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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.
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.
The SARS-CoV-2 virus is the cause of the respiratory disease COVID-19. As of today, therapeutic interventions in severe COVID-19 cases are still not available as no effective therapeutics have been developed so far. Despite the ongoing development of a number of effective vaccines, therapeutics to fight the disease once it has been contracted will still be required. Promising targets for the development of antiviral agents against SARS-CoV-2 can be found in the viral RNA genome. The 5′- and 3′-genomic ends of the 30 kb SCoV-2 genome are highly conserved among Betacoronaviruses and contain structured RNA elements involved in the translation and replication of the viral genome. The 40 nucleotides (nt) long highly conserved stem-loop 4 (5_SL4) is located within the 5′-untranslated region (5′-UTR) important for viral replication. 5_SL4 features an extended stem structure disrupted by several pyrimidine mismatches and is capped by a pentaloop. Here, we report extensive 1H, 13C, 15N and 31P resonance assignments of 5_SL4 as the basis for in-depth structural and ligand screening studies by solution NMR spectroscopy.
The current outbreak of the highly infectious COVID-19 respiratory disease is caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). To fight the pandemic, the search for promising viral drug targets has become a cross-border common goal of the international biomedical research community. Within the international Covid19-NMR consortium, scientists support drug development against SARS-CoV-2 by providing publicly available NMR data on viral proteins and RNAs. The coronavirus nucleocapsid protein (N protein) is an RNA-binding protein involved in viral transcription and replication. Its primary function is the packaging of the viral RNA genome. The highly conserved architecture of the coronavirus N protein consists of an N-terminal RNA-binding domain (NTD), followed by an intrinsically disordered Serine/Arginine (SR)-rich linker and a C-terminal dimerization domain (CTD). Besides its involvement in oligomerization, the CTD of the N protein (N-CTD) is also able to bind to nucleic acids by itself, independent of the NTD. Here, we report the near-complete NMR backbone chemical shift assignments of the SARS-CoV-2 N-CTD to provide the basis for downstream applications, in particular site-resolved drug binding studies.
The ongoing pandemic caused by the Betacoronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) demonstrates the urgent need of coordinated and rapid research towards inhibitors of the COVID-19 lung disease. The covid19-nmr consortium seeks to support drug development by providing publicly accessible NMR data on the viral RNA elements and proteins. The SARS-CoV-2 genome encodes for approximately 30 proteins, among them are the 16 so-called non-structural proteins (Nsps) of the replication/transcription complex. The 217-kDa large Nsp3 spans one polypeptide chain, but comprises multiple independent, yet functionally related domains including the viral papain-like protease. The Nsp3e sub-moiety contains a putative nucleic acid-binding domain (NAB) with so far unknown function and consensus target sequences, which are conceived to be both viral and host RNAs and DNAs, as well as protein-protein interactions. Its NMR-suitable size renders it an attractive object to study, both for understanding the SARS-CoV-2 architecture and drugability besides the classical virus’ proteases. We here report the near-complete NMR backbone chemical shifts of the putative Nsp3e NAB that reveal the secondary structure and compactness of the domain, and provide a basis for NMR-based investigations towards understanding and interfering with RNA- and small-molecule-binding by Nsp3e.
Many naturally occurring or artificially created RNAs are capable of binding to guanine or guanine derivatives with high affinity and selectivity. They bind their ligands using very different recognition modes involving a diverse set of hydrogen bonding and stacking interactions. Apparently, the potential structural diversity for guanine, guanosine, and guanine nucleotide binding motifs is far from being fully explored. Szostak and coworkers have derived a large set of different GTP-binding aptamer families differing widely in sequence, secondary structure, and ligand specificity. The so-called class V–GTP aptamer from this set binds GTP with very high affinity and has a complex secondary structure. Here we use solution NMR spectroscopy to demonstrate that the class V aptamer binds GTP through the formation of an intermolecular two-layered G-quadruplex structure that directly incorporates the ligand and folds only upon ligand addition. Ligand binding and G-quadruplex formation depend strongly on the identity of monovalent cations present with a clear preference for potassium ions. GTP binding through direct insertion into an intermolecular G-quadruplex is a previously unobserved structural variation for ligand-binding RNA motifs and rationalizes the previously observed specificity pattern of the class V aptamer for GTP analogs.
The neomycin sensing riboswitch is the smallest biologically functional RNA riboswitch, forming a hairpin capped with a U-turn loop—a well-known RNA motif containing a conserved uracil. It was shown previously that a U→C substitution of the eponymous conserved uracil does not alter the riboswitch structure due to C protonation at N3. Furthermore, cytosine is evolutionary permitted to replace uracil in other U-turns. Here, we use molecular dynamics simulations to study the molecular basis of this substitution in the neomycin sensing riboswitch and show that a structure-stabilizing monovalent cation-binding site in the wild-type RNA is the main reason for its negligible structural effect. We then use NMR spectroscopy to confirm the existence of this cation-binding site and to demonstrate its effects on RNA stability. Lastly, using quantum chemical calculations, we show that the cation-binding site is altering the electronic environment of the wild-type U-turn so that it is more similar to the cytosine mutant. The study reveals an amazingly complex and delicate interplay between various energy contributions shaping up the 3D structure and evolution of nucleic acids.
The U-turn is a classical three-dimensional RNA folding motif first identified in the anticodon and T-loops of tRNAs. It also occurs frequently as a building block in other functional RNA structures in many different sequence and structural contexts. U-turns induce sharp changes in the direction of the RNA backbone and often conform to the 3-nt consensus sequence 5'-UNR-3' (N = any nucleotide, R = purine). The canonical U-turn motif is stabilized by a hydrogen bond between the N3 imino group of the U residue and the 3' phosphate group of the R residue as well as a hydrogen bond between the 2'-hydroxyl group of the uridine and the N7 nitrogen of the R residue. Here, we demonstrate that a protonated cytidine can functionally and structurally replace the uridine at the first position of the canonical U-turn motif in the apical loop of the neomycin riboswitch. Using NMR spectroscopy, we directly show that the N3 imino group of the protonated cytidine forms a hydrogen bond with the backbone phosphate 3' from the third nucleotide of the U-turn analogously to the imino group of the uridine in the canonical motif. In addition, we compare the stability of the hydrogen bonds in the mutant U-turn motif to the wild type and describe the NMR signature of the C+-phosphate interaction. Our results have implications for the prediction of RNA structural motifs and suggest simple approaches for the experimental identification of hydrogen bonds between protonated C-imino groups and the phosphate backbone.
Non-ribosomal peptide synthetases (NRPS) produce natural products from amino acid building blocks. They often consist of multiple polypeptide chains which assemble in a specific linear order via specialized N- and C-terminal docking domains (N/CDDs). Typically, docking domains function independently from other domains in NRPS assembly. Thus, docking domain replacements enable the assembly of “designer” NRPS from proteins that normally do not interact. The multiprotein “peptide-antimicrobial-Xenorhabdus” (PAX) peptide-producing PaxS NRPS is assembled from the three proteins PaxA, PaxB and PaxC. Herein, we show that the small CDD of PaxA cooperates with its preceding thiolation (T1) domain to bind the NDD of PaxB with very high affinity, establishing a structural and thermodynamical basis for this unprecedented docking interaction, and we test its functional importance in vivo in a truncated PaxS assembly line. Similar docking interactions are apparently present in other NRPS systems.
Long-range tertiary interactions determine the three-dimensional structure of a number of metabolite-binding riboswitch RNA elements and were found to be important for their regulatory function. For the guanine-sensing riboswitch of the Bacillus subtilis xpt-pbuX operon, our previous NMR-spectroscopic studies indicated pre-formation of long-range tertiary contacts in the ligand-free state of its aptamer domain. Loss of the structural pre-organization in a mutant of this RNA (G37A/C61U) resulted in the requirement of Mg2+ for ligand binding. Here, we investigate structural and stability aspects of the wild-type aptamer domain (Gsw) and the G37A/C61U-mutant (Gswloop) of the guanine-sensing riboswitch and their Mg2+-induced folding characteristics to dissect the role of long-range tertiary interactions, the link between pre-formation of structural elements and ligand-binding properties and the functional stability. Destabilization of the long-range interactions as a result of the introduced mutations for Gswloop or the increase in temperature for both Gsw and Gswloop involves pronounced alterations of the conformational ensemble characteristics of the ligand-free state of the riboswitch. The increased flexibility of the conformational ensemble can, however, be compensated by Mg2+. We propose that reduction of conformational dynamics in remote regions of the riboswitch aptamer domain is the minimal pre-requisite to pre-organize the core region for specific ligand binding.