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Nature Communications Jun 2023Cyclic di-AMP is the only known essential second messenger in bacteria and archaea, regulating different proteins indispensable for numerous physiological processes. In...
Cyclic di-AMP is the only known essential second messenger in bacteria and archaea, regulating different proteins indispensable for numerous physiological processes. In particular, it controls various potassium and osmolyte transporters involved in osmoregulation. In Bacillus subtilis, the K/H symporter KimA of the KUP family is inactivated by c-di-AMP. KimA sustains survival at potassium limitation at low external pH by mediating potassium ion uptake. However, at elevated intracellular K concentrations, further K accumulation would be toxic. In this study, we reveal the molecular basis of how c-di-AMP binding inhibits KimA. We report cryo-EM structures of KimA with bound c-di-AMP in detergent solution and reconstituted in amphipols. By combining structural data with functional assays and molecular dynamics simulations we reveal how c-di-AMP modulates transport. We show that an intracellular loop in the transmembrane domain interacts with c-di-AMP bound to the adjacent cytosolic domain. This reduces the mobility of transmembrane helices at the cytosolic side of the K binding site and therefore traps KimA in an inward-occluded conformation.
Topics: Cyclic AMP; Protons; Bacterial Proteins; Second Messenger Systems; Membrane Transport Proteins; Potassium; Dinucleoside Phosphates
PubMed: 37344476
DOI: 10.1038/s41467-023-38944-1 -
Nature Aug 2022Stimulator of interferon genes (STING) is an antiviral signalling protein that is broadly conserved in both innate immunity in animals and phage defence in prokaryotes....
Stimulator of interferon genes (STING) is an antiviral signalling protein that is broadly conserved in both innate immunity in animals and phage defence in prokaryotes. Activation of STING requires its assembly into an oligomeric filament structure through binding of a cyclic dinucleotide, but the molecular basis of STING filament assembly and extension remains unknown. Here we use cryogenic electron microscopy to determine the structure of the active Toll/interleukin-1 receptor (TIR)-STING filament complex from a Sphingobacterium faecium cyclic-oligonucleotide-based antiphage signalling system (CBASS) defence operon. Bacterial TIR-STING filament formation is driven by STING interfaces that become exposed on high-affinity recognition of the cognate cyclic dinucleotide signal c-di-GMP. Repeating dimeric STING units stack laterally head-to-head through surface interfaces, which are also essential for human STING tetramer formation and downstream immune signalling in mammals. The active bacterial TIR-STING structure reveals further cross-filament contacts that brace the assembly and coordinate packing of the associated TIR NADase effector domains at the base of the filament to drive NAD hydrolysis. STING interface and cross-filament contacts are essential for cell growth arrest in vivo and reveal a stepwise mechanism of activation whereby STING filament assembly is required for subsequent effector activation. Our results define the structural basis of STING filament formation in prokaryotic antiviral signalling.
Topics: Animals; Antiviral Agents; Bacterial Proteins; Bacteriophages; Cryoelectron Microscopy; Dinucleoside Phosphates; Humans; Immunity, Innate; Membrane Proteins; Operon; Receptors, Interleukin-1; Sphingobacterium; Toll-Like Receptors
PubMed: 35859168
DOI: 10.1038/s41586-022-04999-1 -
Molecules (Basel, Switzerland) May 2020The regulation of multiple bacterial phenotypes was found to depend on different cyclic dinucleotides (CDNs) that constitute intracellular signaling second messenger... (Review)
Review
The regulation of multiple bacterial phenotypes was found to depend on different cyclic dinucleotides (CDNs) that constitute intracellular signaling second messenger systems. Most notably, c-di-GMP, along with proteins related to its synthesis, sensing, and degradation, was identified as playing a central role in the switching from biofilm to planktonic modes of growth. Recently, this research topic has been under expansion, with the discoveries of new CDNs, novel classes of CDN receptors, and the numerous functions regulated by these molecules. In this review, we comprehensively describe the three main bacterial enzymes involved in the synthesis of c-di-GMP, c-di-AMP, and cGAMP focusing on description of their three-dimensional structures and their structural similarities with other protein families, as well as the essential residues for catalysis. The diversity of CDN receptors is described in detail along with the residues important for the interaction with the ligand. Interestingly, genomic data strongly suggest that there is a tendency for bacterial cells to use both c-di-AMP and c-di-GMP signaling networks simultaneously, raising the question of whether there is crosstalk between different signaling systems. In summary, the large amount of sequence and structural data available allows a broad view of the complexity and the importance of these CDNs in the regulation of different bacterial behaviors. Nevertheless, how cells coordinate the different CDN signaling networks to ensure adaptation to changing environmental conditions is still open for much further exploration.
Topics: Bacteria; Bacterial Proteins; Binding Sites; Biofilms; Cyclic GMP; Dinucleoside Phosphates; Gene Expression Regulation, Bacterial; Models, Molecular; Nucleotides, Cyclic; Plankton; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Signal Transduction
PubMed: 32466317
DOI: 10.3390/molecules25102462 -
Sichuan Da Xue Xue Bao. Yi Xue Ban =... Nov 2022Cyclic dimeric adenosine 3',5'-monophosphate (c-di-AMP) is a newly-discovered second messenger in bacteria and archaea. By directly binding to or affecting the... (Review)
Review
Cyclic dimeric adenosine 3',5'-monophosphate (c-di-AMP) is a newly-discovered second messenger in bacteria and archaea. By directly binding to or affecting the expression of target proteins, c-di-AMP regulates the physiological functions of bacteria, including maintaining osmotic pressure, balancing central metabolism, monitoring DNA damage, and controlling biofilm and spore formation. As a new pathogen-associated molecular pattern (PAMP), it binds to the host pattern recognition receptor (PRR), induces cyclic GMP-AMP synthase (cGAS)-STING signal axis to produce type Ⅰ interferon by activating the stimulator of interferon genes (STING), and promotes the secretion of inflammatory factors through nuclear factor κB (NF-κB) signaling pathway, thereby playing an important role in host immunity to bacterial infection and tumorigenesis. Due to its immunogenicity, c-di-AMP could be used as an immune adjuvant to provide new targets for the development of vaccines. However, the specific mechanism of action of c-di-AMP in host immunity awaits further exploration. Herein, we presented the structure and biological characteristics of c-di-AMP, and summarized the possible mechanism of c-di-AMP's regulation of host immune response. In addition, we also reported the latest findings on using c-di-AMP as an immune adjuvant in clinical treatment. Research on the function of c-di-AMP and its mechanism of action on host immune response provides new ideas for finding clinical solutions to bacterial resistance, infection control, tumor prevention, and vaccine development in the future.
Topics: Dinucleoside Phosphates; Bacteria; Biofilms; Signal Transduction
PubMed: 36443059
DOI: 10.12182/20220860102 -
Infection and Immunity Oct 2021Second messenger nucleotides are produced by bacteria in response to environmental stimuli and play a major role in the regulation of processes associated with bacterial...
Second messenger nucleotides are produced by bacteria in response to environmental stimuli and play a major role in the regulation of processes associated with bacterial fitness, including but not limited to osmoregulation, envelope homeostasis, central metabolism, and biofilm formation. In this study, we uncovered the biological significance of c-di-AMP in the opportunistic pathogen Enterococcus faecalis by isolating and characterizing strains lacking genes responsible for c-di-AMP synthesis () and degradation ( and ). Using complementary approaches, we demonstrated that either complete loss of c-di-AMP (Δ strain) or c-di-AMP accumulation (Δ, Δ, and Δ Δ strains) drastically impaired general cell fitness and virulence of E. faecalis. In particular, the Δ strain was highly sensitive to envelope-targeting antibiotics, was unable to multiply and quickly lost viability in human serum or urine , and was virtually avirulent in an invertebrate (Galleria mellonella) and in two catheter-associated mouse infection models that recapitulate key aspects of enterococcal infections in humans. In addition to evidence linking these phenotypes to altered activity of metabolite and peptide transporters and inability to maintain osmobalance, we found that the attenuated virulence of the Δ strain also could be attributed to a defect in Ebp pilus production and activity that severely impaired biofilm formation under both and conditions. Collectively, these results demonstrate that c-di-AMP signaling is essential for E. faecalis pathogenesis and a desirable target for drug development.
Topics: Animals; Biofilms; Dinucleoside Phosphates; Enterococcus faecalis; Fimbriae, Bacterial; Gene Expression Regulation, Bacterial; Gram-Positive Bacterial Infections; Humans; Virulence
PubMed: 34424750
DOI: 10.1128/IAI.00365-21 -
MBio Jan 2021The development of safe and effective vaccines against viruses is central to disease control. With advancements in DNA synthesis technology, the production of synthetic... (Review)
Review
The development of safe and effective vaccines against viruses is central to disease control. With advancements in DNA synthesis technology, the production of synthetic viral genomes has fueled many research efforts that aim to generate attenuated viruses by introducing synonymous mutations. Elucidation of the mechanisms underlying virus attenuation through synonymous mutagenesis is revealing interesting new biology that can be exploited for vaccine development. Here, we review recent advancements in this field of synthetic virology and focus on the molecular mechanisms of attenuation by genetic recoding of viruses. We highlight the action of the zinc finger antiviral protein (ZAP) and RNase L, two proteins involved in the inhibition of viruses enriched for CpG and UpA dinucleotides, that are often the products of virus recoding algorithms. Additionally, we discuss current challenges in the field as well as studies that may illuminate how other host functions, such as translation, are potentially involved in the attenuation of recoded viruses.
Topics: Animals; DNA Viruses; Dinucleoside Phosphates; Endoribonucleases; Genome, Viral; Humans; Silent Mutation; Vaccines, Attenuated; Viral Vaccines; Virus Replication; Viruses
PubMed: 33402534
DOI: 10.1128/mBio.02238-20 -
Viruses Sep 2021An evolutionary arms race occurs between viruses and hosts. Hosts have developed an array of antiviral mechanisms aimed at inhibiting replication and spread of viruses,... (Review)
Review
An evolutionary arms race occurs between viruses and hosts. Hosts have developed an array of antiviral mechanisms aimed at inhibiting replication and spread of viruses, reducing their fitness, and ultimately minimising pathogenic effects. In turn, viruses have evolved sophisticated counter-measures that mediate evasion of host defence mechanisms. A key aspect of host defences is the ability to differentiate between self and non-self. Previous studies have demonstrated significant suppression of CpG and UpA dinucleotide frequencies in the coding regions of RNA and small DNA viruses. Artificially increasing these dinucleotide frequencies results in a substantial attenuation of virus replication, suggesting dinucleotide bias could facilitate recognition of non-self RNA. The interferon-inducible gene, zinc finger antiviral protein (ZAP) is the host factor responsible for sensing CpG dinucleotides in viral RNA and restricting RNA viruses through direct binding and degradation of the target RNA. Herpesviruses are large DNA viruses that comprise three subfamilies, alpha, beta and gamma, which display divergent CpG dinucleotide patterns within their genomes. ZAP has recently been shown to act as a host restriction factor against human cytomegalovirus (HCMV), a beta-herpesvirus, which in turn evades ZAP detection by suppressing CpG levels in the major immediate-early transcript IE1, one of the first genes expressed by the virus. While suppression of CpG dinucleotides allows evasion of ZAP targeting, synonymous changes in nucleotide composition that cause genome biases, such as low GC content, can cause inefficient gene expression, especially in unspliced transcripts. To maintain compact genomes, the majority of herpesvirus transcripts are unspliced. Here we discuss how the conflicting pressures of ZAP evasion, the need to maintain compact genomes through the use of unspliced transcripts and maintaining efficient gene expression may have shaped the evolution of herpesvirus genomes, leading to characteristic CpG dinucleotide patterns.
Topics: Alphaherpesvirinae; Animals; Betaherpesvirinae; Dinucleoside Phosphates; Evolution, Molecular; Gammaherpesvirinae; Gene Expression; Genome, Viral; Herpesviridae; Host-Pathogen Interactions; Humans; Interferons; RNA Splicing; RNA, Viral; RNA-Binding Proteins; Signal Transduction; Viral Proteins
PubMed: 34578438
DOI: 10.3390/v13091857 -
Cyclic di-AMP, a second messenger of primary importance: tertiary structures and binding mechanisms.Nucleic Acids Research Apr 2020Cyclic diadenylate (c-di-AMP) is a widespread second messenger in bacteria and archaea that is involved in the maintenance of osmotic pressure, response to DNA damage,... (Review)
Review
Cyclic diadenylate (c-di-AMP) is a widespread second messenger in bacteria and archaea that is involved in the maintenance of osmotic pressure, response to DNA damage, and control of central metabolism, biofilm formation, acid stress resistance, and other functions. The primary importance of c-di AMP stems from its essentiality for many bacteria under standard growth conditions and the ability of several eukaryotic proteins to sense its presence in the cell cytoplasm and trigger an immune response by the host cells. We review here the tertiary structures of the domains that regulate c-di-AMP synthesis and signaling, and the mechanisms of c-di-AMP binding, including the principal conformations of c-di-AMP, observed in various crystal structures. We discuss how these c-di-AMP molecules are bound to the protein and riboswitch receptors and what kinds of interactions account for the specific high-affinity binding of the c-di-AMP ligand. We describe seven kinds of non-covalent-π interactions between c-di-AMP and its receptor proteins, including π-π, C-H-π, cation-π, polar-π, hydrophobic-π, anion-π and the lone pair-π interactions. We also compare the mechanisms of c-di-AMP and c-di-GMP binding by the respective receptors that allow these two cyclic dinucleotides to control very different biological functions.
Topics: Animals; Dinucleoside Phosphates; Molecular Conformation; Riboswitch; Second Messenger Systems; Signal Transduction
PubMed: 32095817
DOI: 10.1093/nar/gkaa112 -
Angewandte Chemie (International Ed. in... Jul 2022The synthesis of complementary strands is the reaction underlying the replication of genetic information. It is likely that the earliest self-replicating systems used...
The synthesis of complementary strands is the reaction underlying the replication of genetic information. It is likely that the earliest self-replicating systems used RNA as genetic material. How RNA was copied in the absence of enzymes and what sequences were most likely to have supported replication is not clear. Here we show that mixtures of dinucleotides with C and G as bases copy an RNA sequence of up to 12 nucleotides in dilute aqueous solution. Successful enzyme-free copying occurred with in situ activation at 4 °C and pH 6.0. Dimers were incorporated in favor of monomers when both competed as reactants, and little misincorporation was detectable in mass spectra. Simulations using experimental rate constants confirmed that mixed C/G sequences are good candidates for successful replication with dimers. Because dimers are intermediates in the synthesis of longer strands, our results support evolutionary scenarios encompassing formation and copying of RNA strands in enzyme-free fashion.
Topics: Dinucleoside Phosphates; Mass Spectrometry; Nucleotides; RNA
PubMed: 35445525
DOI: 10.1002/anie.202203067 -
Communications Biology Jul 2022Predicting protein-protein interaction and non-interaction are two important different aspects of multi-body structure predictions, which provide vital information about...
Predicting protein-protein interaction and non-interaction are two important different aspects of multi-body structure predictions, which provide vital information about protein function. Some computational methods have recently been developed to complement experimental methods, but still cannot effectively detect real non-interacting protein pairs. We proposed a gene sequence-based method, named NVDT (Natural Vector combine with Dinucleotide and Triplet nucleotide), for the prediction of interaction and non-interaction. For protein-protein non-interactions (PPNIs), the proposed method obtained accuracies of 86.23% for Homo sapiens and 85.34% for Mus musculus, and it performed well on three types of non-interaction networks. For protein-protein interactions (PPIs), we obtained accuracies of 99.20, 94.94, 98.56, 95.41, and 94.83% for Saccharomyces cerevisiae, Drosophila melanogaster, Helicobacter pylori, Homo sapiens, and Mus musculus, respectively. Furthermore, NVDT outperformed established sequence-based methods and demonstrated high prediction results for cross-species interactions. NVDT is expected to be an effective approach for predicting PPIs and PPNIs.
Topics: Animals; Dinucleoside Phosphates; Drosophila melanogaster; Genetic Techniques; Genetic Vectors; Helicobacter pylori; Mice; Saccharomyces cerevisiae
PubMed: 35780196
DOI: 10.1038/s42003-022-03617-0