Model For Ligand Binding Research Articles

Hemoglobin Kirklareli (α H58L), a New Variant Associated with Iron Deficiency and Increased CO Binding

Mutations in hemoglobin can cause a wide range of phenotypic outcomes, including anemia due to protein instability and red cell lysis. Uncovering the biochemical basis for these phenotypes can provide new insights into hemoglobin structure and function as well as identify new therapeutic opportunities. We report here a new hemoglobin α chain variant in a female patient with mild anemia, whose father also carries the trait and is from the Turkish city of Kirklareli. Both the patient and her father had a His-58(E7) → Leu mutation in α1. Surprisingly, the patient's father is not anemic, but he is a smoker with high levels of HbCO (∼16%). To understand these phenotypes, we examined recombinant human Hb (rHb) Kirklareli containing the α H58L replacement. Mutant α subunits containing Leu-58(E7) autoxidize ∼8 times and lose hemin ∼200 times more rapidly than native α subunits, causing the oxygenated form of rHb Kirklareli to denature very rapidly under physiological conditions. The crystal structure of rHb Kirklareli shows that the α H58L replacement creates a completely apolar active site, which prevents electrostatic stabilization of bound O2, promotes autoxidation, and enhances hemin dissociation by inhibiting water coordination to the Fe(III) atom. At the same time, the mutant α subunit has an ∼80,000-fold higher affinity for CO than O2, causing it to rapidly take up and retain carbon monoxide, which prevents denaturation both in vitro and in vivo and explains the phenotypic differences between the father, who is a smoker, and his daughter.

An integrated perspective on RNA aptamer ligand-recognition models: clearing muddy waters.

Riboswitches are short RNA motifs that sensitively and selectively bind cognate ligands to modulate gene expression. Like protein receptor-ligand pairs, their binding dynamics are traditionally categorized as following one of two paradigmatic mechanisms: conformational selection and induced fit. In conformational selection, ligand binding stabilizes a particular state already present in the receptor's dynamic ensemble. In induced fit, ligand-receptor interactions enable the system to overcome the energetic barrier into a previously inaccessible state. In this article, we question whether a polarized division of RNA binding mechanisms truly meets the conceptual needs of the field. We will review the history behind this classification of RNA-ligand interactions, and the way induced fit in particular has been rehabilitated by single-molecule studies of RNA aptamers. We will highlight several recent results from single-molecule experimental studies of riboswitches that reveal gaps or even contradictions between common definitions of the two terms, and we will conclude by proposing a more robust framework that considers the range of RNA behaviors unveiled in recent years as a reality to be described, rather than an increasingly unwieldy set of exceptions to the traditional models.

Binding characteristics of Cu2+ to natural humic acid fractions sequentially extracted from the lake sediments.

Humic acids (HAs) determine the distribution, toxicity, bioavailability, and ultimate fate of heavy metals in the environment. In this work, ten HA fractions (F1-F10) were used as adsorbent, which were sequentially extracted from natural sediments of Lake Wuliangsuhai, to investigate the binding characteristics of Cu2+ to HA. On the basis of the characterization results, differences were found between the ten extracted HA fractions responding to their elemental compositions and acidic functional groups. The characterization results reveal that the responses of ten extracted HA fractions to their elemental compositions and acidic functional groups were different. The O/C and (O+N)/C ratio of F1-F8 approximately ranged from 0.66 to 0.53 and from 0.72 to 0.61, respectively; the measured results showed that the contents of phenolic groups and carboxyl groups decreased from 4.46 to 2.60mmol/g and 1.60 to 0.58mmol/g, respectively. The binding characteristics of Cu2+ to the ten HA fractions were well modeled by the bi-Langmuir model; the binding behavior of Cu2+ to all the ten HA fractions were strongly impacted by pH and ionic strength. The FTIR and SEM-EDX image of HA fractions (pre- and post-adsorption) revealed that carboxyl and phenolic groups were responsible for the Cu2+ sorption on the ten sequentially extracted HA fractions process, which is the same with the analysis of the ligand binding and bi-Langmuir models Accordingly, the adsorption capacity of the former HA fractions on Cu2+ were higher than the latter ones, which may be attributed to the difference of carboxyl and phenolic group contents between the former and latter extracted HA fractions. Additionally, the functional groups with N and S should not be neglected. This work is hopeful to understand the environmental effect of humic substances, environmental geochemical behavior, and bioavailability of heavy metals in lakes.

Fluorescence-Based Strategies to Investigate the Structure and Dynamics of Aptamer-Ligand Complexes.

In addition to the helical nature of double-stranded DNA and RNA, single-stranded oligonucleotides can arrange themselves into tridimensional structures containing loops, bulges, internal hairpins and many other motifs. This ability has been used for more than two decades to generate oligonucleotide sequences, so-called aptamers, that can recognize certain metabolites with high affinity and specificity. More recently, this library of artificially-generated nucleic acid aptamers has been expanded by the discovery that naturally occurring RNA sequences control bacterial gene expression in response to cellular concentration of a given metabolite. The application of fluorescence methods has been pivotal to characterize in detail the structure and dynamics of these aptamer-ligand complexes in solution. This is mostly due to the intrinsic high sensitivity of fluorescence methods and also to significant improvements in solid-phase synthesis, post-synthetic labeling strategies and optical instrumentation that took place during the last decade. In this work, we provide an overview of the most widely employed fluorescence methods to investigate aptamer structure and function by describing the use of aptamers labeled with a single dye in fluorescence quenching and anisotropy assays. The use of 2-aminopurine as a fluorescent analog of adenine to monitor local changes in structure and fluorescence resonance energy transfer (FRET) to follow long-range conformational changes is also covered in detail. The last part of the review is dedicated to the application of fluorescence techniques based on single-molecule microscopy, a technique that has revolutionized our understanding of nucleic acid structure and dynamics. We finally describe the advantages of monitoring ligand-binding and conformational changes, one molecule at a time, to decipher the complexity of regulatory aptamers and summarize the emerging folding and ligand-binding models arising from the application of these single-molecule FRET microscopy techniques.

Protein Binding Pocket Dynamics.

The dynamics of protein binding pockets are crucial for their interaction specificity. Structural flexibility allows proteins to adapt to their individual molecular binding partners and facilitates the binding process. This implies the necessity to consider protein internal motion in determining and predicting binding properties and in designing new binders. Although accounting for protein dynamics presents a challenge for computational approaches, it expands the structural and physicochemical space for compound design and thus offers the prospect of improved binding specificity and selectivity. A cavity on the surface or in the interior of a protein that possesses suitable properties for binding a ligand is usually referred to as a binding pocket. The set of amino acid residues around a binding pocket determines its physicochemical characteristics and, together with its shape and location in a protein, defines its functionality. Residues outside the binding site can also have a long-range effect on the properties of the binding pocket. Cavities with similar functionalities are often conserved across protein families. For example, enzyme active sites are usually concave surfaces that present amino acid residues in a suitable configuration for binding low molecular weight compounds. Macromolecular binding pockets, on the other hand, are located on the protein surface and are often shallower. The mobility of proteins allows the opening, closing, and adaptation of binding pockets to regulate binding processes and specific protein functionalities. For example, channels and tunnels can exist permanently or transiently to transport compounds to and from a binding site. The influence of protein flexibility on binding pockets can vary from small changes to an already existent pocket to the formation of a completely new pocket. Here, we review recent developments in computational methods to detect and define binding pockets and to study pocket dynamics. We introduce five different classes of protein pocket dynamics: (1) appearance/disappearance of a subpocket in an existing pocket; (2) appearance/disappearance of an adjacent pocket on the protein surface in the direct vicinity of an already existing pocket; (3) pocket breathing, which may be caused by side-chain fluctuations or backbone or interdomain vibrational motion; (4) opening/closing of a channel or tunnel, connecting a pocket inside the protein with solvent, including lid motion; and (5) the appearance/disappearance of an allosteric pocket at a site on a protein distinct from an already existing pocket with binding of a ligand to the allosteric binding site affecting the original pocket. We suggest that the class of pocket dynamics, as well as the type and extent of protein motion affecting the binding pocket, should be factors considered in choosing the most appropriate computational approach to study a given binding pocket. Furthermore, we examine the relationship between pocket dynamics classes and induced fit, conformational selection, and gating models of ligand binding on binding kinetics and thermodynamics. We discuss the implications of protein binding pocket dynamics for drug design and conclude with potential future directions for computational analysis of protein binding pocket dynamics.

Binding characteristics of Pb2+ to natural fulvic acid extracted from the sediments in Lake Wuliangsuhai, Inner Mongolia plateau, P. R. China

Humic substance (HS) is the determining factor in controlling the geochemistry behavior and progress of heavy metals. Using fulvic acid (FA) extracted from natural sediment as sorbent, this work studied binding characteristics between FA and Pb2+ considering the influences of temperature, ionic strength and FA concentrations. The results showed the amount of bound-Pb increased with increasing initial Pb2+ concentrations and FA concentrations, but decreased with increasing ionic strengths. Correspondingly, the logarithm conditional distribution coefficients (log K D) of bound-Pb sharply declined as the increase of initial Pb2+, and log K D increase accompanied the increase of FA concentrations and decrease with the ionic strengths. The two-site ligand binding model fit all the binding experimental data very well and proved that two classes of binding sites were involved in the interaction of Pb2+ with the extracted FA. The results clearly confirmed the chemical heterogeneity of the binding sites present in the FA extracted from the lake sediment. Fourier transform infrared spectra indicated that two types of functional groups exist in the extracted FA and participated in the interaction of FA and Pb2+, corresponding to the two-site ligand binding model in description of the characteristics of Pb2+–FA complex. The apparent stability constant (K S) values calculated for the strong binding sites (S1) were higher than for the weak sites (S2) by 1–2 orders of magnitude; however, S1 sites were limited in numbers, as evidenced by lowering the maximum binding capability (B max) values, which were 3–40 times smaller than those for S2 sites. The bound process of Pb2+ to FA is spontaneous and exothermic from the result of the thermodynamic parameters. This work is important to reveal the mechanism of binding characteristics between Pb2+ and FA. It also gives a theoretical and practical direction to understanding the environmental effect of HS, environmental geochemical behavior and bio-availability of heavy metals in lakes.

Detecting O2 binding sites in protein cavities.

Internal cavities are important elements in protein structure, dynamics, stability and function. Here we use NMR spectroscopy to investigate the binding of molecular oxygen (O2) to cavities in a well-studied model for ligand binding, the L99A mutant of T4 lysozyme. On increasing the O2 concentration to 8.9 mM, changes in 1H, 15N, and 13C chemical shifts and signal broadening were observed specifically for backbone amide and side chain methyl groups located around the two hydrophobic cavities of the protein. O2-induced longitudinal relaxation enhancements for amide and methyl protons could be adequately accounted for by paramagnetic dipolar relaxation. These data provide the first experimental demonstration that O2 binds specifically to the hydrophobic, and not the hydrophilic cavities, in a protein. Molecular dynamics simulations visualized the rotational and translational motions of O2 in the cavities, as well as the binding and egress of O2, suggesting that the channel consisting of helices D, E, G, H, and J could be the potential gateway for ligand binding to the protein. Due to strong paramagnetic relaxation effects, O2 gas-pressure NMR measurements can detect hydrophobic cavities when populated to as little as 1%, and thereby provide a general and highly sensitive method for detecting oxygen binding in proteins.

Structural Basis of Species-Dependent Differential Affinity of 6-Alkoxy-5-Aryl-3-Pyridinecarboxamide Cannabinoid-1 Receptor Antagonists.

6-Alkoxy-5-aryl-3-pyridincarboxamides, including the brain-penetrant compound 14G: [5-(4-chlorophenyl)-6-(cyclopropylmethoxy)-N-[(1R,2R)-2-hydroxy-cyclohexyl]-3-pyridinecarboxamide] and its peripherally restricted analog 14H: [5-(4-chlorophenyl)-N-[(1R,2R)-2-hydroxycyclohexyl]-6-(2-methoxyethoxy)-3-pyridinecarboxamide], have been recently introduced as selective, high-affinity antagonists of the human cannabinoid-1 receptor (hCB1R). Binding analyses revealed two orders of magnitude lower affinity of these compounds for mouse and rat versus human CB1R, whereas the affinity of rimonabant is comparable for all three CB1Rs. Modeling of ligand binding to CB1R and binding assays with native and mutant (Ile105Met) hCB1Rs indicate that the Ile105 to Met mutation in rodent CB1Rs accounts for the species-dependent affinity of 14G: and 14H: . Our work identifies Ile105 as a new pharmacophore component for developing better hCB1R antagonists and invalidates rodent models for assessing the antiobesity efficacy of 14G: and 14H: .

Comparison of the kinetics of different Markov models for ligand binding under varying conditions.

We recently derived a Markov model for macromolecular ligand binding dynamics from few physical assumptions and showed that its stationary distribution is the grand canonical ensemble [J. W. R. Martini, M. Habeck, and M. Schlather, J. Math. Chem. 52, 665 (2014)]. The transition probabilities of the proposed Markov process define a particular Glauber dynamics and have some similarity to the Metropolis-Hastings algorithm. Here, we illustrate that this model is the stochastic analog of (pseudo) rate equations and the corresponding system of differential equations. Moreover, it can be viewed as a limiting case of general stochastic simulations of chemical kinetics. Thus, the model links stochastic and deterministic approaches as well as kinetics and equilibrium described by the grand canonical ensemble. We demonstrate that the family of transition matrices of our model, parameterized by temperature and ligand activity, generates ligand binding kinetics that respond to changes in these parameters in a qualitatively similar way as experimentally observed kinetics. In contrast, neither the Metropolis-Hastings algorithm nor the Glauber heat bath reflects changes in the external conditions correctly. Both converge rapidly to the stationary distribution, which is advantageous when the major interest is in the equilibrium state, but fail to describe the kinetics of ligand binding realistically. To simulate cellular processes that involve the reversible stochastic binding of multiple factors, our pseudo rate equation model should therefore be preferred to the Metropolis-Hastings algorithm and the Glauber heat bath, if the stationary distribution is not of only interest.

Real Time Observation of Ligand Binding to a GPCR: Conformational Selection Governs the Ability of Retinals to Bind Opsin

Despite extensive study, how retinal enters and exits the visual G protein-coupled receptor (GPCR) rhodopsin remains unclear. One clue may lie in two openings between TM1/TM7 and TM5/TM6 in the active receptor state. Recently, it has been proposed that retinal enters the inactive apoprotein (ops) when the receptor transiently adopts the open, active (ops∗) conformation. Here, we directly tested this “transient activation” hypothesis (TAH), using a fluorescence-based approach to measure rates of retinal binding to samples containing differing amounts of ops and ops∗. In contrast to what the TAH would predict, we found the inverse agonist, 11-cis retinal (11CR), binding is slower to a sample that contained more ops∗ (produced using the constitutively active mutant M257Y_M257Y-CAM). Interestingly, the increased presence of ops∗ allowed for binding of the agonist, all-trans retinal (ATR) faster, whereas WT opsin showed none. Further weighting the conformational equilibrium towards even more ops∗ using a G protein peptide mimic (either free in solution or fused to the receptor) accelerated the rate of ATR binding and slowed 11CR binding. Similar results were obtained using a peptide mimetic of arrestin. The TM5/TM6 hole does not appear to be sufficient for this conformational selection as increasing the size of this opening by mutagenesis does not enable ATR binding. Unexpectedly, the larger hole slows 11CR binding, suggesting a role in trapping the inverse agonist. In summary, our results indicate that the TAH must be modified to accommodate the key role conformational selection plays in stable retinal binding. Additionally, we show that rhodopsin behaves similarly to ligand-binding GPCRs with respect to exogenous ligand interactions, indicating a role for the visual receptor as a model for hydrophobic ligand binding.

Rational design of partial agonists for the muscarinic m1 acetylcholine receptor.

Aiming to design partial agonists for a G-protein-coupled receptor based on dynamic ligand binding, we synthesized three different series of bipharmacophoric ligands composed of the orthosteric building blocks iperoxo and 1 linked to allosteric modulators (BQCA-derived compounds, BQCAd; TBPB-derived compound, TBPBd). Their interactions were studied with the human muscarinic acetylcholine M1-receptor (hM1) with respect to receptor binding and Gq-protein signaling. Results demonstrate that iperoxo/BQCAd (2, 3) and 1/BQCAd hybrids (4) act as M1 partial agonists, whereas 1/TBPBd hybrids (5) did not activate M1-receptors. Among the iperoxo/BQCAd-hybrids, spacer length in conjunction with the pattern of substitution tuned efficacy. Most interestingly, a model of dynamic ligand binding revealed that the spacer length of 2a and 3a controlled the probability of switch between the inactive purely allosteric and the active bitopic orthosteric/allosteric binding pose. In summary, dynamic ligand binding can be exploited in M1 receptors to design partial agonists with graded efficacy.

Activation of PPARδ: from computer modelling to biological effects.

PPARδ is a ligand-activated receptor that dimerizes with another nuclear receptor of the retinoic acid receptor family. The dimers interact with other co-activator proteins and form active complexes that bind to PPAR response elements and promote transcription of genes involved in lipid metabolism. It appears that various natural fatty acids and their metabolites serve as endogenous activators of PPARδ; however, there is no consensus in the literature on the nature of the prime activators of the receptor. In vitro and cell-based assays of PPARδ activation by fatty acids and their derivatives often produce conflicting results. The search for synthetic and selective PPARδ agonists, which may be pharmacologically useful, is intense. Current rational modelling used to obtain such compounds relies mostly on crystal structures of synthetic PPARδ ligands with the recombinant ligand binding domain (LBD) of the receptor. Here, we introduce an original computational prediction model for ligand binding to PPARδ LBD. The model was built based on EC50 data of 16 ligands with available crystal structures and validated by calculating binding probabilities of 82 different natural and synthetic compounds from the literature. These compounds were independently tested in cell-free and cell-based assays for their capacity to bind or activate PPARδ, leading to prediction accuracy of between 70% and 93% (depending on ligand type). This new computational tool could therefore be used in the search for natural and synthetic agonists of the receptor.

Bound Flavin Model Suggests Similar Electron‐Transfer Mechanisms in Shewanella and Geobacter

AbstractCertain microbes are capable of transporting electrons from the cell interior‐respiratory electron chain to insoluble electron acceptors located outside of the cell, a process referred to as extracellular electron transport (EET). Bacteria capable of EET are currently utilized as “living anode catalysts” in microbial fuel cells. Several EET mechanisms have been proposed, yet they lack molecular‐level consistency. Here, we review our recent work, presenting a “bound‐flavin cofactor” model, which we believe provides a suitable explanation for all of the published data to date for the model EET microbes of Shewanella oneidensis and Geobacter sulfurreducens. We discuss the interaction between free flavin and outer‐membrane c‐type cytochromes based on a protein–ligand binding model, the accumulation of cell‐secreted flavins in nanostructured electrodes, and EET through intermittent direct contacts or conductive extracellular appendages.

Experimental determination and modeling of arsenic complexation with humic and fulvic acids

The complexation of humic acid (HA) and fulvic acid (FA) with arsenic (As) in water was studied. Experimental results indicate that arsenic may form complexes with HA and FA with a higher affinity for arsenate than for arsenite. With the presence of iron oxide based adsorbents, binding of arsenic to HA/FA in water was significantly suppressed, probably due to adsorption of As and HA/FA. A two-site ligand binding model, considering only strong and weak site types of binding affinity, was successfully developed to describe the complexation of arsenic on the two natural organic fractions. The model showed that the numbers of weak sites were more than 10 times those of strong sites on both HA and FA for both arsenic species studied. The numbers of both types of binding sites were found to be proportional to the HA concentrations, while the apparent stability constants, defined for describing binding affinity between arsenic and the sites, are independent of the HA concentrations. To the best of our knowledge, this is the first study to characterize the impact of HA concentrations on the applicability of the ligand binding model, and to extrapolate the model to FA. The obtained results may give insights on the complexation of arsenic in HA/FA laden groundwater and on the selection of more effective adsorption-based treatment methods for natural waters.

Differential control of heme reactivity in alpha and beta subunits of hemoglobin: a combined Raman spectroscopic and computational study.

The use of hybrid hemoglobin (Hb), with mesoheme substituted for protoheme, allows separate monitoring of the α or β hemes along the allosteric pathway. Using resonance Raman (rR) spectroscopy in silica gel, which greatly slows protein motions, we have observed that the Fe–histidine stretching frequency, νFeHis, which is a monitor of heme reactivity, evolves between frequencies characteristic of the R and T states, for both α or β chains, prior to the quaternary R–T and T–R shifts. Computation of νFeHis, using QM/MM and the conformational search program PELE, produced remarkable agreement with experiment. Analysis of the PELE structures showed that the νFeHis shifts resulted from heme distortion and, in the α chain, Fe–His bond tilting. These results support the tertiary two-state model of ligand binding (Henry et al., Biophys. Chem.2002, 98, 149). Experimentally, the νFeHis evolution is faster for β than for α chains, and pump–probe rR spectroscopy in solution reveals an inflection in the νFeHis time course at 3 μs for β but not for α hemes, an interval previously shown to be the first step in the R–T transition. In the α chain νFeHis dropped sharply at 20 μs, the final step in the R–T transition. The time courses are fully consistent with recent computational mapping of the R–T transition via conjugate peak refinement by Karplus and co-workers (Fischer et al., Proc. Natl. Acad. Sci. U. S. A.2011, 108, 5608). The effector molecule IHP was found to lower νFeHis selectively for α chains within the R state, and a binding site in the α1α2 cleft is suggested.

Modeling of ligand binding to dopamine D2 receptor

The dopaminic receptors have been for long time the major targets for developing new small molecules with high affinity and selectivity to treat psychiatric disorders, neurodegeneration, drug abuse, and other therapeutic areas. In the absence of a 3D structure for the human D2 dopamine (HDD2) receptor, the efforts for discovery and design of new potential drugs rely on comparative models generation, docking and pharmacophore development studies. To get a better understanding of the HDD2 receptor binding site and the ligand-receptor interactions a homology model of HDD2 receptor based on the X-ray structure of ?2-adrenergic receptor has been built and used to dock a set of partial agonists of HDD2 receptor. The main characteristics of the binding mode for the HDD2 partial agonists set are given by the ligand particular folding and a complex network of contacts represented by stacking interactions, salt bridge and hydrogen bond formation. The characterization of the partial agonist binding mode at HDD2 receptor provide the needed information to generate pharmacophore models which represent essential information in the future virtual screening studies in order to identify new potential HDD2 partial agonists.

Novel protein–protein interaction inhibitor of Nrf2–Keap1 discovered by structure-based virtual screening

Herein we first reported hierarchical structure-based virtual screening utilizing the receptor–ligand binding model of Nrf2–Keap1. The most promising compound, 15, which is one of the most potent direct PPI inhibitors of Nrf2–Keap1 reported so far, can effectively disrupt the Nrf2–Keap1 interaction with the in vitro EC50 of 9.80 μM in the fluorescence polarization (FP) assay. 15 can also activate the Nrf2 transcription activity in the cell-based ARE–luciferase reporter assays in a dose-dependent manner. The compound can serve as a promising starting point for the discovery of potent inhibitors of Nrf2–Keap1 interaction.

Homology modeling of Kv1.5 channel block by cationic and electroneutral ligands

The inner pore of potassium channels is targeted by many ligands of intriguingly different chemical structures. Previous studies revealed common and diverse characteristics of action of ligands including cooperativity of ligand binding, voltage- and use-dependencies, and patterns of ligand-sensing residues. Not all these data are rationalized in published models of ligand-channel complexes. Here we have used energy calculations with experimentally defined constraints to dock flecainide, ICAGEN-4, benzocaine, vernakalant, and AVE0118 into the inner pore of Kv1.5 channel. We arrived at ligand-binding models that suggest possible explanations for different values of the Hill coefficient, different voltage dependencies of ligands action, and effects of mutations of residues in subunit interfaces. Two concepts were crucial to build the models. First, the inner-pore block of a potassium channel requires a cationic “blocking particle”. A ligand, which lacks a positively charged group, blocks the channel in a complex with a permeant ion. Second, hydrophobic moieties of a flexible ligand have a tendency to bind in hydrophobic subunit interfaces.

A Fluorescence-Based Thermal Shift Assay Identifies Inhibitors of Mitogen Activated Protein Kinase Kinase 4

Prostate cancer (PCa) is the second highest cause of cancer death in United States males. If the metastatic movement of PCa cells could be inhibited, then mortality from PCa could be greatly reduced. Mitogen-activated protein kinase kinase 4 (MAP2K4) has previously been shown to activate pro-invasion signaling pathways in human PCa. Recognizing that MAP2K4 represents a novel and validated therapeutic target, we sought to develop and characterize an efficient process for the identification of small molecules that target MAP2K4. Using a fluorescence-based thermal shift assay (FTS) assay, we first evaluated an 80 compound library of known kinase inhibitors, thereby identifying 8 hits that thermally stabilized MAP2K4 in a concentration dependent manner. We then developed an in vitro MAP2K4 kinase assay employing the biologically relevant downstream substrates, JNK1 and p38 MAPK, to evaluate kinase inhibitory function. In this manner, we validated the performance of our initial FTS screen. We next applied this approach to a 2000 compound chemically diverse library, identified 7 hits, and confirmed them in the in vitro kinase assay. Finally, by coupling our structure-activity relationship data to MAP2K4's crystal structure, we constructed a model for ligand binding. It predicts binding of our identified inhibitory compounds to the ATP binding pocket. Herein we report the creation of a robust inhibitor-screening platform with the ability to inform the discovery and design of new and potent MAP2K4 inhibitors.

Differences between cotranscriptional and free riboswitch folding

Riboswitches are part of noncoding regions of messenger RNA (mRNA) that act as RNA sensors regulating gene expression of the downstream gene. Typically, one out of two distinct conformations is formed depending on ligand binding when the transcript leaves RNA polymerase (RNAP). Elongation of the RNA chain by RNAP, folding and binding all occurs simultaneously and interdependently on the seconds’ timescale. To investigate the effect of transcript elongation velocity on folding for the S-adenosylmethionine (SAM)-I and adenine riboswitches we employ two complementary coarse-grained in silico techniques. Native structure-based molecular dynamics simulations provide a 3D, atomically resolved model of folding with homogenous energetics. Energetically more detailed kinetic Monte Carlo simulations give access to longer timescale by describing folding on the secondary structure level and feature the incorporation of competing aptamer conformations and a ligand-binding model. Depending on the extrusion scenarios, we observe and quantify different pathways in structure formation with robust agreements between the two techniques. In these scenarios, free-folding riboswitches exhibit different folding characteristics compared with transcription-rate limited folding. The critical transcription rate distinguishing these cases is higher than physiologically relevant rates. This result suggests that in vivo folding of the analyzed SAM-I and adenine riboswitches is transcription-rate limited.