-
Nucleic Acids Research Feb 2021Regulatory protein access to the DNA duplex 'interior' depends on local DNA 'breathing' fluctuations, and the most fundamental of these are thermally-driven base...
Regulatory protein access to the DNA duplex 'interior' depends on local DNA 'breathing' fluctuations, and the most fundamental of these are thermally-driven base stacking-unstacking interactions. The smallest DNA unit that can undergo such transitions is the dinucleotide, whose structural and dynamic properties are dominated by stacking, while the ion condensation, cooperative stacking and inter-base hydrogen-bonding present in duplex DNA are not involved. We use dApdA to study stacking-unstacking at the dinucleotide level because the fluctuations observed are likely to resemble those of larger DNA molecules, but in the absence of constraints introduced by cooperativity are likely to be more pronounced, and thus more accessible to measurement. We study these fluctuations with a combination of Molecular Dynamics simulations on the microsecond timescale and Markov State Model analyses, and validate our results by calculations of circular dichroism (CD) spectra, with results that agree well with the experimental spectra. Our analyses show that the CD spectrum of dApdA is defined by two distinct chiral conformations that correspond, respectively, to a Watson-Crick form and a hybrid form with one base in a Hoogsteen configuration. We find also that ionic structure and water orientation around dApdA play important roles in controlling its breathing fluctuations.
Topics: Circular Dichroism; DNA; Dinucleoside Phosphates; Ions; Markov Chains; Models, Molecular; Sodium Chloride; Water
PubMed: 33503257
DOI: 10.1093/nar/gkab015 -
Chembiochem : a European Journal of... Mar 2021Cyclic dinucleotide signaling systems, which are found ubiquitously throughout nature, allow organisms to rapidly and dynamically sense and respond to alterations in...
Cyclic dinucleotide signaling systems, which are found ubiquitously throughout nature, allow organisms to rapidly and dynamically sense and respond to alterations in their environments. In recent years, the second messenger, cyclic di-(3',5')-adenosine monophosphate (c-di-AMP), has been identified as an essential signaling molecule in a diverse array of bacterial genera. We and others have shown that defects in c-di-AMP homeostasis result in severe physiological defects and virulence attenuation in many bacterial species. Despite significant advancements in the field, there is still a major gap in the understanding of the environmental and cellular factors that influence c-di-AMP dynamics due to a lack of tools to sensitively and rapidly monitor changes in c-di-AMP levels. To address this limitation, we describe here the development of a luciferase-based coupled enzyme assay that leverages the cyclic nucleotide phosphodiesterase, CnpB, for the sensitive and high-throughput quantification of 3'3'-c-di-AMP. We also demonstrate the utility of this approach for the quantification of the cyclic oligonucleotide-based anti-phage signaling system (CBASS) effector, 3'3'-cGAMP. These findings establish CDA-Luc as a more affordable and sensitive alternative to conventional c-di-AMP detection tools with broad utility for the study of bacterial cyclic dinucleotide physiology.
Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Adenosine Monophosphate; Bacteria; Bacterial Proteins; Dinucleoside Phosphates; Enzyme Assays; High-Throughput Screening Assays; Hydrolysis; Luciferases; Luminescent Measurements; Mycobacterium tuberculosis
PubMed: 33142009
DOI: 10.1002/cbic.202000667 -
Biochemistry Aug 2023Adenylate kinases play a crucial role in cellular energy homeostasis through the interconversion of ATP, AMP, and ADP in all living organisms. Here, we explore how...
Adenylate kinases play a crucial role in cellular energy homeostasis through the interconversion of ATP, AMP, and ADP in all living organisms. Here, we explore how adenylate kinase (AdK) from interacts with diadenosine tetraphosphate (AP4A), a putative alarmone associated with transcriptional regulation, stress, and DNA damage response. From a combination of EPR and NMR spectroscopy together with X-ray crystallography, we found that AdK interacts with AP4A with two distinct modes that occur on disparate time scales. First, AdK dynamically interconverts between open and closed states with equal weights in the presence of AP4A. On a much slower time scale, AdK hydrolyses AP4A, and we suggest that the dynamically accessed substrate-bound open AdK conformation enables this hydrolytic activity. The partitioning of the enzyme into open and closed states is discussed in relation to a recently proposed linkage between active site dynamics and collective conformational dynamics.
Topics: Escherichia coli; Adenylate Kinase; Hydrolysis; Dinucleoside Phosphates; Catalysis; Catalytic Domain
PubMed: 37418448
DOI: 10.1021/acs.biochem.3c00189 -
Bacterial Second Messenger Cyclic di-AMP Modulates the Competence State in Streptococcus pneumoniae.Journal of Bacteriology Jan 2020(the pneumococcus) is a naturally competent organism that causes diseases such as pneumonia, otitis media, and bacteremia. The essential bacterial second messenger...
(the pneumococcus) is a naturally competent organism that causes diseases such as pneumonia, otitis media, and bacteremia. The essential bacterial second messenger cyclic di-AMP (c-di-AMP) is an emerging player in the stress responses of many pathogens. In , c-di-AMP is produced by a diadenylate cyclase, CdaA, and cleaved by phosphodiesterases Pde1 and Pde2. c-di-AMP binds a transporter of K (Trk) family protein, CabP, which subsequently halts K uptake via the transporter TrkH. Recently, it was reported that Pde1 and Pde2 are essential for pneumococcal virulence in mouse models of disease. To elucidate c-di-AMP-mediated transcription that may lead to changes in pathogenesis, we compared the transcriptomes of wild-type (WT) and Δ Δ strains by transcriptome sequencing (RNA-Seq) analysis. Notably, we found that many competence-associated genes are significantly upregulated in the Δ Δ strain compared to the WT. These genes play a role in DNA uptake, recombination, and autolysis. Competence is induced by a quorum-sensing mechanism initiated by the secreted factor competence-stimulating peptide (CSP). Surprisingly, the Δ Δ strain exhibited reduced transformation efficiency compared to WT bacteria, which was c-di-AMP dependent. Transformation efficiency was also directly related to the [K] in the medium, suggesting a link between c-di-AMP function and the pneumococcal competence state. We found that a strain that possesses a V76G variation in CdaA produced less c-di-AMP and was highly susceptible to CSP. Deletion of and restored the growth of these bacteria in medium with CSP. Overall, our study demonstrates a novel role for c-di-AMP in the competence program of Genetic competence in bacteria leads to horizontal gene transfer, which can ultimately affect antibiotic resistance, adaptation to stress conditions, and virulence. While the mechanisms of pneumococcal competence signaling cascades have been well characterized, the molecular mechanism behind competence regulation is not fully understood. The bacterial second messenger c-di-AMP has previously been shown to play a role in bacterial physiology and pathogenesis. In this study, we provide compelling evidence for the interplay between c-di-AMP and the pneumococcal competence state. These findings not only attribute a new biological function to this dinucleotide as a regulator of competence, transformation, and survival under stress conditions in pneumococci but also provide new insights into how pneumococcal competence is modulated.
Topics: Bacterial Proteins; DNA-Binding Proteins; Dinucleoside Phosphates; Glycine; Hydrogen-Ion Concentration; Potassium; Second Messenger Systems; Sequence Analysis, RNA; Streptococcus pneumoniae; Transcriptome
PubMed: 31767779
DOI: 10.1128/JB.00691-19 -
Nature Communications Feb 2021Many bacteria use cyclic di-AMP as a second messenger to control potassium and osmotic homeostasis. In Bacillus subtilis, several c-di-AMP binding proteins and RNA...
Many bacteria use cyclic di-AMP as a second messenger to control potassium and osmotic homeostasis. In Bacillus subtilis, several c-di-AMP binding proteins and RNA molecules have been identified. Most of these targets play a role in controlling potassium uptake and export. In addition, c-di-AMP binds to two conserved target proteins of unknown function, DarA and DarB, that exclusively consist of the c-di-AMP binding domain. Here, we investigate the function of the c-di-AMP-binding protein DarB in B. subtilis, which consists of two cystathionine-beta synthase (CBS) domains. We use an unbiased search for DarB interaction partners and identify the (p)ppGpp synthetase/hydrolase Rel as a major interaction partner of DarB. (p)ppGpp is another second messenger that is formed upon amino acid starvation and under other stress conditions to stop translation and active metabolism. The interaction between DarB and Rel only takes place if the bacteria grow at very low potassium concentrations and intracellular levels of c-di-AMP are low. We show that c-di-AMP inhibits the binding of DarB to Rel and the DarB-Rel interaction results in the Rel-dependent accumulation of pppGpp. These results link potassium and c-di-AMP signaling to the stringent response and thus to the global control of cellular physiology.
Topics: Bacillus subtilis; Bacterial Proteins; Dinucleoside Phosphates; Guanosine Pentaphosphate; Hydrolases; Models, Biological; Protein Binding; Protein Domains; Second Messenger Systems; Signal Transduction
PubMed: 33619274
DOI: 10.1038/s41467-021-21306-0 -
Angewandte Chemie (International Ed. in... Feb 2023Diadenosine polyphosphates (Ap As) are non-canonical nucleotides whose cellular concentrations increase during stress and are therefore termed alarmones, signaling...
Diadenosine polyphosphates (Ap As) are non-canonical nucleotides whose cellular concentrations increase during stress and are therefore termed alarmones, signaling homeostatic imbalance. Their cellular role is poorly understood. In this work, we assessed Ap As for their usage as cosubstrates for protein AMPylation, a post-translational modification in which adenosine monophosphate (AMP) is transferred to proteins. In humans, AMPylation mediated by the AMPylator FICD with ATP as a cosubstrate is a response to ER stress. Herein, we demonstrate that Ap A is proficiently consumed for AMPylation by FICD. By chemical proteomics using a new chemical probe, we identified new potential AMPylation targets. Interestingly, we found that AMPylation targets of FICD may differ depending on the nucleotide cosubstrate. These results may suggest that signaling at elevated Ap A levels during cellular stress differs from when Ap A is present at low concentrations, allowing response to extracellular cues.
Topics: Humans; Guanosine Pentaphosphate; Proteins; Adenosine Monophosphate; Dinucleoside Phosphates; Protein Processing, Post-Translational
PubMed: 36524454
DOI: 10.1002/anie.202213279 -
Journal of Biomolecular Structure &... 2022Human stimulator of interferon genes (STING) is a signaling adaptor protein that triggers innate immune system by response to cytosolic DNA and second messenger cyclic...
Human stimulator of interferon genes (STING) is a signaling adaptor protein that triggers innate immune system by response to cytosolic DNA and second messenger cyclic dinucleotides (CDNs). Natural CDNs contain purine nucleobase with different phosphodiester linkage types (3'-3', 2'-2' or mixed 2'-3'-linkages) and exhibit different binding affinity towards STING, ranging from micromolar to nanomolar. High-affinity CDNs are considered as suitable candidates for treatment of chronic hepatitis B and cancer. We have used molecular dynamics simulations to investigate dynamical aspects of binding of natural CDNs (specifically, 2'-2'-cGAMP, 2'-3'-cGAMP, 3'-3'-cGAMP, 3'-3'-c-di-AMP, and 3'-3'-c-di-GMP) with STING protein. Our results revealed that CDN/STING interactions are controlled by the balance between fluctuations (conformational changes) in the CDN ligand and the protein dynamics. Binding of different CDNs induces different degrees of conformational/dynamics changes in STING ligand binding cavity, especially in α-helices, the so-called lid region and α-tails. The ligand residence time in STING protein pocket depends on different contribution of R232 and R238 residues interacting with oxygen atoms of phosphodiester groups in ligand, water distribution around interacting charged centers (in protein residues and ligand) and structural stability of closed conformation state of STING protein. These findings may perhaps guide design of new compounds modulating STING activity.Communicated by Ramaswamy H. Sarma.
Topics: Humans; Molecular Dynamics Simulation; Ligands; Dinucleoside Phosphates; DNA; Oligonucleotides
PubMed: 34187319
DOI: 10.1080/07391102.2021.1942213 -
Scientific Reports Feb 2022The zinc finger antiviral protein (ZAP) is known to restrict viral replication by binding to the CpG rich regions of viral RNA, and subsequently inducing viral RNA...
The zinc finger antiviral protein (ZAP) is known to restrict viral replication by binding to the CpG rich regions of viral RNA, and subsequently inducing viral RNA degradation. This enzyme has recently been shown to be capable of restricting SARS-CoV-2. These data have led to the hypothesis that the low abundance of CpG in the SARS-CoV-2 genome is due to an evolutionary pressure exerted by the host ZAP. To investigate this hypothesis, we performed a detailed analysis of many coronavirus sequences and ZAP RNA binding preference data. Our analyses showed neither evidence for an evolutionary pressure acting specifically on CpG dinucleotides, nor a link between the activity of ZAP and the low CpG abundance of the SARS-CoV-2 genome.
Topics: Animals; Base Sequence; Binding Sites; COVID-19; Dinucleoside Phosphates; Evolution, Molecular; Genome, Viral; Host-Pathogen Interactions; Humans; Nucleotide Motifs; Protein Binding; RNA, Viral; RNA-Binding Proteins; SARS-CoV-2; Virus Replication
PubMed: 35165300
DOI: 10.1038/s41598-022-06046-5 -
International Journal of Molecular... Jun 2022AppA, the periplasmic phytase of clade 2 of the histidine phosphatase (HP2) family, has been well-characterized and successfully engineered for use as an animal feed...
AppA, the periplasmic phytase of clade 2 of the histidine phosphatase (HP2) family, has been well-characterized and successfully engineered for use as an animal feed supplement. AppA is a 1D-6-phytase and highly stereospecific but transiently accumulates 1D--Ins(2,3,4,5)P and other lower phosphorylated intermediates. If this bottleneck in liberation of orthophosphate is to be obviated through protein engineering, an explanation of its rather rigid preference for the initial site and subsequent cleavage of phytic acid is required. To help explain this behaviour, the role of the catalytic proton donor residue in determining AppA stereospecificity was investigated. Four variants were generated by site-directed mutagenesis of the active site HDT amino acid sequence motif containing the catalytic proton donor, D304. The identity and position of the prospective proton donor residue was found to strongly influence stereospecificity. While the wild-type enzyme has a strong preference for 1D-6-phytase activity, a marked reduction in stereospecificity was observed for a D304E variant, while a proton donor-less mutant (D304A) displayed exclusive 1D-1/3-phytase activity. High-resolution X-ray crystal structures of complexes of the mutants with a non-hydrolysable substrate analogue inhibitor point to a crucial role played by D304 in stereospecificity by influencing the size and polarity of specificity pockets A and B. Taken together, these results provide the first evidence for the involvement of the proton donor residue in determining the stereospecificity of HP2 phytases and prepares the ground for structure-informed engineering studies targeting the production of animal feed enzymes capable of the efficient and complete dephosphorylation of dietary phytic acid.
Topics: 6-Phytase; Acid Phosphatase; Animals; Dinucleoside Phosphates; Escherichia coli; Escherichia coli Proteins; Phytic Acid; Prospective Studies; Protons
PubMed: 35683026
DOI: 10.3390/ijms23116346 -
Nature Communications May 2020CRISPR-associated (Cas) DNA-endonucleases are remarkably effective tools for genome engineering, but have limited target ranges due to their protospacer adjacent motif...
CRISPR-associated (Cas) DNA-endonucleases are remarkably effective tools for genome engineering, but have limited target ranges due to their protospacer adjacent motif (PAM) requirements. We demonstrate a critical expansion of the targetable sequence space for a type II-A CRISPR-associated enzyme through identification of the natural 5[Formula: see text]-NAAN-3[Formula: see text] PAM preference of Streptococcus macacae Cas9 (SmacCas9). To achieve efficient editing activity, we graft the PAM-interacting domain of SmacCas9 to its well-established ortholog from Streptococcus pyogenes (SpyCas9), and further engineer an increased efficiency variant (iSpyMac) for robust genome editing activity. We establish that our hybrids can target all adenine dinucleotide PAM sequences and possess robust and accurate editing capabilities in human cells.
Topics: Adenine; Amino Acid Sequence; CRISPR-Associated Protein 9; Dinucleoside Phosphates; Gene Editing; HEK293 Cells; Humans; Nucleotide Motifs; Reproducibility of Results; Streptococcus
PubMed: 32424114
DOI: 10.1038/s41467-020-16117-8