The ART of RNAylation: covalent RNA-protein linkage in bacteriophage infection.

Bacteriophages have been a treasure trove for the discovery of fundamental biological principles and the expansion of our enzymatic toolkit since the dawn of molecular biology. In a recent study by Wolfram-Schauerte et al. these ubiquitous bacteria-infecting viruses reveal yet another new biological concept: post-translational modification through covalent RNA-protein linkages.

Arid5a uses disordered extensions of its core ARID domain for distinct DNA- and RNA-recognition and gene regulation.

AT-rich interacting domain (ARID)-containing proteins, Arids, are a heterogeneous DNA-binding protein family involved in transcription regulation and chromatin processing. For the member Arid5a, no exact DNA-binding preference has been experimentally defined so far. Additionally, the protein binds to mRNA motifs for transcript stabilization, supposedly through the DNA-binding ARID domain. To date, however, no unbiased RNA motif definition and clear dissection of nucleic acid-binding through the ARID domain have been undertaken. Using NMR-centered biochemistry, we here define the Arid5a DNA preference. Further, high-throughput in vitro binding reveals a consensus RNA-binding motif engaged by the core ARID domain. Finally, transcriptome-wide binding (iCLIP2) reveals that Arid5a has a weak preference for (A)U-rich regions in pre-mRNA transcripts of factors related to RNA processing. We find that the intrinsically disordered regions flanking the ARID domain modulate the specificity and affinity of DNA binding, while they appear crucial for RNA interactions. Ultimately, our data suggest that Arid5a uses its extended ARID domain for bifunctional gene regulation and that the involvement of IDR extensions is a more general feature of Arids in interacting with different nucleic acids at the chromatin-mRNA interface.

Insight into the Structural Basis for Dual Nucleic Acid-Recognition by the Scaffold Attachment Factor B2 Protein.

The family of scaffold attachment factor B (SAFB) proteins comprises three members and was first identified as binders of the nuclear matrix/scaffold. Over the past two decades, SAFBs were shown to act in DNA repair, mRNA/(l)ncRNA processing and as part of protein complexes with chromatin-modifying enzymes. SAFB proteins are approximately 100 kDa-sized dual nucleic acid-binding proteins with dedicated domains in an otherwise largely unstructured context, but whether and how they discriminate DNA and RNA binding has remained enigmatic. We here provide the SAFB2 DNA- and RNA-binding SAP and RRM domains in their functional boundaries and use solution NMR spectroscopy to ascribe DNA- and RNA-binding functions. We give insight into their target nucleic acid preferences and map the interfaces with respective nucleic acids on sparse data-derived SAP and RRM domain structures. Further, we provide evidence that the SAP domain exhibits intra-domain dynamics and a potential tendency to dimerize, which may expand its specifically targeted DNA sequence range. Our data provide a first molecular basis of and a starting point towards deciphering DNA- and RNA-binding functions of SAFB2 on the molecular level and serve a basis for understanding its localization to specific regions of chromatin and its involvement in the processing of specific RNA species.

Structures and nucleic acid-binding preferences of the eukaryotic ARID domain.

The DNA-binding AT-rich interactive domain (ARID) exists in a wide range of proteins throughout eukaryotic kingdoms. ARID domain-containing proteins are involved in manifold biological processes, such as transcriptional regulation, cell cycle control and chromatin remodeling. Their individual domain composition allows for a sub-classification within higher mammals. ARID is categorized as binder of double-stranded AT-rich DNA, while recent work has suggested ARIDs as capable of binding other DNA motifs and also recognizing RNA. Despite a broad variability on the primary sequence level, ARIDs show a highly conserved fold, which consists of six α-helices and two loop regions. Interestingly, this minimal core domain is often found extended by helices at the N- and/or C-terminus with potential roles in target specificity and, subsequently function. While high-resolution structural information from various types of ARIDs has accumulated over two decades now, there is limited access to ARID-DNA complex structures. We thus find ourselves left at the beginning of understanding ARID domain target specificities and the role of accompanying domains. Here, we systematically summarize ARID domain conservation and compare the various types with a focus on their structural differences and DNA-binding preferences, including the context of multiple other motifs within ARID domain containing proteins.

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

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.

LanI-Mediated Lantibiotic Immunity in Bacillus subtilis: Functional Analysis.

Lantibiotics subtilin and nisin are produced by Bacillus subtilis and Lactococcus lactis, respectively. To prevent toxicity of their own lantibiotic, both bacteria express specific immunity proteins, called SpaI and NisI. In addition, ABC transporters SpaFEG and NisFEG prevent lantibiotic toxicity by transporting the respective peptides to the extracellular space. Although the three-dimensional structures of SpaI and NisI have been solved, very little is known about the molecular function of either lipoprotein. Using laser-induced liquid bead ion desorption (LILBID)-mass spectrometry, we show here that subtilin interacts with SpaI monomers. The expression of either SpaI or NisI in a subtilin-nonproducing B. subtilis strain resulted in the respective strain being more resistant against either subtilin or nisin. Furthermore, pore formation provided by subtilin and nisin was prevented specifically upon the expression of either SpaI or NisI. As shown with a nisin-subtilin hybrid molecule, the C-terminal part of subtilin but not any particular lanthionine ring was needed for SpaI-mediated immunity. With respect to growth, SpaI provided less immunity against subtilin than is provided by the ABC transporter SpaFEG. However, SpaI prevented pore formation much more efficiently than SpaFEG. Taken together, our data show the physiological function of SpaI as a fast immune response to protect the cellular membrane.IMPORTANCE The two lantibiotics nisin and subtilin are produced by Lactococcus lactis and Bacillus subtilis, respectively. Both peptides have strong antimicrobial activity against Gram-positive bacteria, and therefore, appropriate protection mechanisms are required for the producing strains. To prevent toxicity of their own lantibiotic, both bacteria express immunity proteins, called SpaI and NisI, and in addition, ABC transporters SpaFEG and NisFEG. Whereas it has been shown that the ABC transporters protect the producing strains by transporting the toxic peptides to the extracellular space, the exact mode of action and the physiological function of the lipoproteins during immunity are still unknown. Understanding the exact role of lantibiotic immunity proteins is of major importance for improving production rates and for the design of newly engineered peptide antibiotics. Here, we show (i) the specificity of each lipoprotein for its own lantibiotic, (ii) the specific physical interaction of subtilin with its lipoprotein SpaI, (iii) the physiological function of SpaI in protecting the cellular membrane, and (iv) the importance of the C-terminal part of subtilin for its interaction with SpaI.

Specificity of Subtilin-Mediated Activation of Histidine Kinase SpaK.

Autoinduction via two-component systems is a widespread regulatory mechanism that senses environmental and metabolic changes. Although the lantibiotics nisin and subtilin are closely related and share the same lanthionine ring structure, they autoinduce their biosynthesis in a highly specific manner. Subtilin activates only the two-component system SpaRK of Bacillus subtilis, whereas nisin activates solely the two-component system NisRK of Lactococcus lactis To identify components that determine the specificity of subtilin autoinduction, several variants of the respective lantibiotics were analyzed for their autoinductive capacities. Here, we show that amino acid position 20 is crucial for SpaK activation, as an engineered nisin molecule with phenylalanine at position 20 (nisin N20F) was able to activate SpaK in a specific manner. In combination with the N-terminal tryptophan of subtilin (nisin I1W/N20F), SpaK autoinduction reached almost the level of subtilin-mediated autoinduction. Furthermore, the overall structure of subtilin is also important for its association with the histidine kinase. The destruction of the second lanthionine ring (subtilin C11A, ring B), as well as mutations that interfere with the flexibility of the hinge region located between lanthionine rings C and D (subtilin L21P/Q22P), abolished SpaK autoinduction. Although the C-terminal part of subtilin is needed for efficient SpaK autoinduction, the destruction of lanthionine rings D and E had no measurable impact. Based on these findings, a model for the interaction of subtilin with histidine kinase SpaK was established.IMPORTANCE Although two-component systems are important regulatory systems that sense environmental changes, very little information on the molecular mechanism of sensing or the interaction of the sensor with its respective kinase is available. The strong specificity of linear lantibiotics such as subtilin and nisin for their respective kinases provides an excellent model system to unravel the structural needs of these lantibiotics for activating histidine kinases in a specific manner. More than that, the biosyntheses of lantibiotics are autoinduced via two-component systems. Therefore, an understanding of their interactions with histidine kinases is needed for the biosynthesis of newly engineered peptide antibiotics. Using a Bacillus subtilis-based reporter system, we were able to identify the molecular constraints that are necessary for specific SpaK activation and to provide SpaK specificity to nisin with just two point mutations.

The Solution Structure of the Lantibiotic Immunity Protein NisI and Its Interactions with Nisin.

Many Gram-positive bacteria produce lantibiotics, genetically encoded and posttranslationally modified peptide antibiotics, which inhibit the growth of other Gram-positive bacteria. To protect themselves against their own lantibiotics these bacteria express a variety of immunity proteins including the LanI lipoproteins. The structural and mechanistic basis for LanI-mediated lantibiotic immunity is not yet understood. Lactococcus lactis produces the lantibiotic nisin, which is widely used as a food preservative. Its LanI protein NisI provides immunity against nisin but not against structurally very similar lantibiotics from other species such as subtilin from Bacillus subtilis. To understand the structural basis for LanI-mediated immunity and their specificity we investigated the structure of NisI. We found that NisI is a two-domain protein. Surprisingly, each of the two NisI domains has the same structure as the LanI protein from B. subtilis, SpaI, despite the lack of significant sequence homology. The two NisI domains and SpaI differ strongly in their surface properties and function. Additionally, SpaI-mediated lantibiotic immunity depends on the presence of a basic unstructured N-terminal region that tethers SpaI to the membrane. Such a region is absent from NisI. Instead, the N-terminal domain of NisI interacts with membranes but not with nisin. In contrast, the C-terminal domain specifically binds nisin and modulates the membrane affinity of the N-terminal domain. Thus, our results reveal an unexpected structural relationship between NisI and SpaI and shed light on the structural basis for LanI mediated lantibiotic immunity.

Autoinduction Specificities of the Lantibiotics Subtilin and Nisin.

The biosynthesis of the lantibiotics subtilin and nisin is regulated by autoinduction via two-component systems. Although subtilin is structurally closely related to nisin and contains the same lanthionine ring structure, both lantibiotics specifically autoinduce their biosynthesis. Subtilin and also the subtilin-like lantibiotics entianin and ericin autoinduce the two-component system SpaRK of Bacillus subtilis, whereas the biosynthesis of nisin is autoinduced via the two-component system NisRK of Lactococcus lactis. Autoinduction is highly specific for the respective lantibiotic and therefore of major importance for the functional expression of genetically engineered subtilin-like lantibiotics. To identify the structural features required for subtilin autoinduction, subtilin-nisin hybrids and specific point mutations of amino acid position 1 were generated. For subtilin autoinduction, the N-terminal tryptophan is the most important for full SpaK activation. The failure of subtilin to autoinduce the histidine kinase NisK mainly depends on the N-terminal tryptophan, as its single exchange to the aliphatic amino acid residues isoleucine, leucine, and valine provided NisK autoinduction. In addition, the production of subtilin variants which did not autoinduce their own biosynthesis could be rescued upon heterologous coexpression in B. subtilis DSM15029 by the autoinducing subtilin-like lantibiotic entianin.

Activation of Histidine Kinase SpaK Is Mediated by the N-Terminal Portion of Subtilin-Like Lantibiotics and Is Independent of Lipid II.

The biosynthesis of the lantibiotic subtilin is autoinduced in a quorum-sensing mechanism via histidine kinase SpaK. Subtilin-like lantibiotics, such as entianin, ericin S, and subtilin, specifically activated SpaK in a comparable manner, whereas the structurally similar nisin did not provide the signal for SpaK activation at nontoxic concentrations. Surprisingly, nevertheless, nisin if applied together with entianin partly quenched SpaK activation. The N-terminal entianin1-20 fragment (comprising N-terminal amino acids 1 to 20) was sufficient for SpaK activation, although higher concentrations were needed. The N-terminal nisin1-20 fragment also interfered with entianin-mediated activation of SpaK and, remarkably, at extremely high concentrations also activated SpaK. Our data show that the N-terminal entianin1-20 fragment is sufficient for SpaK activation. However, if present, the C-terminal part of the molecule further strongly enhances the activation, possibly by its interference with the cellular membrane. As shown by using lipid II-interfering substances and a lipid II-deficient mutant strain, lipid II is not needed for the sensing mechanism.

Scotopic and Photopic Conventional Visual Acuity and Hyperacuity - Binocular Summation.

Purpose: The purpose of this study was to determine and compare binocular summation (BiS) of conventional visual acuity (cVA) versus hyperacuity (hVA) for photopic and scotopic luminance conditions as a potential biomarker to assess the outcome of interventions on binocular function. Methods: Sixteen young adults (age range [years] = 21-31; 8 women; cVA logMAR < 0.0) participated in this study. The Freiburg Visual Acuity Test (FrACT) was used for VA testing and retested on another day. Both cVA and hVA were determined for dark grey optotypes on light grey background. Participants underwent 40 minutes of dark adaptation prior to scotopic VA testing. Binocular and monocular VA testing was performed. The eye with better VA over the 2 days of testing was selected, the BiS was quantified (binocular VA - better monocular VA) and repeated measures ANOVAs were performed. Results: Binocular VA exceeded monocular VA for all luminance conditions, VA-types, and sessions. We report BiS estimates for photopic and scotopic cVA and hVA, (logMAR BiS ± SEM [decimal BiS]): photopic = -0.01 ± 0.01 [1.03] and -0.06 ± 0.03 [1.15]; and scotopic = -0.05 ± 0.01 [1.12] and -0.11 ± 0.04 [1.28], respectively). Improvement for binocular vision estimates ranged from 0.01 to 0.11 logMAR. A repeated-measures ANOVA (RM ANOVA) did not reveal significant effects of LUMINANCE or VA TYPE on BiS, albeit a trend for strongest BiS for scotopic hVA (15% vs. 28%, photopic versus scotopic, respectively) and weakest for photopic cVA (3% vs. 12%, photopic versus scotopic conditions, respectively). Conclusions: Our results indicate that BiS of VA is relevant to scotopic and photopic hVA and cVA. It appears therefore a plausible candidate biomarker to assess the outcome of retinal therapies restoring rod or cone function on binocular vision. Translational Relevance: Binocular summation of visual acuity might serve as a clinical biomarker to monitor therapy outcome on binocular rod and cone-mediated vision.

1H, 13C, 15N backbone chemical shift assignments of the extended ARID domain in human AT-rich interactive domain protein 5a (Arid5a).

The family of AT-rich interactive domain (ARID) containing proteins -Arids- contains 15 members that have almost exclusively been described as DNA-binding proteins. Interestingly, a decade ago the family member Arid5a was found to bind and stabilize mRNAs of immune system key players and thereby account for driving inflammatory and autoimmune diseases. How exactly binding to DNA and RNA is coordinated by the Arid5a ARID domain remains unknown, mainly due to the lack of atom-resolved information on nucleic acid-binding. This in particular applies to the protein's ARID domain, despite the comfortable size of its core unit for NMR-based investigations. Furthermore, the core domain of ARID domains is found to be extended by functionally relevant, often flexible stretches, but whether such elongations are present and crucial for the versatile Arid5a functions is unknown. We here provide a near-complete NMR backbone resonance assignment of the Arid5a ARID domain with N- and C-terminal extensions, which serves as a basis for further studies of its nucleic acid-binding preferences and targeted inhibition by means of NMR. Our data thus significantly contribute to unravelling mechanisms of Arid5a-mediated gene regulation and diseases.

The preference signature of the SARS-CoV-2 Nucleocapsid NTD for its 5'-genomic RNA elements.

The nucleocapsid protein (N) of SARS-CoV-2 plays a pivotal role during the viral life cycle. It is involved in RNA transcription and accounts for packaging of the large genome into virus particles. N manages the enigmatic balance of bulk RNA-coating versus precise RNA-binding to designated cis-regulatory elements. Numerous studies report the involvement of its disordered segments in non-selective RNA-recognition, but how N organizes the inevitable recognition of specific motifs remains unanswered. We here use NMR spectroscopy to systematically analyze the interactions of N's N-terminal RNA-binding domain (NTD) with individual cis RNA elements clustering in the SARS-CoV-2 regulatory 5'-genomic end. Supported by broad solution-based biophysical data, we unravel the NTD RNA-binding preferences in the natural genome context. We show that the domain's flexible regions read the intrinsic signature of preferred RNA elements for selective and stable complex formation within the large pool of available motifs.

NMR Reveals Specific Tracts within the Intrinsically Disordered Regions of the SARS-CoV-2 Nucleocapsid Protein Involved in RNA Encountering.

The SARS-CoV-2 nucleocapsid (N) protein is crucial for the highly organized packaging and transcription of the genomic RNA. Studying atomic details of the role of its intrinsically disordered regions (IDRs) in RNA recognition is challenging due to the absence of structure and to the repetitive nature of their primary sequence. IDRs are known to act in concert with the folded domains of N and here we use NMR spectroscopy to identify the priming events of N interacting with a regulatory SARS-CoV-2 RNA element. 13C-detected NMR experiments, acquired simultaneously to 1H detected ones, provide information on the two IDRs flanking the N-terminal RNA binding domain (NTD) within the N-terminal region of the protein (NTR, 1-248). We identify specific tracts of the IDRs that most rapidly sense and engage with RNA, and thus provide an atom-resolved picture of the interplay between the folded and disordered regions of N during RNA interaction.

Structures and target RNA preferences of the RNA-binding protein family of IGF2BPs: An overview.

Insulin-like growth factor 2 mRNA-binding proteins (IMPs, IGF2BPs) act in mRNA transport and translational control but are oncofetal tumor marker proteins. The IMP protein family represents a number of bona fide multi-domain RNA-binding proteins with up to six RNA-binding domains, resulting in a high complexity of possible modes of interactions with target mRNAs. Their exact mechanism in stability control of oncogenic mRNAs is only partially understood. Our and other laboratories' recent work has significantly pushed the understanding of IMP protein specificities both toward RNA engagement and between each other from NMR and crystal structures serving the basis for systematic biochemical and functional investigations. We here summarize the known structural and biochemical information about IMP RNA-binding domains and their RNA preferences. The article also touches on the respective roles of RNA secondary and protein tertiary structures for specific RNA-protein complexes, including the limited knowledge about IMPs' protein-protein interactions, which are often RNA mediated.

1H, 13C, and 15N backbone chemical-shift assignments of SARS-CoV-2 non-structural protein 1 (leader protein).

The current COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has become a worldwide health crisis, necessitating coordinated scientific research and urgent identification of new drug targets for treatment of 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 comprises a single RNA of about 30 kb in length, in which 14 open reading frames (ORFs) have been annotated, and encodes approximately 30 proteins. The first two-thirds of the SARS-CoV-2 genome is made up of two large overlapping open-reading-frames (ORF1a and ORF1b) encoding a replicase polyprotein, which is subsequently cleaved to yield 16 so-called non-structural proteins. The non-structural protein 1 (Nsp1), which is considered to be a major virulence factor, suppresses host immune functions by associating with host ribosomal complexes at the very end of its C-terminus. Furthermore, Nsp1 facilitates initiation of viral RNA translation via an interaction of its N-terminal domain with the 5' untranslated region (UTR) of the viral RNA. Here, we report the near-complete backbone chemical-shift assignments of full-length SARS-CoV-2 Nsp1 (19.8 kDa), which reveal the domain organization, secondary structure and backbone dynamics of Nsp1, and which will be of value to further NMR-based investigations of both the biochemical and physiological functions of Nsp1.

1H, 13C, and 15N backbone chemical shift assignments of the C-terminal dimerization domain of SARS-CoV-2 nucleocapsid protein.

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.

Backbone chemical shift spectral assignments of SARS coronavirus-2 non-structural protein nsp9.

As part of an International consortium aiming at the characterization by NMR of the proteins of the SARS-CoV-2 virus, we have obtained the virtually complete assignment of the backbone atoms of the non-structural protein nsp9. This small (12 kDa) protein is encoded by ORF1a, binds to RNA and seems to be essential for viral RNA synthesis. The crystal structures of the SARS-CoV-2 protein and other homologues suggest that the protein is dimeric as also confirmed by analytical ultracentrifugation and dynamic light scattering. Our data constitute the prerequisite for further NMR-based characterization, and provide the starting point for the identification of small molecule lead compounds that could interfere with RNA binding and prevent viral replication.

1H, 13C and 15N backbone chemical shift assignments of SARS-CoV-2 nsp3a.

The non-structural protein nsp3 from SARS-CoV-2 plays an essential role in the viral replication transcription complex. Nsp3a constitutes the N-terminal domain of nsp3, comprising a ubiquitin-like folded domain and a disordered acidic chain. This region of nsp3a has been linked to interactions with the viral nucleoprotein and the structure of double membrane vesicles. Here, we report the backbone resonance assignment of both domains of nsp3a. The study is carried out in the context of the international covid19-nmr consortium, which aims to characterize SARS-CoV-2 proteins and RNAs, providing for example NMR chemical shift assignments of the different viral components. Our assignment will provide the basis for the identification of inhibitors and further functional and interaction studies of this essential protein.

1H, 13C and 15N assignment of stem-loop SL1 from the 5'-UTR of SARS-CoV-2.

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.