosome, in particular protein rS1, induces complete opening of the translation initiation region. Our investigation thus unravels the intricate dynamic network involving RNA regulator, ligand inducer and ribosome protein modulator during translation initiation.
1H, 13C, and 15N backbone chemical shift assignments of coronavirus-2 non-structural protein Nsp10
(2020)
The international Covid19-NMR consortium aims at the comprehensive spectroscopic characterization of SARS-CoV-2 RNA elements and proteins and will provide NMR chemical shift assignments of the molecular components of this virus. The SARS-CoV-2 genome encodes approximately 30 different proteins. Four of these proteins are involved in forming the viral envelope or in the packaging of the RNA genome and are therefore called structural proteins. The other proteins fulfill a variety of functions during the viral life cycle and comprise the so-called non-structural proteins (nsps). Here, we report the near-complete NMR resonance assignment for the backbone chemical shifts of the non-structural protein 10 (nsp10). Nsp10 is part of the viral replication-transcription complex (RTC). It aids in synthesizing and modifying the genomic and subgenomic RNAs. Via its interaction with nsp14, it ensures transcriptional fidelity of the RNA-dependent RNA polymerase, and through its stimulation of the methyltransferase activity of nsp16, it aids in synthesizing the RNA cap structures which protect the viral RNAs from being recognized by the innate immune system. Both of these functions can be potentially targeted by drugs. Our data will aid in performing additional NMR-based characterizations, and provide a basis for the identification of possible small molecule ligands interfering with nsp10 exerting its essential role in viral replication.
One current goal in native mass spectrometry is the assignment of binding affinities to noncovalent complexes. Here we introduce a novel implementation of the existing laser-induced liquid bead ion desorption (LILBID) mass spectrometry method: this new method, LILBID laser dissociation curves, assesses binding strengths quantitatively. In all LILBID applications, aqueous sample droplets are irradiated by 3 µm laser pulses. Variation of the laser energy transferred to the droplet during desorption affects the degree of complex dissociation. In LILBID laser dissociation curves, laser energy transfer is purposely varied, and a binding affinity is calculated from the resulting complex dissociation. A series of dsDNAs with different binding affinities was assessed using LILBID laser dissociation curves. The binding affinity results from the LILBID laser dissociation curves strongly correlated with the melting temperatures from UV melting curves and with dissociation constants from isothermal titration calorimetry, standard solution phase methods. LILBID laser dissociation curve data also showed good reproducibility and successfully predicted the melting temperatures and dissociation constants of three DNA sequences. LILBID laser dissociation curves are a promising native mass spectrometry binding affinity method, with reduced time and sample consumption compared to melting curves or titrations.
The Mycobacterium tuberculosis tyrosine-specific phosphatase MptpA and its cognate kinase PtkA are prospective targets for anti-tuberculosis drugs as they interact with the host defense response within the macrophages. Although both are structurally well-characterized, the functional mechanism regulating their activity remains poorly understood. Here, we investigate the effect of post-translational oxidation in regulating the function of MptpA. Treatment of MptpA with H2O2/NaHCO3, mimicking cellular oxidative stress conditions, leads to oxidation of the catalytic cysteine (C11) and to a conformational rearrangement of the phosphorylation loop (D-loop) by repositioning the conserved tyrosine 128 (Y128) and generating a temporarily inactive preclosed state of the phosphatase. Thus, the catalytic cysteine in the P-loop acts as a redox switch and regulates the phosphatase activity of MptpA.
The p53 protein family is the most studied protein family of all. Sequence analysis and structure determination have revealed a high
similarity of crucial domains between p53, p63 and p73. Functional studies, however, have shown a wide variety of different tasks in
tumor suppression, quality control and development. Here we review the structure and organization of the individual domains of
p63 and p73, the interaction of these domains in the context of full-length proteins and discuss the evolutionary origin of this
protein family.
FACTS:
● Distinct physiological roles/functions are performed by specific isoforms.
● The non-divided transactivation domain of p63 has a constitutively high activity while the transactivation domains of p53/p73
are divided into two subdomains that are regulated by phosphorylation.
● Mdm2 binds to all three family members but ubiquitinates only p53.
● TAp63α forms an autoinhibited dimeric state while all other vertebrate p53 family isoforms are constitutively tetrameric.
● The oligomerization domain of p63 and p73 contain an additional helix that is necessary for stabilizing the tetrameric states.
During evolution this helix got lost independently in different phylogenetic branches, while the DNA binding domain became
destabilized and the transactivation domain split into two subdomains.
OPEN QUESTIONS:
● Is the autoinhibitory mechanism of mammalian TAp63α conserved in p53 proteins of invertebrates that have the same function
of genomic quality control in germ cells?
● What is the physiological function of the p63/p73 SAM domains?
● Do the short isoforms of p63 and p73 have physiological functions?
● What are the roles of the N-terminal elongated TAp63 isoforms, TA* and GTA?
tRNAs are L-shaped RNA molecules of ~ 80 nucleotides that are responsible for decoding the mRNA and for the incorporation of the correct amino acid into the growing peptidyl-chain at the ribosome. They occur in all kingdoms of life and both their functions, and their structure are highly conserved. The L-shaped tertiary structure is based on a cloverleaf-like secondary structure that consists of four base paired stems connected by three to four loops. The anticodon base triplet, which is complementary to the sequence of the mRNA, resides in the anticodon loop whereas the amino acid is attached to the sequence CCA at the 3′-terminus of the molecule. tRNAs exhibit very stable secondary and tertiary structures and contain up to 10% modified nucleotides. However, their structure and function can also be maintained in the absence of nucleotide modifications. Here, we present the assignments of nucleobase resonances of the non-modified 77 nt tRNAIle from the gram-negative bacterium Escherichia coli. We obtained assignments for all imino resonances visible in the spectra of the tRNA as well as for additional exchangeable and non-exchangeable protons and for heteronuclei of the nucleobases. Based on these assignments we could determine the chemical shift differences between modified and non-modified tRNAIle as a first step towards the analysis of the effect of nucleotide modifications on tRNA’s structure and dynamics.
During evolution of an RNA world, the development of enzymatic function was essential. Such enzymatic function was linked to RNA sequences capable of adopting specific RNA folds that possess catalytic pockets to promote catalysis. Within this primordial RNA world, initially evolved self-replicating ribozymes presumably mutated to ribozymes with new functions. Schultes and Bartel (Science 2000, 289, 448–452) investigated such conversion from one ribozyme to a new ribozyme with distinctly different catalytic functions. Within a neutral network that linked these two prototype ribozymes, a single RNA chain could be identified that exhibited both enzymatic functions. As commented by Schultes and Bartel, this system possessing one sequence with two enzymatic functions serves as a paradigm for an evolutionary system that allows neutral drifts by stepwise mutation from one ribozyme into a different ribozyme without loss of intermittent function. Here, we investigated this complex functional diversification of ancestral ribozymes by analyzing several RNA sequences within this neutral network between two ribozymes with class III ligase activity and with self-cleavage reactivity. We utilized rapid RNA sample preparation for NMR spectroscopic studies together with SHAPE analysis and in-line probing to characterize secondary structure changes within the neutral network. Our investigations allowed delineation of the secondary structure space and by comparison with the previously determined catalytic function allowed correlation of the structure-function relation of ribozyme function in this neutral network.
Polo-like kinase 1 (PLK1) is a crucial regulator of cell cycle progression. It is established that the activation of PLK1 depends on the coordinated action of Aurora-A and Bora. Nevertheless, very little is known about the spatiotemporal regulation of PLK1 during G2, specifically, the mechanisms that keep cytoplasmic PLK1 inactive until shortly before mitosis onset. Here, we describe PLK1 dimerization as a new mechanism that controls PLK1 activation. During the early G2 phase, Bora supports transient PLK1 dimerization, thus fine-tuning the timely regulated activation of PLK1 and modulating its nuclear entry. At late G2, the phosphorylation of T210 by Aurora-A triggers dimer dissociation and generates active PLK1 monomers that support entry into mitosis. Interfering with this critical PLK1 dimer/monomer switch prevents the association of PLK1 with importins, limiting its nuclear shuttling, and causes nuclear PLK1 mislocalization during the G2-M transition. Our results suggest a novel conformational space for the design of a new generation of PLK1 inhibitors.
Riboswitches are regulatory RNA elements that undergo functionally important allosteric conformational switching upon binding of specific ligands. The here investigated guanidine-II riboswitch binds the small cation, guanidinium, and forms a kissing loop-loop interaction between its P1 and P2 hairpins. We investigated the structural changes to support previous studies regarding the binding mechanism. Using NMR spectroscopy, we confirmed the structure as observed in crystal structures and we characterized the kissing loop interaction upon addition of Mg2+ and ligand for the riboswitch aptamer from Escherichia coli. We further investigated closely related mutant constructs providing further insight into functional differences between the two (different) hairpins P1 and P2. Formation of intermolecular interactions were probed by small-angle X-ray scattering (SAXS) and NMR DOSY data. All data are consistent and show the formation of oligomeric states of the riboswitch induced by Mg2+ and ligand binding.
1H, 13C and 15N chemical shift assignment of the stem-loops 5b + c from the 5′-UTR of SARS-CoV-2
(2022)
The ongoing pandemic of the respiratory disease COVID-19 is caused by the SARS-CoV-2 (SCoV2) virus. SCoV2 is a member of the Betacoronavirus genus. The 30 kb positive sense, single stranded RNA genome of SCoV2 features 5′- and 3′-genomic ends that are highly conserved among Betacoronaviruses. These genomic ends contain structured cis-acting RNA elements, which are involved in the regulation of viral replication and translation. Structural information about these potential antiviral drug targets supports the development of novel classes of therapeutics against COVID-19. The highly conserved branched stem-loop 5 (SL5) found within the 5′-untranslated region (5′-UTR) consists of a basal stem and three stem-loops, namely SL5a, SL5b and SL5c. Both, SL5a and SL5b feature a 5′-UUUCGU-3′ hexaloop that is also found among Alphacoronaviruses. Here, we report the extensive 1H, 13C and 15N resonance assignment of the 37 nucleotides (nts) long sequence spanning SL5b and SL5c (SL5b + c), as basis for further in-depth structural studies by solution NMR spectroscopy.
Herein, we present a multi-cycle chemoenzymatic synthesis of modified RNA with simplified solid-phase handling to overcome size limitations of RNA synthesis. It combines the advantages of classical chemical solid-phase synthesis and enzymatic synthesis using magnetic streptavidin beads and biotinylated RNA. Successful introduction of light-controllable RNA nucleotides into the tRNAMet sequence was confirmed by gel electrophoresis and mass spectrometry. The methods tolerate modifications in the RNA phosphodiester backbone and allow introductions of photocaged and photoswitchable nucleotides as well as photocleavable strand breaks and fluorophores.
G-quadruplexes (G4), found in numerous places within the human genome, are involved in essential processes of cell regulation. Chromosomal DNA G4s are involved for example, in replication and transcription as first steps of gene expression. Hence, they influence a plethora of downstream processes. G4s possess an intricate structure that differs from canonical B-form DNA. Identical DNA G4 sequences can adopt multiple long-lived conformations, a phenomenon known as G4 polymorphism. A detailed understanding of the molecular mechanisms that drive G4 folding is essential to understand their ambivalent regulatory roles. Disentangling the inherent dynamic and polymorphic nature of G4 structures thus is key to unravel their biological functions and make them amenable as molecular targets in novel therapeutic approaches. We here review recent experimental approaches to monitor G4 folding and discuss structural aspects for possible folding pathways. Substantial progress in the understanding of G4 folding within the recent years now allows drawing comprehensive models of the complex folding energy landscape of G4s that we herein evaluate based on computational and experimental evidence.
Specialized surveillance mechanisms are essential to maintain the genetic integrity of germ cells, which are not only the source of all somatic cells but also of the germ cells of the next generation. DNA damage and chromosomal aberrations are, therefore, not only detrimental for the individual but affect the entire species. In oocytes, the surveillance of the structural integrity of the DNA is maintained by the p53 family member TAp63α. The TAp63α protein is highly expressed in a closed and inactive state and gets activated to the open conformation upon the detection of DNA damage, in particular DNA double-strand breaks. To understand the cellular response to DNA damage that leads to the TAp63α triggered oocyte death we have investigated the RNA transcriptome of oocytes following irradiation at different time points. The analysis shows enhanced expression of pro-apoptotic and typical p53 target genes such as CDKn1a or Mdm2, concomitant with the activation of TAp63α. While DNA repair genes are not upregulated, inflammation-related genes become transcribed when apoptosis is initiated by activation of STAT transcription factors. Furthermore, comparison with the transcriptional profile of the ΔNp63α isoform from other studies shows only a minimal overlap, suggesting distinct regulatory programs of different p63 isoforms.
The function of the p53 transcription factor family is dependent on several folded domains. In addition to a DNA-binding domain, members of this family contain an oligomerization domain. p63 and p73 also contain a C-terminal Sterile α-motif domain. Inhibition of most transcription factors is difficult as most of them lack deep pockets that can be targeted by small organic molecules. Genetic knock-out procedures are powerful in identifying the overall function of a protein, but they do not easily allow one to investigate roles of individual domains. Here we describe the characterization of Designed Ankyrin Repeat Proteins (DARPins) that were selected as tight binders against all folded domains of p63. We determine binding affinities as well as specificities within the p53 protein family and show that DARPins can be used as intracellular inhibitors for the modulation of transcriptional activity. By selectively inhibiting DNA binding of the ΔNp63α isoform that competes with p53 for the same promoter sites, we show that p53 can be reactivated. We further show that inhibiting the DNA binding activity stabilizes p63, thus providing evidence for a transcriptionally regulated negative feedback loop. Furthermore, the ability of DARPins to bind to the DNA-binding domain and the Sterile α-motif domain within the dimeric-only and DNA-binding incompetent conformation of TAp63α suggests a high structural plasticity within this special conformation. In addition, the developed DARPins can also be used to specifically detect p63 in cell culture and in primary tissue and thus constitute a very versatile research tool for studying the function of p63.
NMR structure calculation using NOE-derived distance restraints requires a considerable number of assignments of both backbone and sidechains resonances, often difficult or impossible to get for large or complex proteins. Pseudocontact shifts (PCSs) also play a well-established role in NMR protein structure calculation, usually to augment existing structural, mostly NOE-derived, information. Existing refinement protocols using PCSs usually either require a sizeable number of sidechain assignments or are complemented by other experimental restraints. Here, we present an automated iterative procedure to perform backbone protein structure refinements requiring only a limited amount of backbone amide PCSs. Already known structural features from a starting homology model, in this case modules of repeat proteins, are framed into a scaffold that is subsequently refined by experimental PCSs. The method produces reliable indicators that can be monitored to judge about the performance. We applied it to a system in which sidechain assignments are hardly possible, designed Armadillo repeat proteins (dArmRPs), and we calculated the solution NMR structure of YM4A, a dArmRP containing four sequence-identical internal modules, obtaining high convergence to a single structure. We suggest that this approach is particularly useful when approximate folds are known from other techniques, such as X-ray crystallography, while avoiding inherent artefacts due to, for instance, crystal packing.
The ribosomal S1 protein (rS1) is indispensable for translation initiation in Gram-negative bacteria. rS1 is a multidomain protein that acts as an RNA chaperone and ensures that mRNAs can bind the ribosome in a single-stranded conformation, which could be related to fast recognition. Although many ribosome structures were solved in recent years, a high-resolution structure of a two-domain mRNA-binding competent rS1 construct is not yet available. Here, we present the NMR solution structure of the minimal mRNA-binding fragment of Vibrio Vulnificus rS1 containing the domains D3 and D4. Both domains are homologues and adapt an oligonucleotide-binding fold (OB fold) motif. NMR titration experiments reveal that recognition of miscellaneous mRNAs occurs via a continuous interaction surface to one side of these structurally linked domains. Using a novel paramagnetic relaxation enhancement (PRE) approach and exploring different spin-labeling positions within RNA, we were able to track the location and determine the orientation of the RNA in the rS1–D34 bound form. Our investigations show that paramagnetically labeled RNAs, spiked into unmodified RNA, can be used as a molecular ruler to provide structural information on protein-RNA complexes. The dynamic interaction occurs on a defined binding groove spanning both domains with identical β2-β3-β5 interfaces. Evidently, the 3′-ends of the cis-acting RNAs are positioned in the direction of the N-terminus of the rS1 protein, thus towards the 30S binding site and adopt a conformation required for translation initiation.
The SARS-CoV-2 genome encodes for approximately 30 proteins. Within the international project COVID19-NMR, we distribute the spectroscopic analysis of the viral proteins and RNA. Here, we report NMR chemical shift assignments for the protein Nsp3b, a domain of Nsp3. The 217-kDa large Nsp3 protein contains multiple structurally independent, yet functionally related domains including the viral papain-like protease and Nsp3b, a macrodomain (MD). In general, the MDs of SARS-CoV and MERS-CoV were suggested to play a key role in viral replication by modulating the immune response of the host. The MDs are structurally conserved. They most likely remove ADP-ribose, a common posttranslational modification, from protein side chains. This de-ADP ribosylating function has potentially evolved to protect the virus from the anti-viral ADP-ribosylation catalyzed by poly-ADP-ribose polymerases (PARPs), which in turn are triggered by pathogen-associated sensing of the host immune system. This renders the SARS-CoV-2 Nsp3b a highly relevant drug target in the viral replication process. We here report the near-complete NMR backbone resonance assignment (1H, 13C, 15N) of the putative Nsp3b MD in its apo form and in complex with ADP-ribose. Furthermore, we derive the secondary structure of Nsp3b in solution. In addition, 15N-relaxation data suggest an ordered, rigid core of the MD structure. These data will provide a basis for NMR investigations targeted at obtaining small-molecule inhibitors interfering with the catalytic activity of Nsp3b.
The desensitized channelrhodopsin-2 photointermediate contains 13 -cis, 15 -syn retinal Schiff base
(2021)
Channelrhodopsin-2 (ChR2) is a light-gated cation channel and was used to lay the foundations of optogenetics. Its dark state X-ray structure has been determined in 2017 for the wild-type, which is the prototype for all other ChR variants. However, the mechanistic understanding of the channel function is still incomplete in terms of structural changes after photon absorption by the retinal chromophore and in the framework of functional models. Hence, detailed information needs to be collected on the dark state as well as on the different photointermediates. For ChR2 detailed knowledge on the chromophore configuration in the different states is still missing and a consensus has not been achieved. Using DNP-enhanced solid-state MAS NMR spectroscopy on proteoliposome samples, we unambiguously determined the chromophore configuration in the desensitized state, and we show that this state occurs towards the end of the photocycle.
Polymorphic G-quadruplex (G4) secondary DNA structures have received increasing attention in medicinal chemistry owing to their key involvement in the regulation of the maintenance of genomic stability, telomere length homeostasis and transcription of important proto-oncogenes. Different classes of G4 ligands have been developed for the potential treatment of several human diseases. Among them, the carbazole scaffold with appropriate side chain appendages has attracted much interest for designing G4 ligands. Because of its large and rigid π-conjugation system and ease of functionalization at three different positions, a variety of carbazole derivatives have been synthesized from various natural or synthetic sources for potential applications in G4-based therapeutics and biosensors. Herein, we provide an updated close-up of the literatures on carbazole-based G4 ligands with particular focus given on their detailed binding insights studied by NMR spectroscopy. The structure-activity relationships and the opportunities and challenges of their potential applications as biosensors and therapeutics are also discussed. This review will provide an overall picture of carbazole ligands with remarkable G4 topological preference, fluorescence properties and significant bioactivity; portraying carbazole as a very promising scaffold for assembling G4 ligands with a range of novel functional applications.
SARS-CoV-2 contains a positive single-stranded RNA genome of approximately 30 000 nucleotides. Within this genome, 15 RNA elements were identified as conserved between SARS-CoV and SARS-CoV-2. By nuclear magnetic resonance (NMR) spectroscopy, we previously determined that these elements fold independently, in line with data from in vivo and ex-vivo structural probing experiments. These elements contain non-base-paired regions that potentially harbor ligand-binding pockets. Here, we performed an NMR-based screening of a poised fragment library of 768 compounds for binding to these RNAs, employing three different 1H-based 1D NMR binding assays. The screening identified common as well as RNA-element specific hits. The results allow selection of the most promising of the 15 RNA elements as putative drug targets. Based on the identified hits, we derive key functional units and groups in ligands for effective targeting of the RNA of SARS-CoV-2.
The bacteriophage ΦX174 causes large pore formation in Escherichia coli and related bacteria. Lysis is mediated by the small membrane-bound toxin ΦX174-E, which is composed of a transmembrane domain and a soluble domain. The toxin requires activation by the bacterial chaperone SlyD and inhibits the cell wall precursor forming enzyme MraY. Bacterial cell wall biosynthesis is an important target for antibiotics; therefore, knowledge of molecular details in the ΦX174-E lysis pathway could help to identify new mechanisms and sites of action. In this study, cell-free expression and nanoparticle technology were combined to avoid toxic effects upon ΦX174-E synthesis, resulting in the efficient production of a functional full-length toxin and engineered derivatives. Pre-assembled nanodiscs were used to study ΦX174-E function in defined lipid environments and to analyze its membrane insertion mechanisms. The conformation of the soluble domain of ΦX174-E was identified as a central trigger for membrane insertion, as well as for the oligomeric assembly of the toxin. Stable complex formation of the soluble domain with SlyD is essential to keep nascent ΦX174-E in a conformation competent for membrane insertion. Once inserted into the membrane, ΦX174-E assembles into high-order complexes via its transmembrane domain and oligomerization depends on the presence of an essential proline residue at position 21. The data presented here support a model where an initial contact of the nascent ΦX174-E transmembrane domain with the peptidyl-prolyl isomerase domain of SlyD is essential to allow a subsequent stable interaction of SlyD with the ΦX174-E soluble domain for the generation of a membrane insertion competent toxin.
We report here the in-cell NMR-spectroscopic observation of the binding of the cognate ligand 2′-deoxyguanosine to the aptamer domain of the bacterial 2′-deoxyguanosine-sensing riboswitch in eukaryotic cells, namely Xenopus laevis oocytes and in human HeLa cells. The riboswitch is sufficiently stable in both cell types to allow for detection of binding of the ligand to the riboswitch. Most importantly, we show that the binding mode established by in vitro characterization of this prokaryotic riboswitch is maintained in eukaryotic cellular environment. Our data also bring important methodological insights: Thus far, in-cell NMR studies on RNA in mammalian cells have been limited to investigations of short (<15 nt) RNA fragments that were extensively modified by protecting groups to limit their degradation in the intracellular space. Here, we show that the in-cell NMR setup can be adjusted for characterization of much larger (≈70 nt) functional and chemically non-modified RNA.
The stem-loop (SL1) is the 5'-terminal structural element within the single-stranded SARS-CoV-2 RNA genome. It is formed by nucleotides 7–33 and consists of two short helical segments interrupted by an asymmetric internal loop. This architecture is conserved among Betacoronaviruses. SL1 is present in genomic SARS-CoV-2 RNA as well as in all subgenomic mRNA species produced by the virus during replication, thus representing a ubiquitous cis-regulatory RNA with potential functions at all stages of the viral life cycle. We present here the 1H, 13C and 15N chemical shift assignment of the 29 nucleotides-RNA construct 5_SL1, which denotes the native 27mer SL1 stabilized by an additional terminal G-C base-pair.
The genome of the halophilic archaeon Haloferax volcanii encodes more than 40 one-domain zinc finger µ-proteins. Only one of these, HVO_2753, contains four C(P)XCG motifs, suggesting the presence of two zinc binding pockets (ZBPs). Homologs of HVO_2753 are widespread in many euryarchaeota. An in frame deletion mutant of HVO_2753 grew indistinguishably from the wild-type in several media, but had a severe defect in swarming and in biofilm formation. For further analyses, the protein was produced homologously as well as heterologously in Escherichia coli. HVO_2753 was stable and folded in low salt, in contrast to many other haloarchaeal proteins. Only haloarchaeal HVO_2753 homologs carry a very hydrophilic N terminus, and NMR analysis showed that this region is very flexible and not part of the core structure. Surprisingly, both NMR analysis and a fluorimetric assay revealed that HVO_2753 binds only one zinc ion, despite the presence of two ZBPs. Notably, the analysis of cysteine to alanine mutant proteins by NMR as well by in vivo complementation revealed that all four C(P)XCG motifs are essential for folding and function. The NMR solution structure of the major conformation of HVO_2753 was solved. Unexpectedly, it was revealed that ZBP1 was comprised of C(P)XCG motifs 1 and 3, and ZBP2 was comprised of C(P)XCG motifs 2 and 4. There are several indications that ZBP2 is occupied by zinc, in contrast to ZBP1. To our knowledge, this study represents the first in-depth analysis of a zinc finger µ-protein in all three domains of life.
We report here the nuclear magnetic resonance 19F screening of 14 RNA targets with different secondary and tertiary structure to systematically assess the druggability of RNAs. Our RNA targets include representative bacterial riboswitches that naturally bind with nanomolar affinity and high specificity to cellular metabolites of low molecular weight. Based on counter-screens against five DNAs and five proteins, we can show that RNA can be specifically targeted. To demonstrate the quality of the initial fragment library that has been designed for easy follow-up chemistry, we further show how to increase binding affinity from an initial fragment hit by chemistry that links the identified fragment to the intercalator acridine. Thus, we achieve low-micromolar binding affinity without losing binding specificity between two different terminator structures.
The structure and flexibility of RNA depends sensitively on the microenvironment. Using pulsed electron-electron double-resonance (PELDOR)/double electron-electron resonance (DEER) spectroscopy combined with advanced labeling techniques, we show that the structure of double-stranded RNA (dsRNA) changes upon internalization into Xenopus lævis oocytes. Compared to dilute solution, the dsRNA A-helix is more compact in cells. We recapitulate this compaction in a densely crowded protein solution. Atomic-resolution molecular dynamics simulations of dsRNA semi-quantitatively capture the compaction, and identify non-specific electrostatic interactions between proteins and dsRNA as a possible driver of this effect.