BusR senses bipartite DNA binding motifs by a unique molecular ruler architecture.

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-di-AMP 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.

c-di-AMP hydrolysis by the phosphodiesterase AtaC promotes differentiation of multicellular bacteria.

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-di-AMP 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.

Chemical Synthesis of the Fluorescent, Cyclic Dinucleotides cth GAMP.

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'-cth GAMP and 3'3'-cth 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'-cth GAMP in THP-1 monocytes. Two-photon imaging revealed successful cellular uptake of 2'3'-cth GAMP in THP-1 cells.

A Click-Chemistry Linked 2'3'-cGAMP Analogue.

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.

Assessment of Diadenylate Cyclase and c-di-AMP-phosphodiesterase Activities Using Thin-layer and Ion Exchange Chromatography.

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.

C-Terminal Motifs of the MTW1 Complex Cooperatively Stabilize Outer Kinetochore Assembly in Budding Yeast.

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.

Probing the modularity of megasynthases by rational engineering of a fatty acid synthase Type I.

Modularity is a fundamental property of megasynthases such as polyketide synthases (PKSs). In this study, we exploit the close resemblance between PKSs and animal fatty acid synthase (FAS) to re-engineer animal FAS to probe the modularity of the FAS/PKS family. Guided by sequence and structural information, we truncate and dissect animal FAS into its components, and reassemble them to generate new PKS-like modules as well as bimodular constructs. The novel re-engineered modules resemble all four common types of PKSs and demonstrate that this approach can be a powerful tool to deliver products with higher catalytic efficiency. Our data exemplify the inherent plasticity and robustness of the overall FAS/PKS fold, and open new avenues to explore FAS-based biosynthetic pathways for custom compound design.

Structural and Biophysical Analysis of the Soluble DHH/DHHA1-Type Phosphodiesterase TM1595 from Thermotoga maritima.

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.