S-adenosylmethionine Binding Site Research Articles

Involvement of LaeA and Velvet Proteins in Regulating the Production of Mycotoxins and Other Fungal Secondary Metabolites.

Fungi are rich sources of secondary metabolites of agrochemical, pharmaceutical, and food importance, such as mycotoxins, antibiotics, and antitumor agents. Secondary metabolites play vital roles in fungal pathogenesis, growth and development, oxidative status modulation, and adaptation/resistance to various environmental stresses. LaeA contains an S-adenosylmethionine binding site and displays methyltransferase activity. The members of velvet proteins include VeA, VelB, VelC, VelD and VosA for each member with a velvet domain. LaeA and velvet proteins can form multimeric complexes such as VosA-VelB and VelB-VeA-LaeA. They belong to global regulators and are mainly impacted by light. One of their most important functions is to regulate gene expressions that are responsible for secondary metabolite biosynthesis. The aim of this mini-review is to represent the newest cognition of the biosynthetic regulation of mycotoxins and other fungal secondary metabolites by LaeA and velvet proteins. In most cases, LaeA and velvet proteins positively regulate production of fungal secondary metabolites. The regulated fungal species mainly belong to the toxigenic fungi from the genera of Alternaria, Aspergillus, Botrytis, Fusarium, Magnaporthe, Monascus, and Penicillium for the production of mycotoxins. We can control secondary metabolite production to inhibit the production of harmful mycotoxins while promoting the production of useful metabolites by global regulation of LaeA and velvet proteins in fungi. Furthermore, the regulation by LaeA and velvet proteins should be a practical strategy in activating silent biosynthetic gene clusters (BGCs) in fungi to obtain previously undiscovered metabolites.

Structural and Biochemical Studies on Product Inhibition of S-Adenosylmethionine Synthetase from Corynebacterium glutamicum.

S-Adenosylmethionine (SAM) acts as a methyl donor in living organisms, and S-adenosylmethionine synthetase (MetK) is an essential enzyme for cells, as it synthesizes SAM from methionine and adenosine triphosphate (ATP). This study determined the crystal structures of the apo form and adenosine/triphosphate complex form of MetK from Corynebacterium glutamicum (CgMetK). Results showed that CgMetK has an allosteric inhibitor binding site for the SAM product in the vicinity of the active site and is inhibited by SAM both competitively and noncompetitively. Through structure-guided protein engineering, the CgMetKE68A variant was developed that exhibited an almost complete release of inhibition by SAM with rather enhanced enzyme activity. The crystal structure of the CgMetKE68A variant revealed that the formation of a new hydrogen bond between Tyr66 and Glu102 by the E68A mutation disrupted the allosteric SAM binding site and also improved the protein thermal stability by strengthening the tetramerization of the enzyme.

Structures of SARS-CoV-2 N7-methyltransferase with DOT1L and PRMT7 inhibitors provide a platform for new antivirals.

The RNA N7-methyltransferase (MTase) activity of SARS-CoV-2's nsp14 protein is essential for viral replication and is a target for the development of new antivirals. Nsp14 uses S-adenosyl methionine (SAM) as the methyl donor to cap the 5' end of the SARS-CoV-2 mRNA and generates S-adenosyl homocysteine (SAH) as the reaction byproduct. Due to the central role of histone MTases in cancer, many SAM/SAH analogs with properties of cell permeability have recently been developed for the inhibition of these MTases. We have succeeded in identifying two such compounds (SGC0946 and SGC8158) that display significant antiviral activity and bind to the SARS-CoV-2 nsp14 N7-MTase core. Unexpectedly, crystal structures of SGC0946 and SGC8158 with the SARS-CoV-2 nsp14 N7-MTase core identify them as bi-substrate inhibitors of the viral MTase, co-occupying both the SAM and RNA binding sites; positing novel features that can be derivatized for increased potency and selectivity for SARS-CoV-2 nsp14. Taken together, the high-resolution structures and the accompanying biophysical and viral replication data provide a new avenue for developing analogs of SGC0946 and SGC8158 as antivirals.

Protein Arginine Methyltransferase 5 (PRMT5) Mutations in Cancer Cells.

Arginine methylation is a form of posttranslational modification that regulates many cellular functions such as development, DNA damage repair, inflammatory response, splicing, and signal transduction, among others. Protein arginine methyltransferase 5 (PRMT5) is one of nine identified methyltransferases, and it can methylate both histone and non-histone targets. It has pleiotropic functions, including recruitment of repair machinery to a chromosomal DNA double strand break (DSB) and coordinating the interplay between repair and checkpoint activation. Thus, PRMT5 has been actively studied as a cancer treatment target, and small molecule inhibitors of its enzymatic activity have already been developed. In this report, we analyzed all reported PRMT5 mutations appearing in cancer cells using data from the Catalogue of Somatic Mutations in Cancers (COSMIC). Our goal is to classify mutations as either drivers or passengers to understand which ones are likely to promote cellular transformation. Using gold standard artificial intelligence algorithms, we uncovered several key driver mutations in the active site of the enzyme (D306H, L315P, and N318K). In silico protein modeling shows that these mutations may affect the affinity of PRMT5 for S-adenosylmethionine (SAM), which is required as a methyl donor. Electrostatic analysis of the enzyme active site shows that one of these mutations creates a tunnel in the vicinity of the SAM binding site, which may allow interfering molecules to enter the enzyme active site and decrease its activity. We also identified several non-coding mutations that appear to affect PRMT5 splicing. Our analyses provide insights into the role of PRMT5 mutations in cancer cells. Additionally, since PRMT5 single molecule inhibitors have already been developed, this work may uncover future directions in how mutations can affect targeted inhibition.

Structural-Based Virtual Screening of FDA-Approved Drugs Repository for NSP16 Inhibitors, Essential for SARS-COV-2 Invasion Into Host Cells: Elucidation From MM/PBSA Calculation.

NSP16 is one of the structural proteins of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) necessary for its entrance to the host cells. It exhibits 2'O-methyl-transferase (2'O-MTase) activity of NSP16 using methyl group from S-adenosyl methionine (SAM) by methylating the 5-end of virally encoded mRNAs and shields viral RNA, and also controls its replication as well as infection. In the present study, we used in silico approaches of drug repurposing to target and inhibit the SAM binding site in NSP16 using Food and Drug Administration (FDA)-approved small molecules set from Drug Bank database. Among the 2 456 FDA-approved molecules, framycetin, paromomycin, and amikacin were found to be significant binders against the SAM binding cryptic pocket of NSP16 with docking score of -13.708, -14.997 and -15.841 kcal/mol, respectively. Classical molecular dynamics (MD) simulation and molecular mechanics Poisson-Boltzmann surface area (MM/PBSA)-based binding free energy calculation depicted that all these three framycetin, paromomycin, and amikacin might be promising therapeutic leads towards SARS-CoV-2 infections via host immune escape inhibition pathway.

Mycobacterium tuberculosis Methyltransferase Rv1515c Can Suppress Host Defense Mechanisms by Modulating Immune Functions Utilizing a Multipronged Mechanism.

Mycobacterium tuberculosis (M. tb) gene Rv1515c encodes a conserved hypothetical protein exclusively present within organisms of MTB complex and absent in non-pathogenic mycobacteria. In silico analysis revealed that Rv1515c contain S-adenosylmethionine binding site and methyltransferase domain. The DNA binding and DNA methyltransferase activity of Rv1515c was confirmed in vitro. Knock-in of Rv1515c in a model mycobacteria M. smegmatis (M. s_Rv1515c) resulted in remarkable physiological and morphological changes and conferred the recombinant strain with an ability to adapt to various stress conditions, including resistance to TB drugs. M. s_Rv1515c was phagocytosed at a greater rate and displayed extended intra-macrophage survival in vitro. Recombinant M. s_Rv1515c contributed to enhanced virulence by suppressing the host defense mechanisms including RNS and ROS production, and apoptotic clearance. M. s_Rv1515c, while suppressing the phagolysosomal maturation, modulated pro-inflammatory cytokine production and also inhibited antigen presentation by downregulating the expression of MHC-I/MHC-II and co-stimulatory signals CD80 and CD86. Mice infected with M. s_Rv1515c produced more Treg cells than vector control (M. s_Vc) and exhibited reduced effector T cell responses, along-with reduced expression of macrophage activation markers in the chronic phase of infection. M. s_Rv1515c was able to survive in the major organs of mice up to 7 weeks post-infection. These results indicate a crucial role of Rv1515c in M. tb pathogenesis.

Development of Improved Mumps Vaccine Candidates by Mutating Viral mRNA Cap Methyltransferase Sites in the Large Polymerase Protein.

Although a live attenuated vaccine is available for controlling mumps virus (MuV), mumps still outbreaks frequently worldwide. The attenuated MuV vaccine strain S79 is widely used in mumps vaccination in China, but still with many shortcomings, among which the most prominent are the side effects and decreased immunity. Therefore, there is a need to further improve the safety and efficacy of the current MuV vaccine. In the present study, we further attenuated MuV S79 vaccine strain by inhibiting viral mRNA methyltransferase (MTase). We generated a panel of eight recombinant MuVs (rMuVs) carrying mutations in the MTase catalytic site or S-adenosylmethionine (SAM) binding site in the large (L) polymerase protein. These rMuVs are genetically stable and seven rMuVs are more attenuated in replication in cell culture and five rMuVs are more attenuated in replication in lungs of cotton rats compared with the parental vaccine strain S79. Importantly, cotton rats vaccinated with these seven rMuV mutants produced high levels of serum neutralizing antibodies and were completely protected against challenge with a wild-type MuV strain (genotype F). Therefore, our results demonstrate that alteration in the MTase catalytic site or SAM binding site in MuV L protein improves the safety or the immunogenicity of the MuV vaccine and thus mRNA cap MTase may be an effective target for the development of new vaccine candidates for MuV.Electronic supplementary materialThe online version of this article (10.1007/s12250-020-00326-y) contains supplementary material, which is available to authorized users.

Deciphering the role of a unique methyltransferase in Mycobacterial infection

Tuberculosis is a life threatening disease caused by the intracellular pathogen Mycobacterium tuberculosis (Mtb), resulting in around two million deaths annually worldwide. Mtb is known to have acquired certain features that help in establishing infection and persisting for longer durations in the host. Recent studies have revealed the acquisition of certain unique methyltransferases by Mtb as a function of phylogenetic evolution which points towards their potential involvement in mycobacterial pathogenesis. In the present study, we identified one such hypothetical methyltransferase (Rvxxxx) gene unique to Mtb complex i.e., absent in non‐pathogenic Mycobacteria. Our in‐silico analysis revealed that Rvxxxx gene possesses an S‐adenosylmethionine binding site and a methyltransferase domain, which was further confirmed through in vitro studies. The introduction of Rvxxxx in the genome of Mycobacterium smegmatis lead to significant perturbations in the physiological and morphological features of the bacilli as well as markedly enhanced its ability to resist and adapt to hostile conditions. Furthermore, recombinant M. smegmatis showed increase in infectivity and survival within host macrophages ex vivo. In addition, Rvxxxx was also found to modulate the proteome profile of recombinant M. smegmatis. Cumulatively, the results of this study indicate that the identified hypothetical methyltransferase Rvxxxx may play a significant role in Mtb pathogenesis and niche adaptation.Support or Funding InformationThe Council of Scientific and Industrial Research, Govt. of India

Structural Insights into Stimulation of Ash1L's H3K36 Methyltransferase Activity through Mrg15 Binding

The evolutionarily conserved Trithorax group protein Ash1 is a SET domain histone methyltransferase that mono- and dimethylates lysine 36 of histone H3 (H3K36). Ash1 forms a complex with Mrg15 and Nurf55, and the binding of Mrg15 greatly stimulates the catalytic activity of Ash1, yet the underlying molecular mechanisms remain unknown. Here we report the crystal structure of the tandem Mrg15-interacting and SET domains of human Ash1L in complex with Mrg15. Ash1L interacts with Mrg15 principally via a segment located N-terminal to the catalytic SET domain. Surprisingly, an autoinhibitory loop in the post-SET region of Ash1L is destabilized on Mrg15 binding despite no direct contact. Dynamics of the autoinhibitory loop can be attributed to subtle structural changes of the S-adenosylmethionine (SAM) binding pocket induced by Mrg15 binding, implicating a mechanism of conformational coupling between SAM and substrate binding sites. The findings broaden the understanding of regulation of H3K36 methyltransferases.

Structural Basis for Regulation of METTL16, an S-Adenosylmethionine Homeostasis Factor

S-adenosylmethionine (SAM) is an essential metabolite that acts as a cofactor for most methylation events in the cell. The N6-methyladenosine (m6A) methyltransferase METTL16 controls SAM homeostasis by regulating the abundance of SAM synthetase MAT2A mRNA in response to changing intracellular SAM levels. Here we present crystal structures of METTL16 in complex with MAT2A RNA hairpins to uncover critical molecular mechanisms underlying the regulated activity of METTL16. The METTL16-RNA complex structures reveal atomic details of RNA substrates that drive productive methylation by METTL16. In addition, we identify a polypeptide loop in METTL16 near the SAM binding site with an autoregulatory role. We show that mutations that enhance or repress METTL16 activity invitro correlate with changes in MAT2A mRNA levels in cells. Thus, we demonstrate the structural basis for the specific activity of METTL16 and further suggest the molecular mechanisms by which METTL16 efficiency is tuned to regulate SAM homeostasis.

Investigating the Mechanism of Action of Diketopiperazines Inhibitors of the Burkholderia cenocepacia Quorum Sensing Synthase CepI: A Site-Directed Mutagenesis Study.

Quorum sensing (QS) is a bacterial intercellular communication process which controls the production of major virulence factors, such as proteases, siderophores, and toxins, as well as biofilm formation. Since the inhibition of this pathway reduces bacterial virulence, QS is considered a valuable candidate drug target, particularly for the treatment of opportunistic infections, such as those caused by Burkholderia cenocepacia in cystic fibrosis patients. Diketopiperazine inhibitors of the acyl homoserine lactone synthase CepI have been recently described. These compounds are able to impair the ability of B. cenocepacia to produce proteases, siderophores, and to form biofilm, being also active in a Caenorhabditis elegans infection model. However, the precise mechanism of action of the compounds, as well as their effect on the cell metabolism, fundamental for candidate drug optimization, are still not completely defined. Here, we performed a proteomic analysis of B. cenocepacia cells treated with one of these inhibitors, and compared it with a cepI deleted strain. Our results demonstrate that the effects of the compound are similar to the deletion of cepI, clearly confirming that these molecules function as inhibitors of the acyl homoserine lactone synthase. Moreover, to deepen our knowledge about the binding mechanisms of the compound to CepI, we exploited previously published in silico structural insights about this enzyme structure and validated different candidate binding pockets on the enzyme surface using site-directed mutagenesis and biochemical analyses. Our experiments identified a region near the predicted S-adenosylmethionine binding site critically involved in interactions with the inhibitor. These results could be useful for future structure-based optimization of these CepI inhibitors.

The Structure of an As(III) S-Adenosylmethionine Methyltransferase with 3-Coordinately Bound As(III) Depicts the First Step in Catalysis.

Arsenic is a ubiquitous environmental toxic substance and a Class 1 human carcinogen. Arsenic methylation by the enzyme As(III) S-adenosylmethionine (SAM) methyltransferase (ArsM in microbes or AS3MT in animals) detoxifies As(III) in microbes but transforms it into more toxic and potentially more carcinogenic methylated species in humans. We previously proposed a reaction pathway for ArsM/AS3MT that involves initial 3-coordinate binding of As(III). To date, reported structures have had only 2-coordinately bound trivalent arsenicals. Here we report a crystal structure of CmArsM from Cyanidioschyzon sp.5508 in which As(III) is 3-coordinately bound to three conserved cysteine residues with a molecule of the product S-adenosyl-l-homocysteine bound in the SAM binding site. We propose that this structure represents the first step in the catalytic cycle. In a previously reported SAM-bound structure, a disulfide bond is formed between two conserved cysteine residues. Comparison of these two structures indicates that there is a conformational change in the N-terminal domain of CmArsM that moves a loop to allow formation of the 3-coordinate As(III) binding site. We propose that this conformational change is an initial step in the As(III) SAM methyltransferase catalytic cycle.

Enhancement of safety and immunogenicity of the Chinese Hu191 measles virus vaccine by alteration of the S-adenosylmethionine (SAM) binding site in the large polymerase protein

The live-attenuated measles virus (MV) vaccine based on the Hu191 strain has played a significant role in controlling measles in China. However, it has considerable adverse effects that may cause public health burden. We hypothesize that the safety and efficacy of MV vaccine can be improved by altering the S-adenosylmethionine (SAM) binding site in the conserved region VI of the large polymerase protein. To test this hypothesis, we established an efficient reverse genetics system for the rMV-Hu191 strain and generated two recombinant MV-Hu191 carrying mutations in the SAM binding site. These two mutants grew to high titer in Vero cells, were genetically stable, and were significantly more attenuated in vitro and in vivo compared to the parental rMV-Hu191 vaccine strain. Importantly, both MV-Hu191 mutants triggered a higher neutralizing antibody than rMV-Hu191 vaccine and provided complete protection against MV challenge. These results demonstrate its potential for an improved MV vaccine candidate.

Inhibition of histone lysine methyltransferases G9a and GLP by ejection of structural Zn(II)

Histone lysine methyltransferases G9a and GLP are validated targets for the development of new epigenetic drugs. Most, if not all, inhibitors of G9a and GLP target the histone substrate binding site or/and the S-adenosylmethionine cosubstrate binding site. Here, we report an alternative approach for inhibiting the methyltransferase activity of G9a and GLP. For proper folding and enzymatic activity, G9a and GLP contain structural zinc fingers, one of them being adjacent to the S-adenosylmethionine binding site. Our work demonstrates that targeting these labile zinc fingers with electrophilic small molecules results in ejection of structural zinc ions, and consequently inhibition of the methyltransferase activity. Very effective Zn(II) ejection and inhibition of G9a and GLP was observed with clinically used ebselen, disulfiram and cisplatin.

Interaction assessments of the first S-adenosylmethionine competitive inhibitor and the essential interacting partner methylosome protein 50 with protein arginine methyltransferase 5 by combined computational methods

Protein arginine methyltransferase 5 (PRMT5) is the most promising anticancer target in PRMT family. In this study, based on the first S-adenosylmethionine (SAM) competitive small molecule inhibitor (17, compound number is from original paper) of PRMT5 reported in our recent paper, we determined the molecular mechanism of 17 interacting with PRMT5 by computational methods. Previously reported CMP5 was also thought of as a SAM competitive inhibitor of PRMT5, but the direct inhibition activity against PRMT5 at enzymatic level was not provided. Therefore, we tested the half-maximal inhibitory concentration (IC50) of CMP5 against PRMT5 at enzymatic level for the purpose of summarizing the interaction characteristics of SAM binding site inhibitors with PRMT5. Additionally, as the essential interacting partner of PRMT5, the binding attributes of the WD-repeat-containing protein MEP50 (methylosome protein 50) was investigated, and nine key residues that contribute most to PRMT5:MEP50 interaction were identified. These results could be helpful in discovering new potent and specific inhibitors of PRMT5, as well as in designing mutant residue assay to modulate the catalytic activity of PRMT5.

Naturally occurring anti-cancer agents targeting EZH2

Natural products are considered as promising tools for the prevention and treatment of cancer. The enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase unit of polycomb repressor complexes such as PRC2 complex that has oncogenic roles through interference with growth and metastatic potential. Several agents targeting EZH2 has been discovered but they often induce side effects in clinical trials. Recently, EZH2 has emerged as a potential target of natural products with documented anti-cancer effects and this discloses a new scenario for the development of EZH2 inhibitory strategies with agents with low cytotoxic detrimental effects. In fact, several natural products such as curcumin, triptolide, ursolic acid, sulforaphane, davidiin, tanshindiols, gambogic acid, berberine and Alcea rosea have been shown to serve as EZH2 modulators. Mechanisms like inhibition of histone H3K4, H3K27 and H3K36 trimethylation, down-regulation of matrix metalloproteinase expression, competitive binding to the S-adenosylmethionine binding site of EZH2 and modulation of tumor-suppressive microRNAs have been demonstrated to mediate the EZH2-inhibitory activity of the mentioned natural products. This review summarizes the pathways that are regulated by various natural products resulting in the suppression of EZH2, and provides a plausible molecular mechanism for the putative anti-cancer effects of these compounds.

Structural and kinetic studies on RosA, the enzyme catalysing the methylation of 8-demethyl-8-amino-d-riboflavin to the antibiotic roseoflavin.

N,N‐8‐demethyl‐8‐amino‐d‐riboflavin dimethyltransferase (RosA) catalyses the final dimethylation of 8‐demethyl‐8‐amino‐d‐riboflavin (AF) to the antibiotic roseoflavin (RoF) in Streptomyces davawensis. In the present study, we solved the X‐ray structure of RosA, and determined the binding properties of substrates and products. Moreover, we used steady‐state and rapid reaction kinetic studies to obtain detailed information on the reaction mechanism. The structure of RosA was found to be similar to that of previously described S‐adenosylmethionine (SAM)‐dependent methyltransferases, featuring two domains: a mainly α‐helical ‘orthogonal bundle’ and a Rossmann‐like domain (α/β twisted open sheet). Bioinformatics studies and molecular modelling enabled us to predict the potential SAM and AF binding sites in RosA, suggesting that both substrates, AF and SAM, bind independently to their respective binding pocket. This finding was confirmed by kinetic experiments that demonstrated a random‐order ‘bi‐bi’ reaction mechanism. Furthermore, we determined the dissociation constants for substrates and products by either isothermal titration calorimetry or UV/Vis absorption spectroscopy, revealing that both products, RoF and S‐adenosylhomocysteine (SAH), bind more tightly to RosA compared with the substrates, AF and SAM. This suggests that RosA may contribute to roseoflavin resistance in S. davawensis. The tighter binding of products is also reflected by the results of inhibition experiments, in which RoF and SAH behave as competitive inhibitors for AF and SAM, respectively. We also showed that formation of a ternary complex of RosA, RoF and SAH (or SAM) leads to drastic spectral changes that are indicative of a hydrophobic environment.DatabaseStructural data are available in the Protein Data Bank under accession number 4D7K.

Design of a fluorescent ligand targeting the S-adenosylmethionine binding site of the histone methyltransferase MLL1.

The histone methyltransferase MLL1 has been linked to translocation-associated gene fusion in childhood leukemias and is an attractive drug target. High-throughput biochemical analysis of MLL1 methyltransferase activity requires the production of at least a trimeric complex of MLL1, RbBP5 and WDR5 to elicit robust activity. Production of trimeric and higher order MLL1 complexes in the quantities and reproducibility required for high-throughput screening presents a significant impediment to MLL1 drug discovery efforts. We present here a small molecule fluorescent ligand (FL-NAH, 6) that is able to bind to the S-adenosylmethionine (SAM) binding site of MLL1 in a manner independent of the associated complex members. We have used FL-NAH to develop a fluorescence polarization-based SAM displacement assay in a 384-well format targeting the MLL1 SET domain in the absence of associated complex members. FL-NAH competes with SAM and is displaced from the MLL1 SET domain by other SAM-binding site ligands with Kdisp values similar to the higher-order complexes, but is unaffected by the H3 peptide substrate. This assay enables screening for SAM-competitive MLL1 inhibitors without requiring the use of trimeric or higher order MLL1 complexes, significantly reducing screening time and cost.

Identification of Small Molecule Inhibitors of Human As(III) S-Adenosylmethionine Methyltransferase (AS3MT).

Arsenic is the most ubiquitous environmental toxin and carcinogen. Long-term exposure to arsenic is associated with human diseases including cancer, cardiovascular disease, and diabetes. Human As(III) S-adenosylmethionine (SAM) methyltransferases (hAS3MT) methylates As(III) to trivalent mono- and dimethyl species that are more toxic and potentially more carcinogenic than inorganic arsenic. Modulators of hAS3MT activity may be useful for the prevention or treatment of arsenic-related diseases. Using a newly developed high-throughput assay for hAS3MT activity, we identified 10 novel noncompetitive small molecule inhibitors. In silico docking analysis with the crystal structure of an AS3MT orthologue suggests that the inhibitors bind in a cleft between domains that is distant from either the As(III) or SAM binding sites. This suggests the presence of a possible allosteric and regulatory site in the enzyme. These inhibitors may be useful tools for future research in arsenic metabolism and are the starting-point for the development of drugs against hAS3MT.

A Highly Coupled Network of Tertiary Interactions in the SAM-I Riboswitch and Their Role in Regulatory Tuning

RNA folding in vivo is significantly influenced by transcription, which is not necessarily recapitulated by Mg2+-induced folding of the corresponding full-length RNA in vitro. Riboswitches that regulate gene expression at the transcriptional level are an ideal system for investigating this aspect of RNA folding as ligand-dependent termination is obligatorily co-transcriptional, providing a clear readout of the folding outcome. The folding of representative members of the SAM-I family of riboswitches has been extensively analyzed using approaches focusing almost exclusively upon Mg2+ and/or S-adenosylmethionine (SAM)-induced folding of full-length transcripts of the ligand binding domain. To relate these findings to co-transcriptional regulatory activity, we have investigated a set of structure-guided mutations of conserved tertiary architectural elements of the ligand binding domain using an in vitro single-turnover transcriptional termination assay, complemented with phylogenetic analysis and isothermal titration calorimetry data. This analysis revealed a conserved internal loop adjacent to the SAM binding site that significantly affects ligand binding and regulatory activity. Conversely, most single point mutations throughout key conserved features in peripheral tertiary architecture supporting the SAM binding pocket have relatively little impact on riboswitch activity. Instead, a secondary structural element in the peripheral subdomain appears to be the key determinant in observed differences in regulatory properties across the SAM-I family. These data reveal a highly coupled network of tertiary interactions that promote high-fidelity co-transcriptional folding of the riboswitch but are only indirectly linked to regulatory tuning.