Flavin Mononucleotide Riboswitch Research Articles

Similarities and Differences in Ligand Binding to Protein and RNA Targets: The Case of Riboflavin.

It is nowadays clear that RNA molecules can play active roles in several biological processes. As a result, an increasing number of RNAs are gradually being identified as potentially druggable targets. In particular, noncoding RNAs can adopt highly organized conformations that are suitable for drug binding. However, RNAs are still considered challenging targets due to their complex structural dynamics and high charge density. Thus, elucidating relevant features of drug-RNA binding is fundamental for advancing drug discovery. Here, by using Molecular Dynamics simulations, we compare key features of ligand binding to proteins with those observed in RNA. Specifically, we explore similarities and differences in terms of (i) conformational flexibility of the target, (ii) electrostatic contribution to binding free energy, and (iii) water and ligand dynamics. As a test case, we examine binding of the same ligand, namely riboflavin, to protein and RNA targets, specifically the riboflavin (RF) kinase and flavin mononucleotide (FMN) riboswitch. The FMN riboswitch exhibited enhanced fluctuations and explored a wider conformational space, compared to the protein target, underscoring the importance of RNA flexibility in ligand binding. Conversely, a similar electrostatic contribution to the binding free energy of riboflavin was found. Finally, greater stability of water molecules was observed in the FMN riboswitch compared to the RF kinase, possibly due to the different shape and polarity of the pockets.

Context-dependence of T-loop Mediated Long-range RNA Tertiary Interactions

The architecture and folding of complex RNAs is governed by a limited set of highly recurrent structural motifs that form long-range tertiary interactions. One of these motifs is the T-loop, which was first identified in tRNA but is broadly distributed across biological RNAs. While the T-loop has been examined in detail in different biological contexts, the various receptors that it interacts with are not as well defined. In this study, we use a cell-based genetic screen in concert with bioinformatic analysis to examine three different, but related, T-loop receptor motifs found in the flavin mononucleotide (FMN) and cobalamin (Cbl) riboswitches. As a host for different T-loop receptors, we employed the env8 class-II Cbl riboswitch, an RNA that uses two T-loop motifs for both folding and supporting the ligand binding pocket. A set of libraries was created in which select nucleotides that participate in the T-loop/T-loop receptor (TL/TLR) interaction were fully randomized. Library members were screened for their ability to support Cbl-dependent expression of a reporter gene. While T-loops appear to be variable in sequence, we find that the functional sequence space is more restricted in the Cbl riboswitch, suggesting that TL/TLR interactions are context dependent. Our data reveal clear sequence signatures for the different types of receptor motifs that align with phylogenic analysis of these motifs in the FMN and Cbl riboswitches. Finally, our data suggest the functional contribution of various nucleobase-mediated long-range interactions within the riboswitch subclass of TL/TLR interactions that are distinct from those found in other RNAs.

Direct evolution of riboflavin kinase significantly enhance flavin mononucleotide synthesis by design and optimization of flavin mononucleotide riboswitch

Flavin mononucleotide (FMN) is the active form of riboflavin. It has a wide range of application scenarios in the pharmaceutical and food additives. However, there are limitations in selecting generic high-throughput screening platforms that improve the properties of enzymes. First, the biosensor in response to FMN concentration was constructed using the FMN riboswitch and confirmed the function of this sensor. Next, the FMN binding site of the sensor was saturated with a mutation that increased its fluorescence range by approximately 127%. Then, the biosensor and the base editing system based on T7RNAP were combined to construct a platform for rapid mutation and screening of riboflavin kinase gene ribC mutants. The mutants screened using this platform increased the yield of FMN by 8-fold. These results indicate that the high-throughput screening platform can rapidly and effectively improve the activity of target enzymes, and provide a new route for screening industrial enzymes.

Affinity-Based Profiling of the Flavin Mononucleotide Riboswitch.

Riboswitches are structural RNA elements that control gene expression. These naturally occurring RNA sensors are of continued interest as antibiotic targets, molecular sensors, and functional elements of synthetic circuits. Here, we describe affinity-based profiling of the flavin mononucleotide (FMN) riboswitch to characterize ligand binding and structural folding. We designed and synthesized photoreactive ligands and used them for photoaffinity labeling. We showed selective labeling of the FMN riboswitch and used this covalent interaction to quantitatively measure ligand binding, which we demonstrate with the naturally occurring antibiotic roseoflavin. We measured conditional riboswitch folding as a function of temperature and cation concentration. Furthermore, combining photoaffinity labeling with reverse transcription revealed ligand binding sites within the aptamer domain with single-nucleotide resolution. The photoaffinity probe was applied to cellular extracts of Bacillus subtilis to demonstrate conditional folding of the endogenous low-abundant ribD FMN riboswitch in biologically derived samples using quantitative PCR. Lastly, binding of the riboswitch-targeting antibiotic roseoflavin to the FMN riboswitch was measured in live bacteria using the photoaffinity probe.

Synthesis and evaluation of antimycobacterial activity of riboflavin derivatives

The riboflavin biosynthetic pathway is a promising target for the development of novel antimycobacterial drugs given the lack of riboflavin transporter in M. tuberculosis. Herein, a series of riboflavin derivatives was designed, synthesized and screened for their antimycobacterial and antibacterial activity. The compounds 1a, 1b, 2a, 3a and 5a displayed noticeable antitubercular activity against M. tuberculosis with minimum inhibitory concentration (MIC99) in the range of 6.25 to 25 μM. The lead compound 5a had a selectivity index of 10.7 in the present study. The compounds 2a, 2b, 2c, 4c and 4d showed relatively low to moderate antibacterial activity (MIC = 100–200 μM) against gram-positive strains. Notably, the compounds do not show any inhibition against gram-negative strains even at 200 μM concentration. Further, molecular docking and binding experiments with representative flavin mononucleotide (FMN) riboswitch suggested that the riboflavin analogs exhibited antimycobacterial activity plausibly through FMN riboswitch-mediated repression of riboflavin biosynthesis. In addition to FMN riboswitch, flavoproteins involved in the flavin biosynthesis could also be target of riboflavin derivatives. In conclusion, the potency and low toxicity of riboflavin analogs particularly 5a (MIC99 = 6.25) make it a lead compound for the synthesis of new analogs for antimycobacterial therapy.

RLDOCK method for predicting RNA-small molecule binding modes.

RNA molecules play critical roles in cellular functions at the level of gene expression and regulation. The intricate 3D structures and the functional roles of RNAs make RNA molecules ideal targets for therapeutic drugs. The rational design of RNA-targeted drug requires accurate modeling of RNA-ligand interactions. Recently a new computational tool, RLDOCK, was developed to predict ligand binding sites and binding poses. Using an iterative multiscale sampling and search algorithm and a energy-based evaluation of ligand poses, the method enables efficient and accurate predictions for RNA-ligand interactions. Here we present a detailed illustration of the computational procedure for the practical implementation of the RLDOCK method. Using Flavin mononucleotide (FMN) docking to F. nucleatum FMN riboswitch as an example, we illustrate the computational protocol for RLDOCK-based prediction of RNA- ligand interactions. The RLDOCK software is freely accessible at http://https://github.com/Vfold-RNA/RLDOCK.

Gram-Negative Antibiotic Active Through Inhibition of an Essential Riboswitch.

Multidrug-resistant Gram-negative (GN) infections for which there are few available treatment options are increasingly common. The development of new antibiotics for these pathogens is challenging because of the inability of most small molecules to accumulate inside GN bacteria. Using recently developed predictive guidelines for compound accumulation in Escherichia coli, we have converted the antibiotic Ribocil C, which targets the flavin mononucleotide (FMN) riboswitch, from a compound lacking whole-cell activity against wild-type GN pathogens into a compound that accumulates to a high level in E. coli, is effective against Gram-negative clinical isolates, and has efficacy in mouse models of GN infections. This compound allows for the first assessment of the translational potential of FMN riboswitch binders against wild-type Gram-negative bacteria.

FMN riboswitch aptamer symmetry facilitates conformational switching through mutually exclusive coaxial stacking configurations

Knowledge of both apo and holo states of riboswitches aid in elucidating the various mechanisms of ligand-induced conformational “switching” that underpin their gene-regulating capabilities. Previous structural studies on the flavin mononucleotide (FMN)-binding aptamer of the FMN riboswitch, however, have revealed minimal conformational changes associated with ligand binding that do not adequately explain the basis for the switching behavior. We have determined a 2.7-Å resolution crystal structure of the ligand-free FMN riboswitch aptamer that is distinct from previously reported structures, particularly in the conformation and orientation of the P1 and P4 helices. The nearly symmetrical tertiary structure provides a mechanism by which one of two pairs of adjacent helices (P3/P4 or P1/P6) undergo collinear stacking in a mutually exclusive manner, in the absence or presence of ligand, respectively. Comparison of these structures suggests the stem-loop that includes P4 and L4 is important for maintaining a global conformational state that, in the absence of ligand, disfavors formation of the P1 regulatory helix. Together, these results provide further insight to the structural basis for conformational switching of the FMN riboswitch.

Structural insights into the interactions of flavin mononucleotide (FMN) and riboflavin with FMN riboswitch: a molecular dynamics simulation study

Antibiotics resistance is becoming a serious problem associated with fatalities and suffering patients. New antibiotics that can target the broader spectrum of cellular processes are warranted. One of the recent approaches in this regard is to target the special type of RNA riboswitches in bacteria. In this report, we have explored the mechanistic pathways of ligand-dependent conformational changes of flavin mononucleotide (FMN) riboswitch using molecular dynamics (MD) simulation studies. Cognate ligands FMN and riboflavin (RBF) have shown very different behavior with FMN riboswitch in terms of their role in the gene regulation process. These two ligands have similar scaffold, except the terminal phosphate group in FMN ligand. The MD simulations reveal that the binding of FMN ligand with the riboswitch does not lead to global folding of structure, rather lead to local changes in riboswitch structure. The binding free energy calculated with molecular mechanics Poisson–Boltzmann surface area method suggests the stronger binding of FMN than RBF to the riboswitch and electrostatic energy contributes chiefly to stabilize the complex. Further, the hydrogen bonding analysis identified the key binding site residues G11, G32, G62 of the riboswitch with FMN and RBF. The critical role of the phosphate group in the FMN ligand for binding with the active site of a riboswitch is also borne out in this study. These results unravel the importance of functional groups in natural ligands on designing newer ligands for FMN riboswitch as new antibiotics in the future.Communicated by Ramaswamy H. Sarma

Mutation-Induced Conformational Changes and Energetics for Binding of FMN Ligand in Flavin Mononucleotide Riboswitch by Molecular Dynamics Simulations

Riboswitches are the type of regulatory elements present in the untranslated region of mRNA and specifically bind to the natural ligand to regulate gene expression. This binding specificity can be affected by even single point mutation incorporated in the core of the riboswitch. In this work, we have examined the mutations at the binding site residue in Flavin Mononucleotide (FMN) riboswitch structure with 30ns molecular dynamics simulations. The interaction of ligand (FMN) with riboswitch has been characterized using root mean square deviation, hydrogen bonding analysis, and the calculated binding affinities. Mutation at A48G and G62U show the enhanced binding energy however, the mutation at A85G, are energetically unfavorable compared to the wild type. This work gives valuable insight into the structures and energetics of the mutated FMN riboswitch to design new hits for biological applications.

Rare variants of the FMN riboswitch class in Clostridium difficile and other bacteria exhibit altered ligand specificity.

Many bacteria use flavin mononucleotide (FMN) riboswitches to control the expression of genes responsible for the biosynthesis and transport of this enzyme cofactor or its precursor, riboflavin. Rare variants of FMN riboswitches found in strains of Clostridium difficile and some other bacteria typically control the expression of proteins annotated as transporters, including multidrug efflux pumps. These RNAs no longer recognize FMN, and differ from the original riboswitch consensus sequence at nucleotide positions normally involved in binding of the ribityl and phosphate moieties of the cofactor. Representatives of one of the two variant subtypes were found to bind the FMN precursor riboflavin and the FMN degradation products lumiflavin and lumichrome. Although the biologically relevant ligand sensed by these variant FMN riboswitches remains uncertain, our findings suggest that many strains of C. difficile might use rare riboswitches to sense flavin degradation products and activate transporters for their detoxification.

Structure-Activity Relationship of Flavin Analogues That Target the Flavin Mononucleotide Riboswitch.

The flavin mononucleotide (FMN) riboswitch is an emerging target for the development of novel RNA-targeting antibiotics. We previously discovered an FMN derivative, 5FDQD, that protects mice against diarrhea-causing Clostridium difficile bacteria. Here, we present the structure-based drug design strategy that led to the discovery of this fluoro-phenyl derivative with antibacterial properties. This approach involved the following stages: (1) structural analysis of all available free and bound FMN riboswitch structures; (2) design, synthesis, and purification of derivatives; (3) in vitro testing for productive binding using two chemical probing methods; (4) in vitro transcription termination assays; and (5) resolution of the crystal structures of the FMN riboswitch in complex with the most mature candidates. In the process, we delineated principles for productive binding to this riboswitch, thereby demonstrating the effectiveness of a coordinated structure-guided approach to designing drugs against RNA.

Crowding Shifts the FMN Recognition Mechanism of Riboswitch Aptamer from Conformational Selection to Induced Fit.

In bacteria, the binding between the riboswitch aptamer domain and ligand is regulated by environmental cues, such as low Mg2+ in macrophages during pathogenesis to ensure spatiotemporal expression of virulence genes. Binding was investigated between the flavin mononucleotide (FMN) riboswitch aptamer and its anionic ligand in the presence of molecular crowding agent without Mg2+ ion, which mimics pathogenic conditions. Structural, kinetic, and thermodynamic analyses under the crowding revealed more dynamic conformational rearrangements of the FMN riboswitch aptamer compared to dilute Mg2+ -containing solution. It is hypothesized that under crowding conditions FMN binds through an induced fit mechanism in contrast to the conformational selection mechanism previously demonstrated in dilute Mg2+ solution. Since these two mechanisms involve different conformational intermediates and rate constants, these findings have practical significance in areas such as drug design and RNA engineering.

Crowding Shifts the FMN Recognition Mechanism of Riboswitch Aptamer from Conformational Selection to Induced Fit

AbstractIn bacteria, the binding between the riboswitch aptamer domain and ligand is regulated by environmental cues, such as low Mg2+ in macrophages during pathogenesis to ensure spatiotemporal expression of virulence genes. Binding was investigated between the flavin mononucleotide (FMN) riboswitch aptamer and its anionic ligand in the presence of molecular crowding agent without Mg2+ ion, which mimics pathogenic conditions. Structural, kinetic, and thermodynamic analyses under the crowding revealed more dynamic conformational rearrangements of the FMN riboswitch aptamer compared to dilute Mg2+‐containing solution. It is hypothesized that under crowding conditions FMN binds through an induced fit mechanism in contrast to the conformational selection mechanism previously demonstrated in dilute Mg2+solution. Since these two mechanisms involve different conformational intermediates and rate constants, these findings have practical significance in areas such as drug design and RNA engineering.

Discovery of Selective RNA-Binding Small Molecules by Affinity-Selection Mass Spectrometry.

Recent advances in understanding the relevance of noncoding RNA (ncRNA) to disease have increased interest in drugging ncRNA with small molecules. The recent discovery of ribocil, a structurally distinct synthetic mimic of the natural ligand of the flavin mononucleotide (FMN) riboswitch, has revealed the potential chemical diversity of small molecules that target ncRNA. Affinity-selection mass spectrometry (AS-MS) is theoretically applicable to high-throughput screening (HTS) of small molecules binding to ncRNA. Here, we report the first application of the Automated Ligand Detection System (ALIS), an indirect AS-MS technique, for the selective detection of small molecule-ncRNA interactions, high-throughput screening against large unbiased small-molecule libraries, and identification and characterization of novel compounds (structurally distinct from both FMN and ribocil) that target the FMN riboswitch. Crystal structures reveal that different compounds induce various conformations of the FMN riboswitch, leading to different activity profiles. Our findings validate the ALIS platform for HTS screening for RNA-binding small molecules and further demonstrate that ncRNA can be broadly targeted by chemically diverse yet selective small molecules as therapeutics.

Validation and Development of an Escherichia coli Riboflavin Pathway Phenotypic Screen Hit as a Small-Molecule Ligand of the Flavin Mononucleotide Riboswitch.

A riboflavin biosynthesis pathway-specific phenotypic screen using a library of compounds, all with unspecified antibiotic activity, identified one small molecule later named ribocil, for which intrinsic antibacterial activity against Escherichia coli was completely suppressed by addition of exogenous riboflavin to the bacterial growth medium. The ability of riboflavin to suppress the activity of ribocil, and further demonstration that ribocil inhibited riboflavin synthesis (IC50=0.3μM), supported that a component of the riboflavin synthesis pathway was the molecular target. Remarkably, resistance mutation selection and whole-genome sequencing showed that the target of ribocil was not an enzyme in the riboflavin biosynthesis pathway, but instead the flavin mononucleotide riboswitch, a noncoding structural RNA element in the ribB gene that encodes a key riboflavin synthesis enzyme. Although ribocil is structurally distinct from the natural riboswitch regulatory ligand flavin mononucleotide, ribocil binding to the riboswitch results in efficient repression of ribB expression and inhibition of riboflavin biosynthesis and bacterial growth. A cell-based riboswitch regulated gene reporter assay as well as an in vitro riboswitch RNA aptamer-binding assay, both of whichare described in detail here along with the riboflavin pathway-specific screen, were developed to further validate the mechanism of action of ribocil and to facilitate the discovery of more potent analogues.

Taxonomic analyses of members of the Streptomyces cinnabarinus cluster, description of Streptomyces cinnabarigriseus sp. nov. and Streptomyces davaonensis sp. nov.

Roseoflavin is the only known riboflavin (vitamin B2) analog with antibiotic properties. It is actively taken up by many micro-organisms and targets flavinmononucleotide riboswitches and flavoproteins. It is described as the product of the tentatively named 'Streptomyces davawensis' JCM 4913. Taxonomic analysis of this strain with a polyphasic approach showed that it is very closely related to Streptomyces cinnabarinus (DSM 40467). The two Streptomyces isolates were obtained from different geographical locations (the Philippines and the Kamchatka Peninsula, respectively), their genomes have been sequenced and the question was whether or not the two isolates were representatives of the same species. As we also worked with another isolate of Streptomyces cinnabarinus JS 360, the producer of the cinnabaramides, we wanted to clarify the taxonomic position of the three isolates by using a polyphasic approach. After analysis of the 16S rRNA gene sequence, we found in total 23 species of the genus Streptomyces that showed a similarity higher than 98.5 % to the three strains. We showed that 'S. davawensis' JCM 4913 and S. cinnabarinus DSM 40467 were very closely related but belong to two different species. Hence, we validate 'S. davawensis' as Streptomyces davaonensis sp. nov. with the type strain JCM 4913T (=DSM 101723T). In addition, the cinnabaramide producer can be clearly differentiated from S. davaonensis and this isolate is described as Streptomyces cinnabarigriseus sp. nov. with strain JS360T (=NCCB 100590T=DSM 101724T) as the type strain.

Dual-Targeting Small-Molecule Inhibitors of the Staphylococcus aureus FMN Riboswitch Disrupt Riboflavin Homeostasis in an Infectious Setting

Riboswitches are bacterial-specific, broadly conserved, non-coding RNA structural elements that control gene expression of numerous metabolic pathways and transport functions essential for cell growth. As such, riboswitch inhibitors represent a new class of potential antibacterial agents. Recently, we identified ribocil-C, a highly selective inhibitor of the flavin mononucleotide (FMN) riboswitch that controls expression of de novo riboflavin (RF, vitamin B2) biosynthesis in Escherichia coli. Here, we provide amechanistic characterization of the antibacterial effects of ribocil-C as well as of roseoflavin (RoF), an antimetabolite analog of RF, among medically significant Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecalis. We provide genetic, biophysical, computational, biochemical, and pharmacological evidence that ribocil-C and RoF specifically inhibit dual FMN riboswitches, separately controlling RF biosynthesis and uptake processes essential for MRSA growth and pathogenesis. Such a dual-targeting mechanism is specifically required to develop broad-spectrum Gram-positive antibacterial agents targeting RF metabolism.

Differential regulation of riboflavin supply genes in Vibrio cholerae

BackgroundRiboflavin is the precursor of important redox cofactors such as flavin mononucleotide (FMN) and flavin adenine dinucleotide, required for several biological processes. Vibrio cholerae, a pathogenic bacterium responsible for the cholera disease, possesses the ability to biosynthesize de novo as well as to uptake riboflavin through the riboflavin biosynthetic pathway (RBP) and the RibN importer, respectively. The intra-organism relationship between riboflavin biosynthesis and uptake functions has not been studied.ResultsThis work determined the transcriptional organization of RBP genes and ribN in V. cholerae through reverse transcription polymerase chain reaction and analyzed their expression when growing with or without extracellular riboflavin using real time PCR. The RBP is organized in three transcriptional units, the major one containing ribD, ribE, ribA and ribH together with genes involved in functions not directly related to riboflavin biosynthesis such as nrdR and nusB. In addition, two independent monocistronic units contain ribA2 and ribB, the later conserving a putative FMN riboswitch. The ribN gene is encoded in operon with a gene coding for a predicted outer membrane protein and a gene encoding a protein with a glutaredoxin domain. Regulation analysis showed that among these transcriptional units, only ribB is negatively regulated by riboflavin and that its repression depends on the RibN riboflavin importer. Moreover, external riboflavin highly induced ribB transcription in a ΔribN strain. Also, a genomic database search found a negative correlation between the presence of nrdR and nusB and the FMN riboswitch in bacterial RBP operons.ConclusionsGrowing in the presence of riboflavin downregulates only a single element among the transcriptional units of riboflavin supply pathways. Thus, endogenous riboflavin biosynthesis seems to be negatively regulated by extracellular riboflavin through its specific effect on transcription of ribB in V. cholerae.

Uptake and Metabolism of Antibiotics Roseoflavin and 8-Demethyl-8-Aminoriboflavin in Riboflavin-Auxotrophic Listeria monocytogenes.

The riboflavin analogs roseoflavin (RoF) and 8-demethyl-8-aminoriboflavin (AF) are produced by the bacteria Streptomyces davawensis and Streptomyces cinnabarinus Riboflavin analogs have the potential to be used as broad-spectrum antibiotics, and we therefore studied the metabolism of riboflavin (vitamin B2), RoF, and AF in the human pathogen Listeria monocytogenes, a bacterium which is a riboflavin auxotroph. We show that the L. monocytogenes protein Lmo1945 is responsible for the uptake of riboflavin, RoF, and AF. Following import, these flavins are phosphorylated/adenylylated by the bifunctional flavokinase/flavin adenine dinucleotide (FAD) synthetase Lmo1329 and adenylylated by the unique FAD synthetase Lmo0728, the first monofunctional FAD synthetase to be described in bacteria. Lmo1329 generates the cofactors flavin mononucleotide (FMN) and FAD, whereas Lmo0728 produces FAD only. The combined activities of Lmo1329 and Lmo0728 are responsible for the intracellular formation of the toxic cofactor analogs roseoflavin mononucleotide (RoFMN), roseoflavin adenine dinucleotide (RoFAD), 8-demethyl-8-aminoriboflavin mononucleotide (AFMN), and 8-demethyl-8-aminoriboflavin adenine dinucleotide (AFAD). In vivo reporter gene assays and in vitro transcription/translation experiments show that the L. monocytogenes FMN riboswitch Rli96, which controls expression of the riboflavin transport gene lmo1945, is negatively affected by riboflavin/FMN and RoF/RoFMN but not by AF/AFMN. Treatment of L. monocytogenes with RoF or AF leads to drastically reduced FMN/FAD levels. We suggest that the reduced flavin cofactor levels in combination with concomitant synthesis of inactive cofactor analogs (RoFMN, RoFAD, AFMN, and AFAD) explain why RoF and AF contribute to antibiotic activity in L. monocytogenes IMPORTANCE: The riboflavin analogs roseoflavin (RoF) and 8-demethyl-8-aminoriboflavin (AF) are small molecules which are produced by Streptomyces davawensis and Streptomyces cinnabarinus RoF and AF were reported to have antibacterial activity, and we studied how these compounds are metabolized by the human bacterial pathogen Listeria monocytogenes We found that the L. monocytogenes protein Lmo1945 mediates uptake of AF and RoF and that the combined activities of the enzymes Lmo1329 and Lmo0728 are responsible for the conversion of AF and RoF to toxic cofactor analogs. Comparative studies with RoF and AF (a weaker antibiotic) suggest that the reduction in FMN/FAD levels and the formation of inactive FMN/FAD analogs explain to a large extent the antibiotic activity of AF and RoF.