Bacterial signaling cascades have recently become of great relevance in the context of bacterial antibiotics resistance. Cyclic diadenylate monophosphate (c-di-AMP) is a key bacterial secondary messenger involved in growth, biofilm formation, virulence gene expression and others. The activation mechanisms of c-di-AMP receptors like the trimeric PII-like proteins upon messenger binding have, however, remained elusive due the pivotal role of highly flexible protein regions. Here, using solution NMR spectroscopy to elucidate the interplay between the ordered and disordered structural elements of the apo and messenger-bound forms of the 44 kDa homotrimeric PII-like signal transduction protein A (PstA), we reveal a sensitive modulation of the conformational ensemble of those extended loops thought to bind the downstream interaction partners by messenger association at the receptor core. The orchestration of the spatial properties of the loops, despite their retained internal dynamics, reveals the importance of allosteric effects even for disordered structural elements, whose steerable ensemble properties have long escaped the classical structural-biology understanding.

Recognition of exogenous RNA by Toll-like receptors (TLRs) is central to pathogen defense. Using two distinct binding pockets, TLR7 and TLR8 recognize RNA degradation products generated by endolysosomal nucleases. RNA modifications present in endogenous RNA prevent TLR activation; notably, pseudouridine-containing RNA lacks immunostimulatory activity. Indeed, this property has been critical to the successful implementation of mRNA technology for medical purposes. However, the molecular mechanism for this immune evasion has remained elusive. Here, we report that RNase T2 and PLD exonucleases do not adequately process pseudouridine-containing RNA to generate TLR-agonistic ligands. As a second safety mechanism, TLR8 neglects pseudouridine as a ligand for its first binding pocket and TLR7 neglects pseudouridine-containing RNA as a ligand for its second pocket. Interestingly, the medically used N1-methylpseudouridine also evades RNase T2, PLD3, and PLD4 processing but is able to directly activate TLR8. Taken together, our findings provide a molecular basis for self-avoidance by RNA-sensing TLRs.

Human Schlafen 11 (SLFN11) is sensitizing cells to DNA damaging agents by irreversibly blocking stalled replication forks, making it a potential predictive biomarker in chemotherapy. Furthermore, SLFN11 acts as a pattern recognition receptor for single-stranded DNA (ssDNA) and functions as an antiviral restriction factor, targeting translation in a codon-usage-dependent manner through its endoribonuclease activity. However, the regulation of the various SLFN11 functions and enzymatic activities remains enigmatic. Here, we present cryo-electron microscopy (cryo-EM) structures of SLFN11 bound to tRNA-Leu and tRNA-Met that give insights into tRNA binding and cleavage, as well as its regulation by phosphorylation at S219 and T230. SLFN11 phosphomimetic mutant S753D adopts a monomeric conformation, shows ATP binding, but loses its ability to bind ssDNA and shows reduced ribonuclease activity. Thus, the phosphorylation site S753 serves as a conformational switch, regulating SLFN11 dimerization, as well as ATP and ssDNA binding, while S219 and T230 regulate tRNA recognition and nuclease activity.

The cGAS-STING pathway is known for its role in sensing cytosolic DNA introduced by a viral infection, bacterial invasion or tumorigenesis. Free DNA is recognized by the cyclic GMP-AMP synthase (cGAS) catalyzing the production of 2',3'-cyclic guanosine monophosphate-adenosine monophosphate (2',3'-cGAMP) in mammals. This cyclic dinucleotide acts as a second messenger, activating the stimulator of interferon genes (STING) that finally triggers the transcription of interferon genes and inflammatory cytokines. Due to the therapeutic potential of this pathway, both the production and the detection of cGAMP via fluorescent moieties for assay development is of great importance. Here, we introduce the paralleled synthetic access to the intrinsically fluorescent, cyclic dinucleotides 2'3'-c(th)GAMP and 3'3'-c(th)GAMP based on phosphoramidite and phosphate chemistry, adaptable for large scale synthesis. We examine their binding properties to murine and human STING and confirm biological activity including interferon induction by 2'3'-c(th)GAMP in THP-1 monocytes. Two-photon imaging revealed successful cellular uptake of 2'3'-c(th)GAMP in THP-1 cells.

2',3'-cGAMP is a cyclic A- and G-containing dinucleotide second messenger, which is formed upon cellular recognition of foreign cytosolic DNA as part of the innate immune response. The molecule binds to the adaptor protein STING, which induces an immune response characterized by the production of type I interferons and cytokines. The development of STING-binding molecules with both agonistic as well as antagonistic properties is currently of tremendous interest to induce or enhance antitumor or antiviral immunity on the one hand, or to treat autoimmune diseases on the other hand. To escape the host innate immune recognition, some viruses encode poxin endonucleases that cleave 2',3'-cGAMP. Here we report that dideoxy-2',3'-cGAMP (1) and analogs thereof, which lack the secondary ribose-OH groups, form a group of poxin-stable STING agonists. Despite their reduced affinity to STING, particularly the compound constructed from two A nucleosides, dideoxy-2',3'-cAAMP (2), features an unusually high antitumor response in mice.

All living cells use cyclic nucleotides as second messengers for signal sensing and transduction. Cyclic di-3',5'-adenosine monophosphate (c-di-AMP) is primarily involved in the control of bacterial and euryarcheal osmoadaptation and is produced by diadenylate cyclases from two molecules of ATP. Specific phosphodiesterases hydrolyze c-di-AMP to the linear phosphoadenylate adenosine 5'-pApA or to AMP. Different methods including high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC) and ion exchange chromatography (IEX) can be used to determine activities of c-di-AMP-synthesizing and degrading enzymes. Here, we describe in detail the TLC and IEX methods adapted for characterization of the diadenylate cyclase DisA and the phosphodiesterase AtaC from Streptomyces venezuelae. TLC allows quick and easy separation of radioactive-labeled substrates and products, while IEX avoids utilization of potentially hazardous radioactive substrates and can be used as a good substitute if an HPLC system is not available. Unlike in TLC assays, samples cannot be analyzed in parallel by using the IEX assay, thus it is more time consuming.

The cyclic dinucleotide second messenger c-di-AMP is a major player in regulation of potassium homeostasis and osmolyte transport in a variety of bacteria. Along with various direct interactions with proteins such as potassium channels, the second messenger also specifically binds to transcription factors, thereby altering the processes in the cell on the transcriptional level. We here describe the structural and biochemical characterization of BusR from the human pathogen Streptococcus agalactiae. BusR is a member of a yet structurally uncharacterized subfamily of the GntR family of transcription factors that downregulates transcription of the genes for the BusA (OpuA) glycine-betaine transporter upon c-diAMP binding. We report crystal structures of full-length BusR, its apo and c-di-AMP bound effector domain, as well as cryo-EM structures of BusR bound to its operator DNA. Our structural data, supported by biochemical and biophysical data, reveal that BusR utilizes a unique domain assembly with a tetrameric coiled-coil in between the binding platforms, serving as a molecular ruler to specifically recognize a 22 bp separated bipartite binding motif. Binding of c-di-AMP to BusR induces a shift in equilibrium from an inactivated towards an activated state that allows BusR to bind the target DNA, leading to transcriptional repression. [GRAPHICS] .

Antibiotic-producing Streptomyces use the diadenylate cyclase DisA to synthesize the nucleotide second messenger c-di-AMP, but the mechanism for terminating c-di-AMP signaling and the proteins that bind the molecule to effect signal transduction are unknown. Here, we identify the AtaC protein as a c-di-AMP-specific phosphodiesterase that is also conserved in pathogens such as Streptococcus pneumoniae and Mycobacterium tuberculosis. AtaC is monomeric in solution and binds Mn2+ to specifically hydrolyze c-di-AMP to AMP via the intermediate 5'-pApA. As an effector of c-di-AMP signaling, we characterize the RCK_C domain protein CpeA. c-diAMP promotes interaction between CpeA and the predicted cation/ proton antiporter, CpeB, linking c-di-AMP signaling to ion homeostasis in Actinobacteria. Hydrolysis of c-di-AMP is critical for normal growth and differentiation in Streptomyces, connecting ionic stress to development. Thus, we present the discovery of two components of c-di-AMP signaling in bacteria and show that precise control of this second messenger is essential for ion balance and coordinated development in Streptomyces.

Cyclic GMP–AMP synthase (cGAS) is an innate immune sensor for cytosolic microbial DNA 1 . After binding DNA, cGAS synthesizes the messenger 2′3′-cyclic GMP–AMP (cGAMP) 2 – 4 , which triggers cell-autonomous defence and the production of type I interferons and pro-inflammatory cytokines via the activation of STING 5 . In addition to responding to cytosolic microbial DNA, cGAS also recognizes mislocalized cytosolic self-DNA and has been implicated in autoimmunity and sterile inflammation 6 , 7 . Specificity towards pathogen- or damage-associated DNA was thought to be caused by cytosolic confinement. However, recent findings place cGAS robustly in the nucleus 8 – 10 , where tight tethering of chromatin is important to prevent autoreactivity to self-DNA 8 . Here we show how cGAS is sequestered and inhibited by chromatin. We provide a cryo-electron microscopy structure of the cGAS catalytic domain bound to a nucleosome, which shows that cGAS does not interact with the nucleosomal DNA, but instead interacts with histone 2A–histone 2B, and is tightly anchored to the ‘acidic patch’. The interaction buries the cGAS DNA-binding site B, and blocks the formation of active cGAS dimers. The acidic patch robustly outcompetes agonistic DNA for binding to cGAS, which suggests that nucleosome sequestration can efficiently inhibit cGAS, even when accessible DNA is nearby, such as in actively transcribed genomic regions. Our results show how nuclear cGAS is sequestered by chromatin and provides a mechanism for preventing autoreactivity to nuclear self-DNA.

Kinetochores are macromolecular protein assemblies at centromeres that mediate accurate chromosome segregation during cell division. The outer kinetochore KNL1SPC105, MIS12MTW1, and NDC80NDC80 complexes assemble the KMN network, which harbors the sites of microtubule binding and spindle assembly checkpoint signaling. The buildup of the KMN network that transmits microtubule pulling forces to budding yeast point centromeres is poorly understood. Here, we identify 225 inter-protein crosslinks by mass spectrometry on KMN complexes isolated from Saccharomyces cerevisiae that delineate the KMN subunit connectivity for outer kinetochore assembly. C-Terminal motifs of Nsl1 and Mtw1 recruit the SPC105 complex through Kre28, and both motifs aid tethering of the NDC80 complex by the previously reported Dsn1 C terminus. We show that a hub of three C-terminal MTW1 subunit motifs mediates the cooperative stabilization of the KMN network, which is augmented by a direct NDC80-SPC105 association.

2'3'-cGAMP is an uncanonical cyclic dinucleotide where one A and one G base are connected via a 3'-5' and a unique 2'-5' linkage. The molecule is produced by the cyclase cGAS in response to cytosolic DNA binding. cGAMP activates STING and hence one of the most powerful pathways of innate immunity. cGAMP analogues with uncharged linkages that feature better cellular penetrability are currently highly desired. Here, the synthesis of a cGAMP analogue with one amide and one triazole linkage is reported. The molecule is best prepared via a first CuI -catalyzed click reaction, which establishes the triazole, while the cyclization is achieved by macrolactamization.

The concentration of messenger molecules in bacterial cells needs to be tightly regulated. This can be achieved by either controlling the synthesis rate, degradation, or export by specific transporters, respectively. The regulation of the essential second messenger c-di-AMP is achieved by modulation of the diadenylate cyclase activity as well as by specific phosphodiesterases that hydrolyze c-di-AMP in the cell. We provide here structural and biochemical data on the DHH-type phosphodiesterase TmPDE (TM1595) from Thermotoga maritima. Our analysis shows that TmPDE is preferentially degrading linear dinucleotides, such as 5'-pApA, 5'-pGpG, and 5'-pApG, compared with cyclic dinucleotide substrates. The high-resolution structural data provided here describe all steps of the PDE reaction: the ligand-free enzyme, two substrate-bound states, and three post-reaction states. We can furthermore show that Pde2 from Streptococcus pneumoniae shares both structural features and substrate specificity based on small-angle X-ray scattering data and biochemical assays.

First discovered in prokaryotes and more recently in eukaryotes, cyclic dinucleotides (CDNs) constitute a unique branch of second messenger signaling systems. Within prokaryotes CDNs regulate a wide array of different biological processes, whereas in the vertebrate system CDN signaling is largely dedicated to activation of the innate immune system. In this book chapter we summarize the occurrence and signaling pathways of these small-molecule second messengers, most importantly in the scope of the mammalian immune system. In this regard, our main focus is the role of the cGAS-STING axis in the context of microbial infection and sterile inflammation and its implications for therapeutic applications.

Andes virus (ANDV) is a human-pathogenic hantavirus. Hantaviruses presumably initiate their mRNA synthesis by using cap structures derived from host cell mRNAs, a mechanism called cap-snatching. A signature for a cap-snatching endonuclease is present in the N terminus of hantavirus L proteins. In this study, we aimed to solve the atomic structure of the ANDV endonuclease and characterize its biochemical features. However, the wild-type protein was refractory to expression in Escherichia coli, presumably due to toxic enzyme activity. To circumvent this problem, we introduced attenuating mutations in the domain that were previously shown to enhance L protein expression in mammalian cells. Using this approach, 13 mutant proteins encompassing ANDV L protein residues 1–200 were successfully expressed and purified. Protein stability and nuclease activity of the mutants was analyzed and the crystal structure of one mutant was solved to a resolution of 2.4 Å. Shape in solution was determined by small angle X-ray scattering. The ANDV endonuclease showed structural similarities to related enzymes of orthobunya-, arena-, and orthomyxoviruses, but also differences such as elongated shape and positively charged patches surrounding the active site. The enzyme was dependent on manganese, which is bound to the active site, most efficiently cleaved single-stranded RNA substrates, did not cleave DNA, and could be inhibited by known endonuclease inhibitors. The atomic structure in conjunction with stability and activity data for the 13 mutant enzymes facilitated inference of structure–function relationships in the protein. In conclusion, we solved the structure of a hantavirus cap-snatching endonuclease, elucidated its catalytic properties, and present a highly active mutant form, which allows for inhibitor screening.

The exceptionally conserved metazoan MAB21 proteins are implicated in cell fate decisions and share considerable sequence homology with the cyclic GMP-AMP synthase. cGAS is the major innate immune sensor for cytosolic DNA and produces the second messenger 2′-5′, 3′-5′ cyclic GMP-AMP. Little is known about the structure and biochemical function of other proteins of the cGAS-MAB21 subfamily, such as MAB21L1, MAB21L2 and MAB21L3. We have determined the crystal structure of human full-length MAB21L1. Our analysis reveals high structural conservation between MAB21L1 and cGAS but also uncovers important differences. Although monomeric in solution, MAB21L1 forms a highly symmetric double-pentameric oligomer in the crystal, raising the possibility that oligomerization could be a feature of MAB21L1. In the crystal, MAB21L1 is in an inactive conformation requiring a conformational change - similar to cGAS - to develop any nucleotidyltransferase activity. Co-crystallization with NTP identified a putative ligand binding site of MAB21 proteins that corresponds to the DNA binding site of cGAS. Finally, we offer a structure-based explanation for the effects of MAB21L2 mutations in patients with eye malformations. The underlying residues participate in fold-stabilizing interaction networks and mutations destabilize the protein. In summary, we provide a first structural framework for MAB21 proteins.

Human cytomegalovirus (HCMV) infections of healthy individuals are mostly unnoticed and result in viral latency. However, HCMV can also cause devastating disease, e.g., upon reactivation in immunocompromised patients. Yet, little is known about human immune cell sensing of DNA-encoded HCMV. Recent studies indicated that during viral infection the cyclic GMP/AMP synthase (cGAS) senses cytosolic DNA and catalyzes formation of the cyclic di-nucleotide cGAMP, which triggers stimulator of interferon genes (STING) and thus induces antiviral type I interferon (IFN-I) responses. We found that plasmacytoid dendritic cells (pDC) as well as monocyte-derived DC and macrophages constitutively expressed cGAS and STING. HCMV infection further induced cGAS, whereas STING expression was only moderately affected. Although pDC expressed particularly high levels of cGAS, and the cGAS/STING axis was functional down-stream of STING, as indicated by IFN-I induction upon synthetic cGAMP treatment, pDC were not susceptible to HCMV infection and mounted IFN-I responses in a TLR9-dependent manner. Conversely, HCMV infected monocyte-derived cells synthesized abundant cGAMP levels that preceded IFN-I production and that correlated with the extent of infection. CRISPR/Cas9- or siRNA-mediated cGAS ablation in monocytic THP-1 cells and primary monocyte-derived cells, respectively, impeded induction of IFN-I responses following HCMV infection. Thus, cGAS is a key sensor of HCMV for IFN-I induction in primary human monocyte-derived DC and macrophages.