Ligand-binding Pocket Research Articles

Mechanism of Ligand Discrimination by the NMT1 Riboswitch.

Riboswitches are conserved functional domains in mRNA that almost exclusively exist in bacteria. They regulate the biosynthesis and transport of amino acids and essential metabolites such as coenzymes, nucleobases, and their derivatives by specifically binding small molecules. Due to their ability to precisely discriminate between different cognate molecules as well as their common existence in bacteria, riboswitches have become potential antibacterial drug targets that could deliver urgently needed antibiotics with novel mechanisms of action. In this work, we report the recognition mechanisms of four oxidization products (XAN, AZA, UAC, and HPA) generated during purine degradation by an RNA motif termed the NMT1 riboswitch. Specifically, we investigated the physical interactions between the riboswitch and the oxidized metabolites by computing the changes in the free energy on mutating key nucleobases in the ligand binding pocket of the riboswitch. We discovered that the electrostatic interactions are central to ligand discrimination by this riboswitch. The relative binding free energies of the mutations further indicated that some of the mutations can also strengthen the binding affinities of the ligands (AZA, UAC, and HPA). These mechanistic details are also potentially relevant in the design of novel compounds targeting riboswitches.

How Neuromembrane Lipids Modulate Membrane Proteins: Insights from G-Protein-Coupled Receptors (GPCRs) and Receptor Tyrosine Kinases (RTKs).

Lipids play a diverse and critical role in cellular processes in all tissues. The unique lipid composition of nerve membranes is particularly interesting because it contains, among other things, polyunsaturated lipids, such as docosahexaenoic acid, which the body only gets through the diet. The crucial role of lipids in neurological processes, especially in receptor-mediated cell signaling, is emphasized by the fact that in many neuropathological diseases there are significant deviations in the lipid composition of nerve membranes compared to healthy individuals. The lipid composition of neuromembranes can significantly affect the function of receptors by regulating the physical properties of the membrane or by affecting specific interactions between receptors and lipids. In addition, it is worth noting that the ligand-binding pocket of many receptors is located inside the cell membrane, due to which lipids can even modulate the binding of ligands to their receptors. These mechanisms highlight the importance of lipids in the regulation of membrane receptor activation and function. In this article, we focus on two major protein families: G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) and discuss how lipids affect their function in neuronal membranes, elucidating the basic mechanisms underlying neuronal function and dysfunction.

μMap Photoproximity Labeling Enables Small Molecule Binding Site Mapping.

The characterization of ligand binding modes is a crucial step in the drug discovery process and is especially important in campaigns arising from phenotypic screening, where the protein target and binding mode are unknown at the outset. Elucidation of target binding regions is typically achieved by X-ray crystallography or photoaffinity labeling (PAL) approaches; yet, these methods present significant challenges. X-ray crystallography is a mainstay technique that has revolutionized drug discovery, but in many cases structural characterization is challenging or impossible. PAL has also enabled binding site mapping with peptide- and amino-acid-level resolution; however, the stoichiometric activation mode can lead to poor signal and coverage of the resident binding pocket. Additionally, each PAL probe can have its own fragmentation pattern, complicating the analysis by mass spectrometry. Here, we establish a robust and general photocatalytic approach toward the mapping of protein binding sites, which we define as identification of residues proximal to the ligand binding pocket. By utilizing a catalytic mode of activation, we obtain sets of labeled amino acids in the proximity of the target protein binding site. We use this methodology to map, in vitro, the binding sites of six protein targets, including several kinases and molecular glue targets, and furthermore to investigate the binding site of the STAT3 inhibitor MM-206, a ligand with no known crystal structure. Finally, we demonstrate the successful mapping of drug binding sites in live cells. These results establish μMap as a powerful method for the generation of amino-acid- and peptide-level target engagement data.

How Cryo-EM Has Expanded Our Understanding of Membrane Transporters.

Over the past two decades, technological advances in membrane protein structural biology have provided insight into the molecular mechanisms that transporters use to move diverse substrates across the membrane. However, the plasticity of these proteins' ligand binding pockets, which allows them to bind a range of substrates, also poses a challenge for drug development. Here we highlight the structure, function, and transport mechanism of ATP-binding cassette/solute carrier transporters that are related to several diseases and multidrug resistance: ABCB1, ABCC1, ABCG2, SLC19A1, and SLC29A1. SIGNIFICANCE STATEMENT: ATP-binding cassette transporters and solute carriers play vital roles in clinical chemotherapeutic outcomes. This paper describes the current understanding of the structure of five pharmacologically relevant transporters and how they interact with their ligands.

Conformational response to ligand binding of TMPRSS2, a protease involved in SARS-CoV-2 infection: Insights through computational modeling.

Thanks to the considerable research which has been undertaken in the last few years to improve our understanding of the biology and mechanism of action of SARS-CoV-2, we know how the virus uses its surface spike protein to infect host cells. The transmembrane prosthesis, serine 2 (TMPRSS2) protein, located on the surface of human cells, recognizes the cleavage site in the spike protein, leading to the release of the fusion peptide and entry of the virus into the host cells. Because of its role, TMPRSS2 has been proposed as a drug target to prevent infection by the virus. In this study, we aim to increase our understanding of TMPRSS2 using long scale microsecond atomistic molecular dynamics simulations, focusing on the conformational changes over time. The comparison between simulations conducted on the protein in the native (apo) and inhibited form (holo), has shown that in the holo form the inhibitor stabilizes the catalytic site and induces rearrangements in the extracellular domain of the protein. In turn, it leads to the formation of a new cavity in the vicinity of the ligand binding pocket that is stable in the microsecond time scale. Given the low specificity of known protease inhibitors, these findings suggest a new potential drug target site that can be used to improve TMPRSS2 specific recognition by newly designed inhibitors.

Computational Analysis Depicting Potency of Phytochemicals to Target MAPK Signalling Pathway in Breast Cancer

Background: Breast cancer has caused significant mortality among women worldwide. A comprehensive analysis of the disease indicated that MAPK signalling pathways are primarily involved in cell proliferation and have substantially induced drug resistance amongst the diseased individuals and henceforth may be considered a potential drug target to combat the disease. Methods: Phytochemicals obtained from medicinal plants were evaluated for their efficacy to target MAPK by molecular docking and were compared with synthetic analogues available in the market to target the disease. The stability of complexes was determined using MD Simulation, and interaction energy was calculated. Ligand binding pockets of interaction were also depicted along with the computation of RMSD. Results: Withanolide belonging to Withania somnifera exhibited the highest BA at both 35 × 57 × 38 Å and 33 × 41 × 41 Å sites of MAPK. Withanolide showed a BA of -10.2 Kcal/mol in comparison to AL8697 and Ralimetinib mesylate, which represented BA of -8.5 and -8.0 Kcal/mol, respectively. The Withanolide- MAPK complex showed stable conformation during MD Simulation. RMSD value for Withanolide was found to be the least, thereby indicating the least fluctuations during the interactions. Conclusion: The present study unveiled that Withanolide may emerge as a potential drug candidate to target MAPK signalling pathway in breast cancer.

A protonated cytidine stabilizes the ligand-binding pocket in the preQ1 riboswitch in thermophilic bacteria.

Riboswitches are bacterial mRNA structure elements regulating either transcription or translation of downstream genes in response to high-affinity binding of a low molecular weight ligand. Among this diverse group of RNA structures, the class-I preQ1sensing riboswitches (QSW) stand out since they are the smallest known natural riboswitches. QSW combine ligand sensing and functional control within a single structural domain that adopts a pseudoknot conformation encapsulating both the cognate ligand and the ribosome binding site. QSW also occur in thermophilic bacteria. In these cases, their tertiary structures have to be stable even at temperatures above 60 °C to be functional at the organism's optimal growth temperatures. Despite the available high-resolution structures of these riboswitches, it is not yet understood which tertiary interactions are primarily responsible for their exceptional temperature stability. Here, we show that an intricate three-dimensional network of non-canonical interactions involving various non-neighboring nucleobases is the origin of the riboswitch's thermostability. An essential part of this network is a so far undetected stably protonated cytidine. It is characterized by an exceptional high pKA value of >9.7 and could be unambiguously identified through the application of modern heteronuclear detected NMR experiments.

Discriminating functional and non-functional nuclear-receptor ligands with a conformational selection-inspired machine learning algorithm

By targeting the ligand-binding pocket (LBP) and stabilizing the corresponding state (agonistic or antagonistic) of a nuclear receptor (NR), functional ligands, namely agonists and antagonists, can regulate the expression of the downstream genes. In addition to the functional ligands, there exists another category of ligands that can target the LBP of an NR, but without downstream functions, which are called non-functional binders (NFBs). In practice, it is difficult to determine the molecular category of an LBP-bound ligand since these molecules bind to the same position in an NR. Therefore, in this study, we propose a series of structure-based machine-learning (ML) models to better characterize the molecular category of the LBP-bound ligands. Our result shows that the best-performing model achieves good performance in predicting these molecules with an accuracy >0.85 and is robust enough to be applied in real-world tasks, such as identification of a ligand’s category targeting out-of-training-set (or external) NRs.

An Atomic Level Investigation of Sodium Ions Regulating Agonist and Antagonist Binding in the Active Site of a Novel Target 5HT2BR Against Drug-Resistant Epilepsy.

The study investigates the movement of sodium ions inside the ligand-binding pocket of the class-A GPCR serotonin receptor (5HT2BR), a primary target for modern drugs. The available PDBs are mutant chimeras, so a 3D structure is modeled and validated by structural similarity (84.05%), Ramachandran favorable residues (93.01%), and clash score. Using MD simulations (500 ns), the ion active site is tracked in the presence and absence of ions and ligands. The ions enter the active site along helices III, VI, and VII, and the primary residue (ASP3.32) interacts with ions via H-bond (stronger- ~2.4 Å). The radial distribution function around ASP3.32 rises promptly at intermediate distances (2 Å < r < 4 Å), suggesting a higher probability of finding sodium ions at these distances. The ions stabilize the receptor at a better RMSD and promote stronger interactions (3-H-bonds, 1-π-bond~3.35 Å) with the agonist, and not the antagonist (no H-bond). Simulating unrestrained ligands further confirms this pattern, suggesting that ions might promote agonist binding but not be a prerequisite for antagonist action. The study highlights the mechanistic evaluation of sodium ions mobility in 5HT2BR modulation and ligand binding, showing potential in drug development.

Efficient biosynthesis of high-value 5‑Hydroxytryptophan using a multienzyme cascade

5-Hydroxytryptophan (5-HTP) is a precursor of serotonin. The increasing demand for 5-HTP is driving the search for a more efficient and cost-effective production method. In this study, a new in vitro multi-enzyme cascade system (MECCS) was developed to convert L-tryptophan (L-trp) to 5-HTP in a single container while achieving the cycling and regeneration of cofactors and coenzymes. The rate-limiting enzyme tryptophan hydroxylase (TPH) was truncated to improve the overall efficiency of the cascade. After screening, the mutant M1 (NΔ143/CΔ26) showed excellent enzymatic performance, with a 2.3-fold increase in catalytic activity. Molecular dynamics simulations showed that the increase in active center binding force and the improvement of substrate tunnel in M1 improved the overall catalytic efficiency. In addition, the rigidity enhancement of the ligand binding pocket surrounding the region improved the thermal stability of the enzyme. Under optimized conditions, the MECCS converted 50 mM L-trp to 36.9 mM 5-HTP, with a conversion rate of 73.8%. This study provides an alternative pathway for the efficient production of 5-HTP.

Structural basis of amine odorant perception by a mammal olfactory receptor.

Odorants are detected as smell in the nasal epithelium of mammals by two G-protein-coupled receptor families, the odorant receptors and the trace amine-associated receptors1,2 (TAARs). TAARs emerged following the divergence of jawed and jawless fish, and comprise a large monophyletic family of receptors that recognize volatile amine odorants to elicit both intraspecific and interspecific innate behaviours such as attraction and aversion3-5. Here we report cryo-electron microscopy structures of mouse TAAR9 (mTAAR9) and mTAAR9-Gs or mTAAR9-Golf trimers in complex with β-phenylethylamine, N,N-dimethylcyclohexylamine or spermidine. The mTAAR9 structures contain a deep and tight ligand-binding pocket decorated with a conserved D3.32W6.48Y7.43 motif, which is essential for amine odorant recognition. In the mTAAR9 structure, a unique disulfide bond connecting the N terminus to ECL2 is required for agonist-induced receptor activation. We identify key structural motifs of TAAR family members for detecting monoamines and polyamines and the shared sequence of different TAAR members that are responsible for recognition of the same odour chemical. We elucidate the molecular basis of mTAAR9 coupling to Gs and Golf by structural characterization and mutational analysis. Collectively, our results provide a structural basis for odorant detection, receptor activation and Golf coupling of an amine olfactory receptor.

Molecular basis and dual ligand regulation of tetrameric estrogen receptor α/14-3-3ζ protein complex

Therapeutic strategies targeting nuclear receptors (NRs) beyond their endogenous ligand binding pocket have gained significant scientific interest driven by a need to circumvent problems associated with drug resistance and pharmacological profile. The hub protein 14-3-3 is an endogenous regulator of various NRs, providing a novel entry point for small molecule modulation of NR activity. Exemplified, 14-3-3 binding to the C-terminal F-domain of the estrogen receptor alpha (ERα), and small molecule stabilization of the ERα/14-3-3ζ protein complex by the natural product Fusicoccin A (FC-A), was demonstrated to downregulate ERα-mediated breast cancer proliferation. This presents a novel drug discovery approach to target ERα; however, structural and mechanistic insights into ERα/14-3-3 complex formation are lacking. Here, we provide an in-depth molecular understanding of the ERα/14-3-3ζ complex by isolating 14-3-3ζ in complex with an ERα protein construct comprising its ligand-binding domain (LBD) and phosphorylated F-domain. Bacterial co-expression and co-purification of the ERα/14-3-3ζ complex, followed by extensive biophysical and structural characterization, revealed a tetrameric complex between the ERα homodimer and the 14-3-3ζ homodimer. 14-3-3ζ binding to ERα, and ERα/14-3-3ζ complex stabilization by FC-A, appeared to be orthogonal to ERα endogenous agonist (E2) binding, E2-induced conformational changes, and cofactor recruitment. Similarly, the ERα antagonist 4-hydroxytamoxifen inhibited cofactor recruitment to the ERα LBD while ERα was bound to 14-3-3ζ. Furthermore, stabilization of the ERα/14-3-3ζ protein complex by FC-A was not influenced by the disease-associated and 4-hydroxytamoxifen resistant ERα-Y537S mutant. Together, these molecular and mechanistic insights provide direction for targeting ERα via the ERα/14-3-3 complex as an alternative drug discovery approach.

Computational drug repurposing of Akt-1 allosteric inhibitors for non-small cell lung cancer

Non-small cell lung carcinomas (NSCLC) are the predominant form of lung malignancy and the reason for the highest number of cancer-related deaths. Widespread deregulation of Akt, a serine/threonine kinase, has been reported in NSCLC. Allosteric Akt inhibitors bind in the space separating the Pleckstrin homology (PH) and catalytic domains, typically with tryptophan residue (Trp-80). This could decrease the regulatory site phosphorylation by stabilizing the PH-in conformation. Hence, in this study, a computational investigation was undertaken to identify allosteric Akt-1 inhibitors from FDA-approved drugs. The molecules were docked at standard precision (SP) and extra-precision (XP), followed by Prime molecular mechanics—generalized Born surface area (MM-GBSA), and molecular dynamics (MD) simulations on selected hits. Post XP-docking, fourteen best hits were identified from a library of 2115 optimized FDA-approved compounds, demonstrating several beneficial interactions such as pi–pi stacking, pi-cation, direct, and water-bridged hydrogen bonds with the crucial residues (Trp-80 and Tyr-272) and several amino acid residues in the allosteric ligand-binding pocket of Akt-1. Subsequent MD simulations to verify the stability of chosen drugs to the Akt-1 allosteric site showed valganciclovir, dasatinib, indacaterol, and novobiocin to have high stability. Further, predictions for possible biological interactions were performed using computational tools such as ProTox-II, CLC-Pred, and PASSOnline. The shortlisted drugs open a new class of allosteric Akt-1 inhibitors for the therapy of NSCLC.

Integration of network pharmacology, transcriptomics and molecular docking reveals two novel hypoglycemic components in snow chrysanthemum

Our previous studies uncovered the glucose-lowering properties of snow chrysanthemum tea, however, the active ingredients and underlying mechanisms were yet to be uncovered. Flavonoids are the most active and abundant components in snow chrysanthemum tea. In this study, we treated leptin-deficient diabetic ob/ob or high-fat diet (HFD)-induced C57BL/6 J obese mice with or without total flavonoids of snow chrysanthemum (TFSC) for 14 weeks. Results indicated that TFSC ameliorated dyslipidemia and fatty liver, thereby reducing hyperlipidemia. Further mechanism experiments, including RNA-seq and experimental validation, revealed TFSC improved glycolipid metabolism primarily by activating the AMPK/Sirt1/PPARγ pathway. Additionally, by integrating UPLC, network pharmacology, transcriptomics, and experimental validation, we identified two novel hypoglycemic compounds, sulfuretin and leptosidin, in TFSC. Treatment with 12.5 μmol/L sulfuretin obviously stimulated cellular glucose consumption, and sulfuretin (3.125, 6.25 and 12.5 μmol/L) significantly mitigated glucose uptake damage and reliably facilitated glucose consumption in insulin-resistant HepG2 cells. Remarkably, sulfuretin interacted with the ligand-binding pocket of PPARγ via three hydrogen bond interactions with the residues LYS-367, GLN-286 and TYR-477. Furthermore, a concentration of 12.5 μmol/L sulfuretin effectively upregulated the expression of PPARγ, exhibiting a comparable potency to a renowned PPARγ agonist at 20 μmol/L. Taken together, our findings have identified two new hypoglycemic compounds and revealed their mechanisms, which significantly expands people’s understanding of the active components in snow chrysanthemum that have hypoglycemic effects.

Hepatocyte Nuclear Factor 4α (HNF4α) Plays a Controlling Role in Expression of the Retinoic Acid Receptor β (RARβ) Gene in Hepatocytes.

HNF4α, a member of the nuclear receptor superfamily, regulates the genes involved in lipid and glucose metabolism. The expression of the RARβ gene in the liver of HNF4α knock-out mice was higher versus wildtype controls, whereas oppositely, RARβ promoter activity was 50% reduced by the overexpression of HNF4α in HepG2 cells, and treatment with retinoic acid (RA), a major metabolite of vitamin A, increased RARβ promoter activity 15-fold. The human RARβ2 promoter contains two DR5 and one DR8 binding motifs, as RA response elements (RARE) proximal to the transcription start site. While DR5 RARE1 was previously reported to be responsive to RARs but not to other nuclear receptors, we show here that mutation in DR5 RARE2 suppresses the promoter response to HNF4α and RARα/RXRα. Mutational analysis of ligand-binding pocket amino acids shown to be critical for fatty acid (FA) binding indicated that RA may interfere with interactions of FA carboxylic acid headgroups with side chains of S190 and R235, and the aliphatic group with I355. These results could explain the partial suppression of HNF4α transcriptional activation toward gene promoters that lack RARE, including APOC3 and CYP2C9, while conversely, HNF4α may bind to RARE sequences in the promoter of the genes such as CYP26A1 and RARβ, activating these genes in the presence of RA. Thus, RA could act as either an antagonist towards HNF4α in genes lacking RAREs, or as an agonist for RARE-containing genes. Overall, RA may interfere with the function of HNF4α and deregulate HNF4α targets genes, including the genes important for lipid and glucose metabolism.

Elucidation of a Thermodynamical Feature Attributed to Substrate Binding to the Prokaryotic H+/Oligopeptide Cotransporter YdgR with Calorimetric Analysis: The Substrate Binding Driven by the Change in Entropy Implies the Release of Bound Water Molecules from the Binding Pocket.

Here, we have elucidated the substrate recognition mechanism by a prokaryotic H+/oligopeptide cotransporter, YdgR, using isothermal titration calorimetry. Under acidic conditions (pH 6.0), the binding of a dipeptide, Val-Ala, to YdgR elicited endothermic enthalpy, which compensated for the increase in entropy due to dipeptide binding. A series of dipeptides were used in the binding titration. The dipeptides represent Val-X and X-Val, where X is Ala, Ser, Val, Tyr, or Phe. Most dipeptides revealed endothermic enthalpy, which was completely compensated by the increase in entropy due to dipeptide binding. The change in enthalpy due to binding correlated well with the change in entropy, whereas the Gibbs free energy involved in the binding of the dipeptide to YdgR remained unchanged irrespective of dipeptide sequences, implying that the binding reaction was driven by entropy, that is, the release of bound water molecules in the binding pocket. It is also important to clarify that, based on the prediction of water molecules in the ligand-binding pocket of YdgR, the release of three bound water molecules in the putative substrate binding pocket occurred through binding to YdgR. In the comparison of Val-X and X-Val dipeptides, the N-terminal region of the binding pocket might contain more bound water molecules than the C-terminal region. In light of these findings, we suggest that bound water molecules might play an important role in substrate recognition and binding by YdgR.

Structural basis for activation of CB1 by an endocannabinoid analog

Endocannabinoids (eCBs) are endogenous ligands of the cannabinoid receptor 1 (CB1), a G protein-coupled receptor that regulates a number of therapeutically relevant physiological responses. Hence, understanding the structural and functional consequences of eCB-CB1 interactions has important implications for designing effective drugs targeting this receptor. To characterize the molecular details of eCB interaction with CB1, we utilized AMG315, an analog of the eCB anandamide to determine the structure of the AMG315-bound CB1 signaling complex. Compared to previous structures, the ligand binding pocket shows some differences. Using docking, molecular dynamics simulations, and signaling assays we investigated the functional consequences of ligand interactions with the “toggle switch” residues F2003.36 and W3566.48. Further, we show that ligand-TM2 interactions drive changes to residues on the intracellular side of TM2 and are a determinant of efficacy in activating G protein. These intracellular TM2 rearrangements are unique to CB1 and are exploited by a CB1-specific allosteric modulator.

Glutamate and GABA receptors in non-neural animals (Placozoa): Preadaptation to neural transmission

Origins of neural system is one of the major transitions in planetary evolution. Many details of these transitions are still unknown. In particular, high diversity of neurotransmitters lacks convincing explanation so far. We analyze homologues of neuronal glutamate and gamma-aminobutyric acid (GABA) receptors of Placozoa – animal phyla lacking neurons but displaying motility and complex behaviour. Phylogenetic analysis and comparison of amino acids in ligand-binding pockets show that glutamate and GABA-like receptors of Placozoa are surprisingly numerous, diverse and fast-evolving. All these traits are characteristic of odorant rather than neurotransmitter receptors of higher animals. We argue that chemoreception system was an important source of diverse receptors for emerging nervous system to recruit, and that amino acid neurotransmitters (glutamate, GABA, glycine) were relevant external stimuli for early animals before the emergence of nervous system.

Androgen receptor signaling and pyrethroids: Potential male infertility consequences.

Infertility is a global health concern inflicting a considerable burden on the global economy and a severe socio-psychological impact. Approximately 15% of couples suffer from infertility globally, with a male factor contribution of approximately 50%. However, male infertility remains largely unexplored, as the burden of infertility is mostly assigned to female people. Endocrine-disrupting chemicals (EDCs) have been proposed as one of the factors causing male infertility. Pyrethroids represent an important class of EDCs, and numerous studies have associated pyrethroid exposure with impaired male reproductive function and development. Therefore, the present study investigated the potentially toxic effects of two common pyrethroids, cypermethrin and deltamethrin, on androgen receptor (AR) signaling. The structural binding characterization of cypermethrin and deltamethrin against the AR ligand-binding pocket was performed using Schrodinger's induced fit docking (IFD) approach. Various parameters were estimated, such as binding interactions, binding energy, docking score, and IFD score. Furthermore, the AR native ligand, testosterone, was subjected to similar experiments against the AR ligand-binding pocket. The results revealed commonality in the amino acid-binding interactions and overlap in other structural parameters between the AR native ligand, testosterone, and the ligands, cypermethrin and deltamethrin. The estimated binding energy values of cypermethrin and deltamethrin were very high and close to those calculated for AR native ligand, testosterone. Taken together, the results of this study suggested potential disruption of AR signaling by cypermethrin and deltamethrin, which may result in androgen dysfunction and subsequent male infertility.

Targeting Riboswitches with Beta-Axial-Substituted Cobalamins.

RNA-targeting small-molecule therapeutics is an emerging field hindered by an incomplete understanding of the basic principles governing RNA-ligand interactions. One way to advance our knowledge in this area is to study model systems where these interactions are better understood, such as riboswitches. Riboswitches bind a wide array of small molecules with high affinity and selectivity, providing a wealth of information on how RNA recognizes ligands through diverse structures. The cobalamin-sensing riboswitch is a particularly useful model system, as similar sequences show highly specialized binding preferences for different biological forms of cobalamin. This riboswitch is also widely dispersed across bacteria and therefore holds strong potential as an antibiotic target. Many synthetic cobalamin forms have been developed for various purposes including therapeutics, but their interaction with cobalamin riboswitches is yet to be explored. In this study, we characterize the interactions of 11 cobalamin derivatives with three representative cobalamin riboswitches using in vitro binding experiments (both chemical footprinting and a fluorescence-based assay) and a cell-based reporter assay. The derivatives show productive interactions with two of the three riboswitches, demonstrating simultaneous plasticity and selectivity within these RNAs. The observed plasticity is partially achieved through a novel structural rearrangement within the ligand binding pocket, providing insight into how similar RNA structures can be targeted. As the derivatives also show in vivo functionality, they serve as several potential lead compounds for further drug development.