Mechanism Of Ligand Binding Research Articles

Elucidating the binding mechanism of paramagnetic amyloid ligands to α-synuclein

Elucidating the binding mechanism of paramagnetic amyloid ligands to α-synuclein

Mechanism of ligand binding to target RNA aptamer

Mechanism of ligand binding to target RNA aptamer

Mechanism of Ligand Binding to Theophylline RNA Aptamer.

Studying RNA-ligand interactions and quantifying their binding thermodynamics and kinetics are of particular relevance in the field of drug discovery. Here, we combined biochemical binding assays and accelerated molecular simulations to investigate ligand binding and dissociation in RNA using the theophylline-binding RNA as a model system. All-atom simulations using a Ligand Gaussian accelerated Molecular Dynamics method (LiGaMD) have captured repetitive binding and dissociation of theophylline and caffeine to RNA. Theophylline's binding free energy and kinetic rate constants align with our experimental data, while caffeine's binding affinity is over 10,000 times weaker, and its kinetics could not be determined. LiGaMD simulations allowed us to identify distinct low-energy conformations and multiple ligand binding pathways to RNA. Simulations revealed a "conformational selection" mechanism for ligand binding to the flexible RNA aptamer, which provides important mechanistic insights into ligand binding to the theophylline-binding model. Our findings suggest that compound docking using a structural ensemble of representative RNA conformations would be necessary for structure-based drug design of flexible RNA.

Fluorescent imidazole derived sensor for selective in vitro and in vivo Fe2+ detection and bioimaging in zebrafish with DFT studies

Herein, we have developed imidazole derivatized fluorescent probes IM-1 and IM-2 for extremely selective detection of Fe2+ with rapid response (LOD: 3.245 μM for IM-1 and 0.297 μM for IM-2) and excellent binding constants (0.214 × 105 M−1 and 1.004 × 105 M−1). Aqueous ethanol system was employed to assess the sensing potency of the probes both in vitro and in vivo in zebrafish is the main highlight of this work. The synthesized fluorophores possess admirable quantum yields of 0.61 and 0.78. The 1:1 binding mechanism of ligands with Fe2+ ions is supported by Job's plot and ESI-Mass spectrum. The synthesized probes demonstrated limited cytotoxicity both in vitro (MDA-MB-231 cells) and in vivo (zebrafish, Danio Rerio) studies. These results prompted us to employ the probes IM-1 and IM-2 to trace out intra cellular Fe2+ ions in zebrafish embryos.

Targeting TRP channels: recent advances in structure, ligand binding, and molecular mechanisms.

Transient receptor potential (TRP) channels are a large and diverse family of transmembrane ion channels that are widely expressed, have important physiological roles, and are associated with many human diseases. These proteins are actively pursued as promising drug targets, benefitting greatly from advances in structural and mechanistic studies of TRP channels. At the same time, the complex, polymodal activation and regulation of TRP channels have presented formidable challenges. In this short review, we summarize recent progresses toward understanding the structural basis of TRP channel function, as well as potential ligand binding sites that could be targeted for therapeutics. A particular focus is on the current understanding of the molecular mechanisms of TRP channel activation and regulation, where many fundamental questions remain unanswered. We believe that a deeper understanding of the functional mechanisms of TRP channels will be critical and likely transformative toward developing successful therapeutic strategies targeting these exciting proteins. This endeavor will require concerted efforts from computation, structural biology, medicinal chemistry, electrophysiology, pharmacology, drug safety and clinical studies.

Computational methods for unlocking the secrets of potassium channels: Structure, mechanism, and drug design

AbstractPotassium (K+) channels play vital roles in various physiological functions, including regulating K+ flow in cell membranes, impacting nervous system signal transduction, neuronal firing, muscle contraction, neurotransmitters, and enzyme secretion. Their activation and switch‐off are directly linked to diseases like arrhythmias, atrial fibrillation, and pain etc. Although the experimental methods play important roles in the studying the structure and function of K+ channels, they are still some limitations to enclose the dynamic molecular processes and the corresponding mechanisms of conformational changes during ion transport, permeation, and gating control. Relatively, computational methods have obvious advantages in studying such problems compared with experimental methods. Recently, more and more three‐dimensional structures of K+ channels have been disclosed based on experimental methods and in silico prediction methods, which provide a good chance to study the molecular mechanism of conformational changes related to the functional regulations of K+ channels. Based on these structural details, molecular dynamics simulations together with related methods such as enhanced sampling and free energy calculations, have been widely used to reveal the conformational dynamics, ion conductance, ion channel gating, and ligand binding mechanisms. Additionally, the accessibility of structures also provides a large space for structure‐based drug design. This review mainly addresses the recent progress of computational methods in the structure, mechanism, and drug design of K+ channels. After summarizing the progress in these fields, we also give our opinion on the future direction in the area of K+ channel research combined with the cutting edge of computational methods.This article is categorized under: Molecular and Statistical Mechanics > Molecular Dynamics and Monte‐Carlo Methods Structure and Mechanism > Computational Biochemistry and Biophysics Data Science > Chemoinformatics

Elucidating Dynamics of Adenylate Kinase from Enzyme Opening to Ligand Release.

This study explores ligand-driven conformational changes in adenylate kinase (AK), which is known for its open-to-close conformational transitions upon ligand binding and release. By utilizing string free energy simulations, we determine the free energy profiles for both enzyme opening and ligand release and compare them with profiles from the apoenzyme. Results reveal a three-step ligand release process, which initiates with the opening of the adenosine triphosphate-binding subdomain (ATP lid), followed by ligand release and concomitant opening of the adenosine monophosphate-binding subdomain (AMP lid). The ligands then transition to nonspecific positions before complete dissociation. In these processes, the first step is energetically driven by ATP lid opening, whereas the second step is driven by ATP release. In contrast, the AMP lid opening and its ligand release make minor contributions to the total free energy for enzyme opening. Regarding the ligand binding mechanism, our results suggest that AMP lid closure occurs via an induced-fit mechanism triggered by AMP binding, whereas ATP lid closure follows conformational selection. This difference in the closure mechanisms provides an explanation with implications for the debate on ligand-driven conformational changes of AK. Additionally, we determine an X-ray structure of an AK variant that exhibits significant rearrangements in the stacking of catalytic arginines, explaining its reduced catalytic activity. In the context of apoenzyme opening, the sequence of events is different. Here, the AMP lid opens first while the ATP lid remains closed, and the free energy associated with ATP lid opening varies with orientation, aligning with the reported AK opening and closing rate heterogeneity. Finally, this study, in conjunction with our previous research, provides a comprehensive view of the intricate interplay between various structural elements, ligands, and catalytic residues that collectively contribute to the robust catalytic power of the enzyme.

Two-Step Covalent Docking with Attracting Cavities.

Due to their various advantages, interest in the development of covalent drugs has been renewed in the past few years. It is therefore important to accurately describe and predict their interactions with biological targets by computer-aided drug design tools such as docking algorithms. Here, we report a covalent docking procedure for our in-house docking code Attracting Cavities (AC), which mimics the two-step mechanism of covalent ligand binding. Ligand binding to the protein cavity is driven by nonbonded interactions, followed by the formation of a covalent bond between the ligand and the protein through a chemical reaction. To test the performance of this method, we developed a diverse, high-quality, openly accessible re-docking benchmark set of 95 covalent complexes bound by 8 chemical reactions to 5 different reactive amino acids. Combination with structures from previous studies resulted in a set of 304 complexes, on which AC obtained a success rate (rmsd ≤ 2 Å) of 78%, outperforming two state-of-the-art covalent docking codes, genetic optimization for ligand docking (GOLD (66%)) and AutoDock (AD (35%)). Using a more stringent success criterion (rmsd ≤ 1.5 Å), AC reached a success rate of 71 vs 55% for GOLD and 26% for AD. We additionally assessed the cross-docking performance of AC on a set of 76 covalent complexes of the SARS-CoV-2 main protease. On this challenging test set of mainly small and highly solvent-exposed ligands, AC yielded success rates of 58 and 28% for re-docking and cross-docking, respectively, compared to 45 and 17% for GOLD.

Calcium’s Role and Signaling in Aging Muscle, Cellular Senescence, and Mineral Interactions

Calcium research, since its pivotal discovery in the early 1800s through the heating of limestone, has led to the identification of its multi-functional roles. These include its functions as a reducing agent in chemical processes, structural properties in shells and bones, and significant role in cells relating to this review: cellular signaling. Calcium signaling involves the movement of calcium ions within or between cells, which can affect the electrochemical gradients between intra- and extracellular membranes, ligand binding, enzyme activity, and other mechanisms that determine cell fate. Calcium signaling in muscle, as elucidated by the sliding filament model, plays a significant role in muscle contraction. However, as organisms age, alterations occur within muscle tissue. These changes include sarcopenia, loss of neuromuscular junctions, and changes in mineral concentration, all of which have implications for calcium’s role. Additionally, a field of study that has gained recent attention, cellular senescence, is associated with aging and disturbed calcium homeostasis, and is thought to affect sarcopenia progression. Changes seen in calcium upon aging may also be influenced by its crosstalk with other minerals such as iron and zinc. This review investigates the role of calcium signaling in aging muscle and cellular senescence. We also aim to elucidate the interactions among calcium, iron, and zinc across various cells and conditions, ultimately deepening our understanding of calcium signaling in muscle aging.

Molecular Insight into Ligand Binding and Transport by the Lentil Lipid Transfer Protein Lc-LTP2: The Role of Basic Amino Acid Residues at Opposite Entrances to the Hydrophobic Cavity.

Lipid transfer proteins (LTPs) realize their functions in plants due to their ability to bind and transport various ligands. Structures of many LTPs have been studied; however, the mechanism of ligand binding and transport is still not fully understood. In this work, we studied the role of Lys61 and Lys81 located near the "top" and "bottom" entrances to the hydrophobic cavity of the lentil lipid transfer protein Lc-LTP2, respectively, in these processes. Using site-directed mutagenesis, we showed that both amino acid residues played a key role in lipid binding to the protein. In experiments with calcein-loaded liposomes, we demonstrated that both the above-mentioned lysine residues participated in the protein interaction with model membranes. According to data obtained from fluorescent spectroscopy and TNS probe displacement, both amino acid residues are necessary for the ability of the protein to transfer lipids between membranes. Thus, we hypothesized that basic amino acid residues located at opposite entrances to the hydrophobic cavity of the lentil Lc-LTP2 played an important role in initial protein-ligand interaction in solution as well as in protein-membrane docking.

Resolving binding pathways and solvation thermodynamics of plant hormone receptors

Plant hormones are small molecules that regulate plant growth, development, and responses to biotic and abiotic stresses. They are specifically recognized by the binding site of their receptors. In this work, we resolved the binding pathways for eight classes of phytohormones (auxin, jasmonate, gibberellin, strigolactone, brassinosteroid, cytokinin, salicylic acid, and abscisic acid) to their canonical receptors using extensive molecular dynamics simulations. Furthermore, we investigated the role of water displacement and reorganization at the binding site of the plant receptors through inhomogeneous solvation theory. Our findings predict that displacement of water molecules by phytohormones contributes to free energy of binding via entropy gain and is associated with significant free energy barriers for most systems analyzed. Also, our results indicate that displacement of unfavorable water molecules in the binding site can be exploited in rational agrochemical design. Overall, this study uncovers the mechanism of ligand binding and the role of water molecules in plant hormone perception, which creates new avenues for agrochemical design to target plant growth and development.

Common scab disease: structural basis of elicitor recognition in pathogenic Streptomyces species.

In Streptomyces scabiei, the main causative agent of common scab disease of root and tuber crops, the interaction between the substrate-binding protein (SBP) CebE (CebEscab) and cellotriose released by the plant host (KD in the nanomolar range) is the first event for the onset of its pathogenic lifestyle. Here, we report the structure of CebEscab in complex with cellotriose at a resolution of 1.55 Å, adopting a general fold of the B subcluster of SBPs. The interaction between CebEscab and cellotriose involves multiple direct or water-mediated hydrogen bonds and hydrophobic interactions, with the glucose monomer at the non-reducing end occupying the most conserved part of the substrate-binding cleft. As main interactions between the two domains of CebE involve cellotriose itself, the closed conformational state of CebE is performed via an induced-fit ligand binding mechanism where cellotriose binding triggers the domain movement. Analysis of regulon predictions revealed that the signaling pathway from CebE-mediated cellotriose transport to the transcriptional activation of thaxtomin phytotoxin biosynthesis is conserved in Streptomyces spp. causing common scab, except for Streptomyces ipomoeae, which specifically colonizes sweet potatoes and responds to other and yet unknown virulence elicitors. Interestingly, strains belonging to the pathogenic species turgidiscabies and caniscabiei have a cellotriose-binding protein orthologous to the CebE protein of the saprophytic species Streptomyces reticuli with lower affinity for its substrate (KD in the micromolar range), suggesting higher cellotriose concentrations for perception of their host. Our work also provides the structural basis for the uptake of cellobiose and cellotriose by non-pathogenic cellulose-decomposing Streptomyces species.IMPORTANCECommon scab is a disease caused by a few Streptomyces species that affects important root and tuber crops including potato, beet, radish, and parsnip, resulting in major economic losses worldwide. In this work, we unveiled the molecular basis of host recognition by these pathogens by solving the structure of the sugar-binding protein CebE of Streptomyces scabiei in complex with cellotriose, the main elicitor of the pathogenic lifestyle of these bacteria. We further revealed that the signaling pathway from CebE-mediated transport of cellotriose is conserved in all pathogenic species except Streptomyces ipomoeae, which causes soft rot disease in sweet potatoes. Our work also provides the structural basis of the uptake of cellobiose and cellotriose in saprophytic Streptomyces species, the first step activating the expression of the enzymatic system degrading the most abundant polysaccharide on earth, cellulose.

Discovery of Nonretinoid Inhibitors of CRBP1: Structural and Dynamic Insights for Ligand-Binding Mechanisms.

The dysregulation of retinoid metabolism has been linked to prevalent ocular diseases including age-related macular degeneration and Stargardt disease. Modulating retinoid metabolism through pharmacological approaches holds promise for the treatment of these eye diseases. Cellular retinol-binding protein 1 (CRBP1) is the primary transporter of all-trans-retinol (atROL) in the eye, and its inhibition has recently been shown to protect mouse retinas from light-induced retinal damage. In this report, we employed high-throughput screening to identify new chemical scaffolds for competitive, nonretinoid inhibitors of CRBP1. To understand the mechanisms of interaction between CRBP1 and these inhibitors, we solved high-resolution X-ray crystal structures of the protein in complex with six selected compounds. By combining protein crystallography with hydrogen/deuterium exchange mass spectrometry, we quantified the conformational changes in CRBP1 caused by different inhibitors and correlated their magnitude with apparent binding affinities. Furthermore, using molecular dynamic simulations, we provided evidence for the functional significance of the "closed" conformation of CRBP1 in retaining ligands within the binding pocket. Collectively, our study outlines the molecular foundations for understanding the mechanism of high-affinity interactions between small molecules and CRBPs, offering a framework for the rational design of improved inhibitors for this class of lipid-binding proteins.

Structural Mechanism and Inhibitors Targeting EGFR Exon 20 Insertion (Ex20ins) Mutations.

Epidermal growth factor receptor (EGFR) targeted therapy is one of the most important and effective strategies to combat EGFR mutant nonsmall-cell lung cancer (NSCLC). However, a substantial number of patients bearing EGFR exon 20 insertion (Ex20ins) mutations respond poorly to common EGFR targeted therapies. This clinical need remained unmet until recently, when the EGFR Ex20ins mutation inhibitor mobocertinib was approved by the FDA. Despite this progress, the structural mechanisms of EGFR Ex20ins mutation resistance and characterization of inhibitor binding modes have not been systematically summarized. Herein, we analyze the structural mechanisms for ligand binding and resistance and summarize recent developments for the reported inhibitors of EGFR Ex20ins mutations. Furthermore, this Perspective aims to provide insights for the design of the next generation of EGFR Ex20ins inhibitors.

Interaction of CYP3A4 with caffeine: First insights into multiple substrate binding

Human cytochrome P450 3A4 (CYP3A4) is a major drug-metabolizing enzyme that shows extreme substrate promiscuity. Moreover, its large and malleable active site can simultaneously accommodate several substrate molecules of the same or different nature, which may lead to cooperative binding and allosteric behavior. Due to difficulty of crystallization of CYP3A4-substrate complexes, it remains unknown how multiple substrates can arrange in the active site. We determined crystal structures of CYP3A4 bound to three and six molecules of caffeine, a psychoactive alkaloid serving as a substrate and modulator of CYP3A4. In the ternary complex, one caffeine binds to the active site suitably for C8-hydroxylation, most preferable for CYP3A4. In the senary complex, three caffeine molecules stack parallel to the heme with the proximal ligand poised for 3-N-demethylation. However, the caffeine stack forms extensive hydrophobic interactions that could preclude product dissociation and multiple turnovers. In both complexes, caffeine is also bound in the substrate channel and on the outer surface known as a peripheral site. At all sites, aromatic stacking with the caffeine ring(s) is likely a dominant interaction, while direct and water-mediated polar contacts provide additional stabilization for the substrate-bound complexes. Protein-ligand interactions via the active site R212, intrachannel T224, and peripheral F219 were experimentally confirmed, and the latter two residues were identified as important for caffeine association. Collectively, the structural, spectral, and mutagenesis data provide valuable insights on the ligand binding mechanism and help better understand how purine-based pharmaceuticals and other aromatic compounds could interact with CYP3A4 and mediate drug-drug interactions.

Binding mechanism of disulfide species to ferric hemeproteins: The case of metmyoglobin

The interactions of the heme iron of hemeproteins with sulfide and disulfide compounds are of potential interest as physiological signaling processes. While the interaction with hydrogen sulfide has been described computationally and experimentally, the reaction with disulfide, and specifically the molecular mechanism for ligand binding has not been studied in detail. In this work, we study the association process for disulfane and its conjugate base disulfanide at different pH conditions. Additionally, by means of advanced sampling techniques based on multiple steered molecular dynamics, we provide free energy profiles for ligand migration for both acid/base species, showing a similar behavior to the previously reported for the related H2S/HS¯ pair. Finally, we studied the ligand interchange reaction (H2O/H2S, HS¯ and H2O/HSSH, HSS¯) by means of hybrid quantum mechanics-molecular mechanics calculations. We show that the anionic species are able to displace more efficiently the H2O bound to the iron, and that the H-bond network in the distal cavity can help the neutral species to perform the reaction. Altogether, we provide a molecular explanation for the experimental information and show that the global association process depends on a fine balance between the migration towards the active site and the ligand interchange reaction.

Molecular Insights on the Conformational Transitions and Activity Regulation of the c-Met Kinase Induced by Ligand Binding.

The tyrosine-protein kinase Met (c-Met) is an important signaling molecule involved in cellular growth and division. The dysregulation of c-Met may induce many fatal diseases, including non-small cell lung cancer, gastrointestinal cancers, hepatocellular carcinoma, etc. The activation of the c-Met kinase is dominant by the structure and dynamics of many important functional motifs, which are regulated by adenosine triphosphate (ATP) binding. c-Met inhibitors bind to the ATP-binding site or the allosteric pocket to compete with ATP molecules or alter the conformation of the function-related domains. Nevertheless, the mechanisms of ligand binding to c-Met are still unclear, especially the regulation of the functional motifs by different inhibitors. These greatly impede the development of novel drugs to overcome the drug tolerance to the currently marketed c-Met inhibitors. In this study, we used enhanced sampling technology to study the binding and regulation of two specific c-Met inhibitors. The results show that the two ligands adopt different binding processes even though with similar binding affinity. More importantly, our results uncovered different protein conformational features and the correlated motions of functional motifs regulated by the inhibitors, providing the structural basis for the functional suppression of the protein kinases.

Characterization, Expression, and Ligand Binding of LGP2 and MDA5 in Largemouth Black Bass Micropterus salmoides (Lacepède, 1802)

Melanoma differentiation-associated gene 5 (MDA5) and the laboratory of genetics and physiology 2 (LGP2) are family members of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), which play important roles in the immune response against pathogens invasion. In the present study, MDA5 and LGP2 genes were identified in largemouth bass (Micropterus salmoides), a fish species with a great economic value. The two proteins contained similar conserved domains and motifs as their counterparts of other vertebrates, including the DExDc domain (the DEAD/DEAH box helicases domain), HELICc domain (helicases superfamily domain), and regulatory domain (RD). Real-time qPCR revealed that the two genes were constitutively expressed in tissues of healthy fish and could be induced in the spleen by polyinosinic and polycytidylic acid (polyI:C) challenge in vivo. Also, selective pressure analysis revealed that the negative selection had roles in the evolutions of the two genes. Furthermore, the dsRNA binding mechanism of msLGP2 and msMDA5 were analyzed by the molecular docking strategy. The amino acids of msLGP2 involved in dsRNA binding were V604, N663, L682, and L684, which were located in the regulatory domain (RD) of msLGP2. The amino acids of msMDA5 involved in dsRNA binding were G429, H434, L842, and L845, which were located in the DExDc domain and the RD domain of msMDA5. These results indicated that fish LGP2 and MDA5 might share similar functions and ligand binding mechanism as their mammalian counterparts.

Molecular Mechanism of Ligand Binding to the Minor Groove of DNA

Although DNA–ligand binding is pervasive in biology, little is known about molecular-level binding mechanisms. Using all-atom, explicit-solvent molecular dynamics simulations in conjunction with weighted ensemble (WE)-enhanced sampling, an ensemble of 2562 binding trajectories of Hoechst 33258 (H33258) to d(CGC AAA TTT GCG) was generated from which the binding mechanism was extracted. In particular, the electrostatic interaction between the positively charged H33258 and the negatively charged DNA backbone drives the formation of initial H33258–DNA contacts. After this initial contact, a hinge-like intermediate state is formed in which one end of H33258 inserts into the minor groove of DNA. Following hinge state formation is a concerted motion whereby the second end of H33258 swings into the minor groove and the spine of hydration along the minor groove causing dehydration. This study illustrates how WE-enhanced simulations of biomolecular ligation processes can offer novel mechanistic insights by generating ensembles of binding events.

FLEXR: automated multi-conformer model building using electron-density map sampling.

Protein conformational dynamics that may inform biology often lie dormant in high-resolution electron-density maps. While an estimated ∼18% of side chains in high-resolution models contain alternative conformations, these are underrepresented in current PDB models due to difficulties in manually detecting, building and inspecting alternative conformers. To overcome this challenge, we developed an automated multi-conformer modeling program, FLEXR. Using Ringer-based electron-density sampling, FLEXR builds explicit multi-conformer models for refinement. Thereby, it bridges the gap of detecting hidden alternate states in electron-density maps and including them in structural models for refinement, inspection and deposition. Using a series of high-quality crystal structures (0.8-1.85 Å resolution), we show that the multi-conformer models produced by FLEXR uncover new insights that are missing in models built either manually or using current tools. Specifically, FLEXR models revealed hidden side chains and backbone conformations in ligand-binding sites that may redefine protein-ligand binding mechanisms. Ultimately, the tool facilitates crystallographers with opportunities to include explicit multi-conformer states in their high-resolution crystallographic models. One key advantage is that such models may better reflect interesting higher energy features in electron-density maps that are rarely consulted by the community at large, which can then be productively used for ligand discovery downstream. FLEXR is open source and publicly available on GitHub at https://github.com/TheFischerLab/FLEXR.