Induced-fit Mechanism Research Articles

A Vaccine Strategy Based on the Identification of an Annular Ganglioside Binding Motif in Monkeypox Virus Protein E8L.

The recent outbreak of Monkeypox virus requires the development of a vaccine specifically directed against this virus as quickly as possible. We propose here a new strategy based on a two-step analysis combining (i) the search for binding domains of viral proteins to gangliosides present in lipid rafts of host cells, and (ii) B epitope predictions. Based on previous studies of HIV and SARS-CoV-2 proteins, we show that the Monkeypox virus cell surface-binding protein E8L possesses a ganglioside-binding motif consisting of several subsites forming a ring structure. The binding of the E8L protein to a cluster of gangliosides GM1 mimicking a lipid raft domain is driven by both shape and electrostatic surface potential complementarities. An induced-fit mechanism unmasks selected amino acid side chains of the motif without significantly affecting the secondary structure of the protein. The ganglioside-binding motif overlaps three potential linear B epitopes that are well exposed on the unbound E8L surface that faces the host cell membrane. This situation is ideal for generating neutralizing antibodies. We thus suggest using these three sequences derived from the E8L protein as immunogens in a vaccine formulation (recombinant protein, synthetic peptides or genetically based) specific for Monkeypox virus. This lipid raft/ganglioside-based strategy could be used for developing therapeutic and vaccine responses to future virus outbreaks, in parallel to existing solutions.

Structural dynamics of DNA strand break sensing by PARP-1 at a single-molecule level

Single-stranded breaks (SSBs) are the most frequent DNA lesions threatening genomic integrity. A highly kinked DNA structure in complex with human PARP-1 domains led to the proposal that SSB sensing in Eukaryotes relies on dynamics of both the broken DNA double helix and PARP-1’s multi-domain organization. Here, we directly probe this process at the single-molecule level. Quantitative smFRET and structural ensemble calculations reveal how PARP-1’s N-terminal zinc fingers convert DNA SSBs from a largely unperturbed conformation, via an intermediate state into the highly kinked DNA conformation. Our data suggest an induced fit mechanism via a multi-domain assembly cascade that drives SSB sensing and stimulates an interplay with the scaffold protein XRCC1 orchestrating subsequent DNA repair events. Interestingly, a clinically used PARP-1 inhibitor Niraparib shifts the equilibrium towards the unkinked DNA conformation, whereas the inhibitor EB47 stabilizes the kinked state.

Molecular Symmetry of Permethylated β-Cyclodextrins upon Complexation

The Cn molecular symmetry implicated by the schemes with which cyclodextrins (CDs), the well-known cyclic oligosaccharides, are introduced in the literature, is not valid. Numerous studies have shown that CDs are rather flexible with their macrocycle adopting various conformations that enable the inclusion complexation of guest molecules of various shapes. In this work, the loss and gain of the C7 symmetry of the heptakis (2, 3, 6-tri-O-methyl)-β-CD (TM-β-CD) is investigated by means of its conformation geometrical features in its hydrated form and upon complexation with molecules of different shapes. For this, the crystal structure of the inclusion complex of a bulky guest molecule (giberellic acid) in TM-β-CD is presented for the first time and compared with the previously determined crystal structures of monohydrated TM-β-CD and the inclusion complex of a linear monoterpenoid (geraniol) in TM-β-CD. The structural investigation was complemented by molecular dynamics simulations in an explicit solvent, based on the crystallographically determined models. The crucial role of the guest, in the symmetry gain of the host, reveals a pronounced induced-fit complexation mechanism for permethylated CDs.

Mechanism of threonine ADP-ribosylation of F-actin by a Tc toxin

Tc toxins deliver toxic enzymes into host cells by a unique injection mechanism. One of these enzymes is the actin ADP-ribosyltransferase TccC3, whose activity leads to the clustering of the cellular cytoskeleton and ultimately cell death. Here, we show in atomic detail how TccC3 modifies actin. We find that the ADP-ribosyltransferase does not bind to G-actin but interacts with two consecutive actin subunits of F-actin. The binding of TccC3 to F-actin occurs via an induced-fit mechanism that facilitates access of NAD+ to the nucleotide binding pocket. The following nucleophilic substitution reaction results in the transfer of ADP-ribose to threonine-148 of F-actin. We demonstrate that this site-specific modification of F-actin prevents its interaction with depolymerization factors, such as cofilin, which impairs actin network turnover and leads to steady actin polymerization. Our findings reveal in atomic detail a mechanism of action of a bacterial toxin through specific targeting and modification of F-actin.

Activation of p53: How phosphorylated Ser15 triggers sequential phosphorylation of p53 at Thr18 by CK1δ.

The N-terminal transactivation domain (TAD) of p53 is a disordered region with multiple phosphorylation sites. Phosphorylation at Thr18 is crucial for the release of p53 from its negative regulator, MDM2. In stressed cells, CK1δ is responsible for phosphorylating Thr18, but requires Ser15 to be phosphorylated. To understand the mechanistic underpinnings of this sequential phosphorylation, molecular modeling and molecular dynamics simulation studies of these phosphorylation events were carried out. Our models suggest that a positively charged region on CK1δ near the adenosine triphosphate (ATP) binding pocket, which is conserved across species, sequesters the negatively charged pSer15, thereby constraining the positioning of the rest of the peptide, such that the side chain of Thr18 is positioned close to the γ-phosphate of ATP. Furthermore, our studies show that the phosphorylated p53 TAD1 (p53pSer15) peptide binds more strongly to CK1δ than does p53. p53 adopts a helical structure when bound to CK1δ, which is lost upon phosphorylation at Ser15, thus gaining higher flexibility and ability to morph into the binding site. We propose that upon phosphorylation at Ser15 the p53 TAD1 peptide binds to CK1δ through an electrostatically driven induced fit mechanism resulting in a flanking fuzzy complex.

Cryptic in vitro ubiquitin ligase activity of HDMX towards p53 is probably regulated by an induced fit mechanism.

HDMX and its homologue HDM2 are two essential proteins for the cell; after genotoxic stress, both are phosphorylated near to their RING domain, specifically at serine 403 and 395, respectively. Once phosphorylated, both can bind the p53 mRNA and enhance its translation; however, both recognize p53 protein and provoke its degradation under normal conditions. HDM2 has been well-recognized as an E3 ubiquitin ligase, whereas it has been reported that even with the high similarity between the RING domains of the two homologs, HDMX does not have the E3 ligase activity. Despite this, HDMX is needed for the proper p53 poly-ubiquitination. Phosphorylation at serine 395 changes the conformation of HDM2, helping to explain the switch in its activity, but no information on HDMX has been published. Here, we study the conformation of HDMX and its phospho-mimetic mutant S403D, investigate its E3 ligase activity and dissect its binding with p53. We show that phospho-mutation does not change the conformation of the protein, but HDMX is indeed an E3 ubiquitin ligase in vitro; however, in vivo, no activity was found. We speculated that HDMX is regulated by induced fit, being able to switch activity accordingly to the specific partner as p53 protein, p53 mRNA or HDM2. Our results aim to contribute to the elucidation of the contribution of the HDMX to p53 regulation.

Exploration of Somatostatin Binding Mechanism to Somatostatin Receptor Subtype 4.

Somatostatin (also named as growth hormone-inhibiting hormone or somatotropin release-inhibiting factor) is a regulatory peptide important for the proper functioning of the endocrine system, local inflammatory reactions, mood and motor coordination, and behavioral responses to stress. Somatostatin exerts its effects via binding to G-protein-coupled somatostatin receptors of which the fourth subtype (SSTR4) is a particularly important receptor mediating analgesic, anti-inflammatory, and anti-depressant effects without endocrine actions. Thus, SSTR4 agonists are promising drug candidates. Although the knowledge of the atomic resolution-binding modes of SST would be essential for drug development, experimental elucidation of the structures of SSTR4 and its complexes is still awaiting. In the present study, structures of the somatostatin–SSTR4 complex were produced using an unbiased, blind docking approach. Beyond the static structures, the binding mechanism of SST was also elucidated in the explicit water molecular dynamics (MD) calculations, and key binding modes (external, intermediate, and internal) were distinguished. The most important residues on both receptor and SST sides were identified. An energetic comparison of SST binding to SSTR4 and 2 offered a residue-level explanation of receptor subtype selectivity. The calculated structures show good agreement with available experimental results and indicate that somatostatin binding is realized via prerequisite binding modes and an induced fit mechanism. The identified binding modes and the corresponding key residues provide useful information for future drug design targeting SSTR4.

Induced fit with replica exchange improves protein complex structure prediction.

Despite the progress in prediction of protein complexes over the last decade, recent blind protein complex structure prediction challenges revealed limited success rates (less than 20% models with DockQ score > 0.4) on targets that exhibit significant conformational change upon binding. To overcome limitations in capturing backbone motions, we developed a new, aggressive sampling method that incorporates temperature replica exchange Monte Carlo (T-REMC) and conformational sampling techniques within docking protocols in Rosetta. Our method, ReplicaDock 2.0, mimics induced-fit mechanism of protein binding to sample backbone motions across putative interface residues on-the-fly, thereby recapitulating binding-partner induced conformational changes. Furthermore, ReplicaDock 2.0 clocks in at 150-500 CPU hours per target (protein-size dependent); a runtime that is significantly faster than Molecular Dynamics based approaches. For a benchmark set of 88 proteins with moderate to high flexibility (unbound-to-bound iRMSD over 1.2 Å), ReplicaDock 2.0 successfully docks 61% of moderately flexible complexes and 35% of highly flexible complexes. Additionally, we demonstrate that by biasing backbone sampling particularly towards residues comprising flexible loops or hinge domains, highly flexible targets can be predicted to under 2 Å accuracy. This indicates that additional gains are possible when mobile protein segments are known.

Dichlorophenylpyridine-Based Molecules Inhibit Furin through an Induced-Fit Mechanism.

Inhibitors of the proprotein convertase furin might serve as broad-spectrum antiviral therapeutics. High cellular potency and antiviral activity against acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported for (3,5-dichlorophenyl)pyridine-derived furin inhibitors. Here we characterized the binding mechanism of this inhibitor class using structural, biophysical, and biochemical methods. We established a MALDI-TOF-MS-based furin activity assay, determined IC50 values, and solved X-ray structures of (3,5-dichlorophenyl)pyridine-derived compounds in complex with furin. The inhibitors induced a substantial conformational rearrangement of the active-site cleft by exposing a central buried tryptophan residue. These changes formed an extended hydrophobic surface patch where the 3,5-dichlorophenyl moiety of the inhibitors was inserted into a newly formed binding pocket. Consistent with these structural rearrangements, we observed slow off-rate binding kinetics and strong structural stabilization in surface plasmon resonance and differential scanning fluorimetry experiments, respectively. The discovered furin conformation offers new opportunities for structure-based drug discovery.

Ligandability Assessment of Human Glutathione Transferase M1-1 Using Pesticides as Chemical Probes.

Glutathione transferases (GSTs; EC 2.5.1.18) form a group of multifunctional enzymes that are involved in phase II of the cellular detoxification mechanism and are associated with increased susceptibility to cancer development and resistance to anticancer drugs. The present study aims to evaluate the ligandability of the human GSTM1-1 isoenzyme (hGSTM1-1) using a broad range of structurally diverse pesticides as probes. The results revealed that hGSTM1-1, compared to other classes of GSTs, displays limited ligandability and ligand-binding promiscuity, as revealed by kinetic inhibition studies. Among all tested pesticides, the carbamate insecticide pirimicarb was identified as the strongest inhibitor towards hGSTM1-1. Kinetic inhibition analysis showed that pirimicarb behaved as a mixed-type inhibitor toward glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). To shine a light on the restricted hGSTM1-1 ligand-binding promiscuity, the ligand-free crystal structure of hGSTM1-1 was determined by X-ray crystallography at 1.59 Å-resolution. Comparative analysis of ligand-free structure with the available ligand-bound structures allowed for the study of the enzyme’s plasticity and the induced-fit mechanism operated by hGSTM1-1. The results revealed important structural features of the H-site that contribute to xenobiotic-ligand binding and specificity. It was concluded that hGSTM1-1 interacts preferentially with one-ring aromatic compounds that bind at a discrete site which partially overlaps with the xenobiotic substrate binding site (H-site). The results of the study form a basis for the rational design of new drugs targeting hGSTM1-1.

Genetic code degeneracy is established by the decoding center of the ribosome.

The degeneracy of the genetic code confers a wide array of properties to coding sequences. Yet, its origin is still unclear. A structural analysis has shown that the stability of the Watson–Crick base pair at the second position of the anticodon–codon interaction is a critical parameter controlling the extent of non-specific pairings accepted at the third position by the ribosome, a flexibility at the root of degeneracy. Based on recent cryo-EM analyses, the present work shows that residue A1493 of the decoding center provides a significant contribution to the stability of this base pair, revealing that the ribosome is directly involved in the establishment of degeneracy. Building on existing evolutionary models, we show the evidence that the early appearance of A1493 and A1492 established the basis of degeneracy when an elementary kinetic scheme of translation was prevailing. Logical considerations on the expansion of this kinetic scheme indicate that the acquisition of the peptidyl transferase center was the next major evolutionary step, while the induced-fit mechanism, that enables a sharp selection of the tRNAs, necessarily arose later when G530 was acquired by the decoding center.

Multiple actions of NMS-P715, the monopolar spindle 1 (MPS1) mitotic checkpoint inhibitor in liver fluke-associated cholangiocarcinoma cells

AimNMS-P715 is a potent inhibitor of monopolar spindle 1 (MPS1) mitotic checkpoint kinase. Overexpression of MPS1 is associated with short survival times in patients with cholangiocarcinoma (CCA). This study investigated the anti-cancer effects of NMS-P715 in human CCA cell lines. Main methodsKKU-100 and KKU-213A CCA cell lines were treated with NMS-P715 and cell viability was determined using MTT and colony formation assays. Inhibitory effects of NMS-P715 on cell cycle and apoptosis were evaluated using flow cytometry. Expression of underlying mechanism-related proteins was examined by Western blotting. Mitotic catastrophe was assessed by counting abnormal nuclei. Transwell assays were used to examine cell migration and invasion. Key findingsMolecular docking showed that the NMS-P715/MPS1 complex was driven by an induced-fit mechanism. We provide new evidence that NMS-P715 potently inhibited cell proliferation and colony formation in both CCA cell lines. This was accompanied by induction of G2/M arrest and the consequent induction of mitotic catastrophe, a process that occurs during defective mitosis. The recent study showed that NMS-P715 activated caspase-dependent apoptosis and autophagosome formation with an increase of LC3 A/B-II protein expression in CCA cell lines. NMS-P715 also greatly impeded cell migration and invasion in CCA cell lines. The combination of NMS-P715 and gemcitabine or cisplatin showed synergistic effects on CCA cell proliferation. SignificanceThis study revealed for the first time that NMS-P715 is a promising candidate for combating CCA owing via multiple actions and may be suitable for further development in a clinical study.

Extended Mechanism of the Plasminogen Activator Staphylokinase Revealed by Global Kinetic Analysis: 1000-fold Higher Catalytic Activity than That of Clinically Used Alteplase

The plasminogen activator staphylokinase is a fibrin-specific thrombolytic biomolecule and an attractive target for the development of effective myocardial infarction and stroke therapy. To engineer the protein rationally, a detailed understanding of the biochemical mechanism and limiting steps is essential. Conventional fitting to equations derived on the basis of simplifying approximations may be inaccurate for complex mechanisms such as that of staphylokinase. We employed a modern numerical approach of global kinetic data analysis whereby steady-state kinetics and binding affinity data sets were analyzed in parallel. Our approach provided an extended, revised understanding of the staphylokinase mechanism without simplifying approximations and determined the value of turnover number kcat of 117 s–1 that was 10000-fold higher than that reported in the literature. The model further showed that the rate-limiting step of the catalytic cycle is binding of staphylokinase to plasmin molecules, which occurs via an induced-fit mechanism. The overall staphylokinase effectivity is further influenced by the formation of an inactive staphylokinase·plasminogen complex. Here, we describe a quick and simplified guide for obtaining reliable estimates of key parameters whose determination is critical to fully understand the staphylokinase catalytic functionality and define rational strategies for its engineering. Our study provides an interesting example of how a global numerical analysis of kinetic data can be used to better understand the mechanism and limiting factors of complex biochemical processes. The high catalytic activity of staphylokinase (more than 1000-fold higher than that of the clinically used drug alteplase) determined herein makes this thrombolytic agent a very attractive target for further engineering.

Crystal structures of PigF, an O-methyltransferase involved in the prodigiosin synthetic pathway, reveal an induced-fit substrate-recognition mechanism.

Prodigiosin, a red linear tripyrrole pigment, is a typical secondary metabolite with numerous biological functions, such as anticancer, antibacterial and immunosuppressant activities, and is synthesized through a bifurcated biosynthesis pathway from 4-methoxy-2,2'-bipyrrole-5-carbaldehyde (MBC) and 2-methyl-3-n-amylpyrrole (MAP). The last step in the biosynthetic pathway of MBC is catalysed by PigF, which transfers a methyl group to 4-hydroxy-2,20-bipyrrole-5-carbaldehyde (HBC) to form the final product MBC. However, the catalytic mechanism of PigF is still elusive. In this study, crystal structures of apo PigF and S-adenosylhomocysteine (SAH)-bound PigF were determined. PigF forms a homodimer and each monomer consists of two domains: a C-terminal catalytic domain and an N-terminal dimerization domain. Apo PigF adopts an open conformation, while the structure of the complex with the product SAH adopts a closed conformation. The binding of SAH induces dramatic conformational changes of PigF, suggesting an induced-fit substrate-binding mechanism. Further structural comparison suggests that this induced-fit substrate-recognition mechanism may generally exist in O-methyltransferases. Docking and mutation studies identified three key residues (His98, His247 and Asp248) that are crucial for enzyme activity. The essential function of His247 and Asp248 and structure analysis suggests that both residues are involved in activation of the HBC substrate of PigF. The invariance of Asp248 in PigF further confirmed its essential role. The invariance and essential role of His98 in PigF suggests that it is involved in correctly positioning the substrate. This study provides new insight into the catalytic mechanism of PigF, reveals an induced-fit substrate-recognition model for PigF and broadens the understanding of O-methyltransferases.

Balancing Histone Deacetylase (HDAC) Inhibition and Drug-likeness: Biological and Physicochemical Evaluation of Class I Selective HDAC Inhibitors.

Herein we report the structure‐activity and structure‐physicochemical property relationships of a series of class I selective ortho‐aminoanilides targeting the “foot‐pocket” in HDAC1&2. To balance the structural benefits and the physicochemical disadvantages of these substances, we started with a set of HDACi related to tacedinaline (CI‐994) and evaluated their solubility, lipophilicity (log D7.4) and inhibition of selected HDAC isoforms. Subsequently, we selected the most promising “capless” HDACi and transferred its ZBG to our previously published scaffold featuring a peptoid‐based cap group. The resulting hit compound 10 c (LSH‐A54) showed favorable physicochemical properties and is a potent, selective HDAC1/2 inhibitor. The following evaluation of its slow binding properties revealed that LSH‐A54 binds tightly to HDAC1 in an induced‐fit mechanism. The potent HDAC1/2 inhibitory properties were reflected by attenuated cell migration in a modified wound healing assay and reduced cell viability in a clonogenic survival assay in selected breast cancer cell lines.

Signal Propagation Within the MCL-1/BIM Protein Complex

The protein MCL-1 is a crucial factor in regulating apoptosis, the programmed cell death, and thus plays a major role in numerous cancer types. The allosteric protein MCL-1 is naturally moderated by the BH3-only peptide BIM, which binds at its canonical binding groove. In its isolated form, BIM is disordered but assumes an α-helical shape when bound by MCL-1. The underlying binding mechanism (i.e., induced fit vs conformational selection), as well as the time scales of the signal cascade subsequent to binding, are not understood. Here, an artificially photoswitchable variant of the MCL-1/BIM complex was designed and investigated by transient infrared spectroscopy. By destabilizing the α-helix of BIM with a covalently linked azobenzene photoswitch, the dynamical response of the whole complex upon an ultrafast photo-perturbation was characterized. While the destabilized and partially unfolded BIM still binds to MCL-1, a step-like cascade of structural rearrangements of both, MCL-1 and BIM was detected, spanning a wide range of time scales from pico- to microseconds. The results indicate that BIM binds according to an induced fit mechanism, while the structural adaptations of MCL-1 may constitute an allosteric signal.

Structural and Functional Characterization of Camelus dromedarius Glutathione Transferase M1-1.

Glutathione transferases (GSTs; EC. 2.5.1.18) are a large family of multifunctional enzymes that play crucial roles in the metabolism and inactivation of a broad range of xenobiotic compounds. In the present work, we report the kinetic and structural characterization of the isoenzyme GSTM1-1 from Camelus dromedarius (CdGSTM1-1). The CdGSΤM1-1 was expressed in E. coli BL21 (DE3) and was purified by affinity chromatography. Kinetics analysis showed that the enzyme displays a relative narrow substrate specificity and restricted ability to bind xenobiotic compounds. The crystal structures of CdGSΤM1-1 were determined by X-ray crystallography in complex with the substrate (GSH) or the reaction product (S-p-nitrobenzyl-GSH), providing snapshots of the induced-fit catalytic mechanism. The thermodynamic stability of CdGSTM1-1 was investigated using differential scanning fluorimetry (DSF) in the absence and in presence of GSH and S-p-nitrobenzyl-GSH and revealed that the enzyme’s structure is significantly stabilized by its ligands. The results of the present study advance the understanding of camelid GST detoxification mechanisms and their contribution to abiotic stress adaptation in harsh desert conditions.

Within and Beyond the Nucleotide Addition Cycle of Viral RNA-dependent RNA Polymerases.

Nucleotide addition cycle (NAC) is a fundamental process utilized by nucleic acid polymerases when carrying out nucleic acid biosynthesis. An induced-fit mechanism is usually taken by these polymerases upon NTP/dNTP substrate binding, leading to active site closure and formation of a phosphodiester bond. In viral RNA-dependent RNA polymerases, the post-chemistry translocation is stringently controlled by a structurally conserved motif, resulting in asymmetric movement of the template-product duplex. This perspective focuses on viral RdRP NAC and related mechanisms that have not been structurally clarified to date. Firstly, RdRP movement along the template strand in the absence of catalytic events may be relevant to catalytic complex dissociation or proofreading. Secondly, pyrophosphate or non-cognate NTP-mediated cleavage of the product strand 3′-nucleotide can also play a role in reactivating paused or arrested catalytic complexes. Furthermore, non-cognate NTP substrates, including NTP analog inhibitors, can not only alter NAC when being misincorporated, but also impact on subsequent NACs. Complications and challenges related to these topics are also discussed.

Energy landscape of the SARS-CoV-2 reveals extensive conformational heterogeneity.

Cryo-electron microscopy (cryo-EM) has produced a number of structural models of the SARS-CoV-2 spike, already prompting biomedical outcomes. However, these reported models and their associated electrostatic potential maps represent an unknown admixture of conformations stemming from the underlying energy landscape of the spike protein. As with any protein, some of the spike's conformational motions are expected to be biophysically relevant, but cannot be interpreted only by static models. Using experimental cryo-EM images, we present the energy landscape of the glycosylated spike protein, and identify the diversity of low-energy conformations in the vicinity of its open (so called 1RBD-up) state. The resulting atomic refinement reveal global and local molecular rearrangements that cannot be inferred from an average 1RBD-up cryo-EM model. Here we report varied degrees of “openness” in global conformations of the 1RBD-up state, not revealed in the single-model interpretations of the density maps, together with conformations that overlap with the reported models. We discover how the glycan shield contributes to the stability of these low-energy conformations. Five out of six binding sites we analyzed, including those for engaging ACE2, therapeutic mini-proteins, linoleic acid, two different kinds of antibodies, switch conformations between their known apo- and holo-conformations, even when the global spike conformation is 1RBD-up. This apo-to-holo switching is reminiscent of a conformational preequilibrium. We found only one binding site, namely that of AB-C135 remains in apo state within all the sampled free energy-minimizing models, suggesting an induced fit mechanism for the docking of this antibody to the spike.

Lysine-Dependent Entropy Effects in the B. subtilis Lysine Riboswitch: Insights from Single-Molecule Thermodynamic Studies.

Riboswitches play an important role in RNA-based sensing/gene regulation control for many bacteria. In particular, the accessibility of multiple conformational states at physiological temperatures allows riboswitches to selectively bind a cognate ligand in the aptamer domain, which triggers secondary structural changes in the expression platform, and thereby "switching" between on or off transcriptional or translational states for the downstream RNA. The present work exploits temperature-controlled, single-molecule total internal reflection fluorescence (TIRF) microscopy to study the thermodynamic landscape of such ligand binding/folding processes, specifically for the Bacillus subtilis lysine riboswitch. The results confirm that the riboswitch folds via an induced-fit (IF) mechanism, in which cognate lysine ligand first binds to the riboswitch before structural rearrangement takes place. The transition state to folding is found to be enthalpically favored (ΔHfold‡ < 0), yet with a free-energy barrier that is predominantly entropic (-TΔSfold‡ > 0), which results in folding (unfolding) rate constants strongly dependent (independent) of lysine concentration. Analysis of the single-molecule kinetic "trajectories" reveals this rate constant dependence of kfold on lysine to be predominantly entropic in nature, with the additional lysine conferring preferential advantage to the folding process by the presence of ligands correctly oriented with respect to the riboswitch platform. By way of contrast, van't Hoff analysis reveals enthalpic contributions to the overall folding thermodynamics (ΔH0) to be surprisingly constant and robustly independent of lysine concentration. The results demonstrate the crucial role of hydrogen bonding between the ligand and riboswitch platform but with only a relatively modest fraction (45%) of the overall enthalpy change needed to access the transition state and initiate transcriptional switching.