Differential roles of kinetic on- and off-rates in T-cell receptor signal integration revealed with a modified Fab'-DNA ligand.

Antibody-derived T-cell receptor (TCR) agonists are commonly used to activate T cells. While antibodies can trigger TCRs regardless of clonotype, they bypass native T cell signal integration mechanisms that rely on monovalent, membrane-associated, and relatively weakly binding ligand in the context of cellular adhesion. Commonly used antibodies and their derivatives bind much more strongly than native peptide major histocompatibility complex (pMHC) ligands bind their cognate TCRs. Because ligand dwell time is a critical parameter that tightly correlates with physiological function of the TCR signaling system, there is a general need, both in research and therapeutics, for universal TCR ligands with controlled kinetic binding parameters. To this end, we have introduced point mutations into recombinantly expressed α-TCRß H57 Fab to modulate the dwell time of monovalent Fab binding to TCR. When tethered to a supported lipid bilayer via DNA complementation, these monovalent Fab'-DNA ligands activate T cells with potencies well-correlated with their TCR binding dwell time. Single-molecule tracking studies in live T cells reveal that individual binding events between Fab'-DNA ligands and TCRs elicit local signaling responses closely resembling native pMHC. The unique combination of high on- and off-rates of the H57 R97L mutant enables direct observations of cooperative interplay between ligand binding and TCR-proximal condensation of the linker for activation of T cells, which is not readily visualized with pMHC. This work provides insights into how T cells integrate kinetic information from TCR ligands and introduces a method to develop affinity panels for polyclonal T cells, such as cells from a human patient.

How arginine inhibits substrate-binding domain 2 elucidated using molecular dynamics simulations.

The substrate-binding domain 2 (SBD2) is an important part of the bacterial glutamine (GLN) transporter and mediates binding and delivery of GLN to the transporter translocation subunit. The SBD2 consists of two domains, D1 and D2, that bind GLN in the space between domains in a closed structure. In the absence of ligand, the SBD2 adopts an open conformation with larger space between domains. The GLN binding and closing are essential for the subsequent transport into the cell. Arginine (ARG) can also bind to SBD2 but does not induce closing and inhibits GLN transport. We use atomistic molecular dynamics (MD) simulations in explicit solvent to study ARG binding in the presence of the open SBD2 structure and observed reversible binding to the native GLN binding site with similar contacts but no transition to a closed SBD2 state. Absolute binding free energy simulations predict a considerable binding affinity of ARG and GLN to the binding site on the D1 domain. Free energy simulations to induce subsequent closing revealed a strong free energy penalty in case of ARG binding in contrast to GLN binding that favors the closed SBD2 state but still retains a free energy barrier for closing. The simulations allowed the identification of the molecular origin of the closing penalty in case of bound ARG and suggested a mutation of lysine at position 373 to alanine that strongly reduced the penalty and allowed closing even in the presence of bound ARG. The study offers an explanation of the molecular mechanism of how ARG competitively inhibits GLN from binding to SBD2 and from triggering the transition to a closed conformation. The proposed Lys373Ala mutation shows promise as a potential tool to validate whether a conformational mismatch between open SBD2 and the translocator is responsible for preventing ARG uptake to the cell.

Epiberberine induced p53/p21-dependent G2/M cell cycle arrest and cell apoptosis in gastric cancer cells by activating γ-aminobutyric acid receptor- ß3.

BACKGROUND AND PURPOSE: Epiberberine (EPI) is one of the most important bioalkaloid found in the rhizome of Coptis chinensis, which has been observed to exhibit pharmaceutical effects against gastric cancer (GC). Nevertheless, the potential mechanism of EPI against GC cells still remains unclear. This study aimed to identify the core receptor on GC cells through which EPI inhibited the growth of GC cells and to explore the underlying inhibitory mechanisms. METHODS: To identify hub receptor targets that respond to EPI treatment, RNA sequencing (RNA-Seq) data from a tumor-bearing mouse model were analyzed using bioinformatics method and molecular docking. The binding interaction between EPI and GABRB3 was validated through western blotting based-cellular thermal shift assay (WB-CETSA). To further verify the binding region between EPI and GABRB3 through circular dichroism (CD) chromatography, fragments of the extracellular and transmembrane domains of the GABRB3 protein were expressed and purified in vitro. Stable cell lines with the overexpression or knockdown of GABRB3 were established using the recombinant lentivirus system. MTT ((3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)) assay, colony formation assay, invasion and migration experiments, and flow cytometry were conducted to validate the inhibitory effect of EPI on the GC cells via GABRB3. Additionally, western blotting was utilized to explore the potential inhibitory mechanisms. RESULTS: Through the combination of multiple bioinformatics methods and molecular docking, we found that the γ-aminobutyric acid type A receptor subunit -ß3 (GABRB3) might be the critical receptor target in response to EPI treatment. The results of WB-CETSA analysis indicated that EPI significantly promoted the thermostability of the GABRB3 protein. Importantly, EPI could directly bind to GABRB3 and alter the secondary structure of GABRB3 fragments similar to the natural agonist, γ-aminobutyric acid (GABA). The EPI-induced suppression of the malignant phenotype of GC cells was dependent on the presence of GABRB3. GABRB3 expression was positively correlated with TP53 in patients with GC. The binding of EPI to GABRB3 stimulated p53 accumulation in GC cells. This activated the p21/CDK1/cyclinB1 pathway, resulting in G2/M cell cycle arrest, and induced the Bcl-2/BAX/Caspase axis-dependent cell apoptosis. CONCLUSION: This study revealed the target receptor for EPI in GC cells and provided new insights into its anticancer mechanisms.

Modelling lipid rafts formation through chemo-mechanical interplay triggered by receptor-ligand binding.

Cell membranes, mediator of many biological mechanisms from adhesion and metabolism up to mutation and infection, are highly dynamic and heterogeneous environments exhibiting a strong coupling between biochemical events and structural re-organisation. This involves conformational changes induced, at lower scales, by lipid order transitions and by the micro-mechanical interplay of lipids with transmembrane proteins and molecular diffusion. Particular attention is focused on lipid rafts, ordered lipid microdomains rich of signalling proteins, that co-localise to enhance substance trafficking and activate different intracellular biochemical pathways. In this framework, the theoretical modelling of the dynamic clustering of lipid rafts implies a full multiphysics coupling between the kinetics of phase changes and the mechanical work performed by transmembrane proteins on lipids, involving the bilayer elasticity. This mechanism produces complex interspecific dynamics in which membrane stresses and chemical potentials do compete by determining different morphological arrangements, alteration in diffusive walkways and coalescence phenomena, with a consequent influence on both signalling potential and intracellular processes. Therefore, after identifying the leading chemo-mechanical interactions, the present work investigates from a modelling perspective the spatio-temporal evolution of raft domains to theoretically explain co-localisation and synergy between proteins' activation and raft formation, by coupling diffusive and mechanical phenomena to observe different morphological patterns and clustering of ordered lipids. This could help to gain new insights into the remodelling of cell membranes and could potentially suggest mechanically based strategies to control their selectivity, by orienting intracellular functions and mechanotransduction.

Activation and characterization of G protein-coupled receptors for CHHs in the mud crab, Scylla paramamosain.

Crustacean hyperglycemic hormone (CHH) superfamily peptides constitute a group of neurohormones, including the crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), and gonad-inhibiting hormone (GIH) or vitellogenesis-inhibiting hormone (VIH), which reportedly play an essential role in regulating various biological activities by binding to their receptors in crustaceans. Although bioinformatics analyses have identified G protein-coupled receptors (GPCRs) as potential CHH receptors, no validation through binding experiments has been carried out. This study employed a eukaryotic expression system, HEK293T cell transient transfection, and ligand-receptor interaction tests to identify the GPCRs of CHHs in the mud crab Scylla paramamosain. We found that four GPCRs (Sp-GPCR-A34-A37) were activated by their corresponding CHHs (Sp-CHH1-v1, Sp-MIH, Sp-VIH) in a dose-dependent manner. Of these, Sp-GPCR-A34 was exclusively activated by Sp-VIH; Sp-GPCR-A35 was activated by Sp-CHH1-v1 and Sp-VIH, respectively; Sp-GPCR-A36 was activated by Sp-CHH1-v1 and Sp-MIH; Sp-GPCR-A37 was exclusively activated by Sp-MIH. The half-maximal effective concentration (EC50) values for all CHHs/GPCRs pairs (both Ca2+ and cAMP signaling) were in the nanomolar range. Overall, our study provided hitherto undocumented evidence of the presence of G protein-coupled receptors of CHH in crustaceans, providing the foothold for further studies on the signaling pathways of CHHs and their corresponding GPCRs.

Kinetic titration method in flow cytometry for quantification of cell receptors.

For the quantitative determination of cell receptors by fluorescence flow cytometry, we proposed a new method, which takes into account the reaction kinetics. The binding reaction of the ligand with receptors begins after placing the cells in the ligand solution. In the proposed method, there are several samples with the same concentration of cells and different initial concentrations of fluorescently labeled ligand, and each sample is measured by a flow cytometer once at the time when the following condition is met: the product of the incubation time (cells with ligand) and the initial concentration of ligand is the same for all samples. The proposed approach eliminates disadvantages and combines advantages of both kinetic and titration methods for quantification of receptors on single cells without the use of traditional calibration fluorescent beads. Practical application of the method was demonstrated in quantification of CD8 and CD14 on peripheral blood human leukocytes. Particularly, we found decreased (by a factor of two) mean number of CD14 on monocytes and granulocytes in patients with atherosclerosis (treated in the hospital) compared to conditionally healthy donors, whereas no difference was found in the mean CD8 expression on leukocytes between the same patient and donor groups.

Fluorescence-Based Measurements of Two-Dimensional Affinity in Membrane Interfaces.

Binding between ligands and receptors across cell contacts influences a range of biological processes including the formation of the immune synapse. The dissociation constant (Kd = 1/affinity) of the interaction corresponds to the concentration of ligands where half of the receptors in the contact have bound a ligand. In this chapter, we outline how to measure this two-dimensional affinity using model cell membranes called supported lipid bilayers (SLBs) functionalized with fluorescently labeled ligands that bind to cells containing the corresponding receptor. The affinity is calculated from the accumulation of ligands at the cell-SLB interface, while the use of different fluorescent tags, and/or unlabeled molecules, makes it possible to include various binding pairs in the contact to better mimic the conditions of binding in vivo.

Jasmonate perception: Ligand-receptor interaction, regulation, and evolution.

Phytohormones integrate external environmental and developmental signals with internal cellular responses for plant survival and multiplication in changing surroundings. Jasmonate (JA), which might originate from prokaryotes and benefit plant terrestrial adaptation, is a vital phytohormone that regulates diverse developmental processes and defense responses against various environmental stresses. In this review, we first provide an overview of ligand-receptor binding techniques used for the characterization of phytohormone-receptor interactions, then introduce the identification of the receptor COI1 and active JA molecules, and finally summarize recent advances on the regulation of JA perception and its evolution.

Diethyl phosphate disrupts hypothalamus-pituitary-adrenal axis endocrine hormones via nuclear receptors GR and Nur77: Integration of evidences from in vivo, in vitro and in silico approaches.

Plenty of population epidemiology and cohort studies have found dialkyl phosphates (DAPs) in the urine were related to endocrine hormone disorders. However, we did not know whether these effects were caused by parent organophosphorus pesticides (OPs) or metabolite DAPs, especially the non-specific metabolite diethyl phosphate (DEP), which was the metabolic end product of most widely used diethyl OPs. In this study, animal experiments (in vivo), cell experiments (in vitro), small molecule-protein binding interaction experiments and computer molecular simulations (in silico) were used to explore the disturbing effects and molecular mechanisms of DEP on the hypothalamic-pituitary-adrenal (HPA) axis endocrine hormones. The animal experiments showed that chronic DEP exposure significantly disturbed the serum contents of HPA axis hormones in adult male rats. The target genes of glucocorticoid receptor (GR) in rat liver, including 11ß-hsd1 and Pepck1 and PEPCK protein expressions, were down-regulated. Moreover, the gluconeogenic abilities of rats were impaired. However, it did not affect the expression of GR in the rat hypothalamus. These results indicated that the physiological functions of glucocorticoids and GR were damaged. Furthermore, spectroscopy experiments, cell experiments, molecular docking and molecular dynamics simulations also suggested that DEP can bind to nuclear receptors GR and Nur77, affecting their transcription factor functions, and the transcriptional expression levels of their downstream target genes were reduced. The biosynthesis and secretion of adrenocorticotropic hormone and glucocorticoids were blocked. Therefore, DEP can inhibit the production and physiological functions of HPA axis endocrine hormones by disrupting these related proteins and antagonizing nuclear receptors. These results were considered to provide a theoretical basis for strictly controlling the residue limits of OPs and their metabolites in foods, agricultural products and the environment. They also revealed new targets for evaluating the toxicities and risks of pesticide metabolites.

The first laminin G-like domain of protein S is essential for binding and activation of Tyro3 receptor and intracellular signalling.

The homologous proteins Gas6 and protein S (ProS1) are both natural ligands for the TAM (Tyro3, Axl, MerTK) receptor tyrosine kinases. ProS1 selectively activates Tyro3; however, the precise molecular interface of the ProS1-Tyro3 contact has not been characterised. We used a set of chimeric proteins in which each of the C-terminal laminin G-like (LG) domains of ProS1 were swapped with those of Gas6, as well as a set of ProS1 mutants with novel added glycosylations within LG1. Alongside wildtype ProS1, only the chimera containing ProS1 LG1 domain stimulated Tyro3 and Erk phosphorylation in human cancer cells, as determined by Western blot. In contrast, Gas6 and chimeras containing minimally the Gas6 LG1 domain stimulated Axl and Akt phosphorylation. We performed in silico homology modelling and molecular docking analysis to construct and evaluate structural models of both ProS1-Tyro3 and Gas6-Axl ligand-receptor interactions. These analyses revealed a contact between the ProS1 LG1 domain and the first immunoglobulin domain of Tyro3, which was similar to the Gas6-Axl interaction, and involved long-range electrostatic interactions that were further stabilised by hydrophobic and polar contacts. The mutant ProS1 proteins, which had added glycosylations within LG1 but which were all outside of the modelled contact region, all activated Tyro3 in cells with no hindrance. In conclusion, we show that the LG1 domain of ProS1 is necessary for activation of the Tyro3 receptor, involving protein-protein interaction interfaces that are homologous to those of the Gas6-Axl interaction.

Chemical control over Asialo-GM1: A dual ligand for pili and Lectin A that activates swarming motility and facilitates adherence of Pseudomonas aeruginosa.

Glycolipid, ganglio-N-tetraosylceramide (asialo-GM1), on the mammalian cells are known to be recognized by type IV pili of Pseudomonas aeruginosa. In this work, we show that asialo-GM1 can also be recognized by Lectin A (LecA), another adhesin protein of the P. aeruginosa, by a fluorescent polarization assay, a label-free bacterial motility enabled binding assay, and bacterial mutant studies. On hydrated semi-solid gel surfaces, asialo-GM1 enables swarming and twitching motilities, while on solid surfaces facilitates the bacterial adherence of P. aeruginosa. These results indicate that asialo-GM1 can modulate bioactivities, adherence, and motilities, that are controlled by opposite signaling pathways. We demonstrate that when a solution of pilin monomers or LecA proteins are spread on hydrated gel surfaces, the asialo-GM1 mediated swarming motility is inhibited. Treatment of artificial liposomes containing asialo-GM1 as a component of lipid bilayer with pilin monomers or LecA proteins caused transient leakage of encapsulated dye from liposomes. These results suggest that pili and LecA proteins not only bind to asialo-GM1 but can also cause asialo-GM1 mediated leakage. We also show that both pili and LecA mutants of P. aeruginosa adhere to asialo-GM1 coated solid surfaces, and that a class of synthetic ligands for pili and LecA inhibits both pili and LecA-mediated adherence of P. aeruginosa on asialo-GM1-coated surfaces.

Supported Lipid Bilayers and the Study of Two-Dimensional Binding Kinetics.

Binding between protein molecules on contacting cells is essential in initiating and regulating several key biological processes. In contrast to interactions between molecules in solution, these events are restricted to the two-dimensional (2D) plane of the meeting cell surfaces. However, converting between the more commonly available binding kinetics measured in solution and the so-called 2D binding kinetics has proven a complicated task since for the latter several factors other than the protein-protein interaction per se have an impact. A few important examples of these are: protein density, membrane fluctuations, force on the bond and the use of auxiliary binding molecules. The development of model membranes, and in particular supported lipid bilayers (SLBs), has made it possible to simplify the studied contact to analyze these effects and to measure 2D binding kinetics of individual protein-protein interactions. We will in this review give an overview of, and discuss, how different SLB systems have been used for this and compare different methods to measure binding kinetics in cell-SLB contacts. Typically, the SLB is functionalized with fluorescently labelled ligands whose interaction with the corresponding receptor on a binding cell can be detected. This interaction can either be studied 1) by an accumulation of ligands in the cell-SLB contact, whose magnitude depends on the density of the proteins and binding affinity of the interaction, or 2) by tracking single ligands in the SLB, which upon interaction with a receptor result in a change of motion of the diffusing ligand. The advantages and disadvantages of other methods measuring 2D binding kinetics will also be discussed and compared to the fluorescence-based methods. Although binding kinetic measurements in cell-SLB contacts have provided novel information on how ligands interact with receptors in vivo the number of these measurements is still limited. This is influenced by the complexity of the system as well as the required experimental time. Moreover, the outcome can vary significantly between studies, highlighting the necessity for continued development of methods to study 2D binding kinetics with higher precision and ease.

Targeted drug delivery of magnetic microbubble for abdominal aortic aneurysm: an in silico study.

Targeted drug delivery (TDD) to abdominal aortic aneurysm (AAA) using a controlled and efficient approach has recently been a significant challenge. In this study, by using magnetic microbubbles (MMBs) under a magnetic field, we investigated the MMBs performance in TDD to AAA based on the amount of surface density of MMBs (SDMM) adhered to the AAA lumen. The results showed that among the types of MMBs studied in the presence of the magnetic field, micromarkers are the best type of microbubble with a -[Formula: see text] increase in SDMM adhered to the critical area of AAA. The results show that applying a magnetic field causes the amount of SDMM adhered to the whole area of AAA to increase -[Formula: see text] times compared to the condition in which the magnetic field is absent. This optimal and maximum value occurs for Definity MMBs with - 3.3 µm diameter. Applying a magnetic field also increases the adhesion surface density by - [Formula: see text], - [Formula: see text], and -[Formula: see text] times for the Micromarker, Optison, and Sonovue microbubbles, respectively, relative to the condition in which the magnetic field is absent. It was shown that using MBBs under magnetic field has the best performance in delivery to AAA for patients with negative inlet blood flow. Also, we have exposed that in an efficient TDD to AAA using MMBs, decreasing the density of MMBs increases drug delivery efficiency and performance. When density is - [Formula: see text], there is the highest difference (about - 75%) between the SDMM adhered to AAA in the presence of a magnetic field and in the absence of a magnetic field.

Molecular Mechanics Study of Flow and Surface Influence in Ligand-Protein Association.

Ligand-protein association is the first and critical step for many biological and chemical processes. This study investigated the molecular association processes under different environments. In biology, cells have different compartments where ligand-protein binding may occur on a membrane. In experiments involving ligand-protein binding, such as the surface plasmon resonance and continuous flow biosynthesis, a substrate flow and surface are required in experimental settings. As compared with a simple binding condition, which includes only the ligand, protein, and solvent, the association rate and processes may be affected by additional ligand transporting forces and other intermolecular interactions between the ligand and environmental objects. We evaluated these environmental factors by using a ligand xk263 binding to HIV protease (HIVp) with atomistic details. Using Brownian dynamics simulations, we modeled xk263 and HIVp association time and probability when a system has xk263 diffusion flux and a non-polar self-assembled monolayer surface. We also examined different protein orientations and accessible surfaces for xk263. To allow xk263 to access to the dimer interface of immobilized HIVp, we simulated the system by placing the protein 20Å above the surface because immobilizing HIVp on a surface prevented xk263 from contacting with the interface. The non-specific interactions increased the binding probability while the association time remained unchanged. When the xk263 diffusion flux increased, the effective xk263 concentration around HIVp, xk263-HIVp association time and binding probability decreased non-linearly regardless of interacting with the self-assembled monolayer surface or not. The work sheds light on the effects of the solvent flow and surface environment on ligand-protein associations and provides a perspective on experimental design.

Accurate Receptor-Ligand Binding Free Energies from Fast QM Conformational Chemical Space Sampling.

Small molecule receptor-binding is dominated by weak, non-covalent interactions such as van-der-Waals hydrogen bonding or electrostatics. Calculating these non-covalent ligand-receptor interactions is a challenge to computational means in terms of accuracy and efficacy since the ligand may bind in a number of thermally accessible conformations. The conformational rotamer ensemble sampling tool (CREST) uses an iterative scheme to efficiently sample the conformational space and calculates energies using the semi-empirical 'Geometry, Frequency, Noncovalent, eXtended Tight Binding' (GFN2-xTB) method. This combined approach is applied to blind predictions of the modes and free energies of binding for a set of 10 drug molecule ligands to the cucurbit[n]urils CB[8] receptor from the recent 'Statistical Assessment of the Modeling of Proteins and Ligands' (SAMPL) challenge including morphine, hydromorphine, cocaine, fentanyl, and ketamine. For each system, the conformational space was sufficiently sampled for the free ligand and the ligand-receptor complexes using the quantum chemical Hamiltonian. A multitude of structures makes up the final conformer-rotamer ensemble, for which then free energies of binding are calculated. For those large and complex molecules, the results are in good agreement with experimental values with a mean error of 3 kcal/mol. The GFN2-xTB energies of binding are validated by advanced density functional theory calculations and found to be in good agreement. The efficacy of the automated QM sampling workflow allows the extension towards other complex molecular interaction scenarios.

Exploration of Ligand-receptor Binding and Mechanisms for Alginate Reduction and Phenotype Reversion by Mucoid Pseudomonas aeruginosa.

Bacteria in general can develop a wide range of phenotypes under different conditions and external stresses. The phenotypes that reside in biofilms, overproduce exopolymers, and show increased motility often exhibit drug tolerance and drug persistence. In this work, we describe a class of small molecules that delay and inhibit the overproduction of alginate by a non-swarming mucoid Pseudomonas aeruginosa. Among these molecules, selected benzophenone-derived alkyl disaccharides cause the mucoid bacteria to swarm on hydrated soft agar gel and revert the mucoid to a nonmucoid phenotype. The sessile (biofilm) and motile (swarming) phenotypes are controlled by opposing signaling pathways with high and low intracellular levels of bis-(3',5')-cyclic diguanosine monophosphate (cdG), respectively. As our molecules control several of these phenotypes, we explored a protein receptor, pilin of the pili appendages, that is consistent with controlling these bioactivities and signaling pathways. To test this binding hypothesis, we developed a bacterial motility-enabled binding assay that uses the interfacial properties of hydrated gels and bacterial motility to conduct label-free ligand-receptor binding studies. The structure-activity correlation and receptor identification reveal a plausible mechanism for reverting mucoid to nonmucoid phenotypes by binding pili appendages with ligands capable of sequestering and neutralizing reactive oxygen species.

Drug delivery performance of nanocarriers based on adhesion and interaction for abdominal aortic aneurysm treatment.

Targeted drug delivery using nanocarriers (NCs) is one of the novel techniques that has recently been used to improve drug delivery to the Abdominal aortic aneurysm (AAA) disease. The purpose of this study is to evaluate the surface density of NCs (SDNC) adhered via ligand-receptor binding to the inner wall of AAA. For this purpose, fluid-structure interaction (FSI) analysis was first performed for the patient-specific and ideal AAA models. Then, by injecting NCs into the aortic artery, the values of SDNC adhered to and interacted with AAA wall were obtained. Two types of NCs, liposomes, and solid particles in four different diameters, were used to investigate the effect of the diameter and the type of NCs on the drug delivery. Additionally, the effect of the number of the injected NCs to the artery on the values of SDNC adhered to and interacted with AAA wall was investigated. The simulation results showed that the interaction and adhesion values of SDNC for Liposome nanoparticles were higher than the ones for the solid particles. Furthermore, as the diameter of NCs increases, the values of SDNC adhered to AAA wall increase, but the values of SDNC interacted with the inner wall of AAA decrease. In the low number of inserted NCs in the artery (1000 NCs), the interaction and adhesion values of SDNC were very slight, and by increasing the number of NCs inserted into the artery, the drug delivery was improved. By examining different AAA models, it was found that the complexity of the shape of AAA has a minor effect on the pattern of increase or decrease of the values of SDNC adhered to and interacted with AAA wall.This study's findings can improve the understanding of NCs design and propose the appropriate amount of their injection into various AAA models.

Measuring ligand-cell surface receptor affinities with axial line-scanning fluorescence correlation spectroscopy.

Development and homeostasis of multicellular organisms is largely controlled by complex cell-cell signaling networks that rely on specific binding of secreted ligands to cell surface receptors. The Wnt signaling network, as an example, involves multiple ligands and receptors to elicit specific cellular responses. To understand the mechanisms of such a network, ligand-receptor interactions should be characterized quantitatively, ideally in live cells or tissues. Such measurements are possible using fluorescence microscopy yet challenging due to sample movement, low signal-to-background ratio and photobleaching. Here, we present a robust approach based on fluorescence correlation spectroscopy with ultra-high speed axial line scanning, yielding precise equilibrium dissociation coefficients of interactions in the Wnt signaling pathway. Using CRISPR/Cas9 editing to endogenously tag receptors with fluorescent proteins, we demonstrate that the method delivers precise results even with low, near-native amounts of receptors.

In-silico analysis of ligand-receptor binding patterns of α-MMC, TCS and MAP30 protein to LRP1 receptor.

Alpha-momorcharin (α-MMC), trichosanthin (TCS), and momordica anti-HIV protein of 30 kD (MAP30) are potential anti-tumor drug candidates but have cytotoxicity to normal cells. The binding of these proteins to LRP1 receptor and the subsequent endocytosis are essential to their cytotoxicity, but this binding process remains largely unknown. This study, in-silico analysis of the binding patterns, was conducted via the protein-protein docking software, ZDOCK 3.0.2 package, to better understand the binding process. Specifically, α-MMC, TCS and MAP30 were selected and bound to binding subunits CR56 and CR17 of LRP1. After docking, the 10 best docking solutions are retained based on the default ZDOCK scores and used for structural assessment. Our results showed that, α-MMC bound to LRP1 stably at the amino acid residues 1-20, at which 8 residues formed 21 hydrogen bonds with 15 residues of CR56 and 10 residues formed 15 hydrogen bonds with 12 residues of CR17. In contrast, TCS and MAP30 bound mainly to LRP1 at the residues 1-57/79-150 and residues 58-102, respectively, which were functional domains of TCS and MAP30. Since residues 1-20 are outside the functional domain of α-MMC, α-MMC is considered more suitable to attenuate by mutating the receptor binding site. Thus, our analysis lays the foundation for future genetic engineering work on α-MMC, and makes important contributions to its potential clinical use in cancer treatment.

Variational implicit-solvent predictions of the dry-wet transition pathways for ligand-receptor binding and unbinding kinetics.

Ligand-receptor binding and unbinding are fundamental biomolecular processes and particularly essential to drug efficacy. Environmental water fluctuations, however, impact the corresponding thermodynamics and kinetics and thereby challenge theoretical descriptions. Here, we devise a holistic, implicit-solvent, multimethod approach to predict the (un)binding kinetics for a generic ligand-pocket model. We use the variational implicit-solvent model (VISM) to calculate the solute-solvent interfacial structures and the corresponding free energies, and combine the VISM with the string method to obtain the minimum energy paths and transition states between the various metastable ("dry" and "wet") hydration states. The resulting dry-wet transition rates are then used in a spatially dependent multistate continuous-time Markov chain Brownian dynamics simulation and the related Fokker-Planck equation calculations of the ligand stochastic motion, providing the mean first-passage times for binding and unbinding. We find the hydration transitions to significantly slow down the binding process, in semiquantitative agreement with existing explicit-water simulations, but significantly accelerate the unbinding process. Moreover, our methods allow the characterization of nonequilibrium hydration states of pocket and ligand during the ligand movement, for which we find substantial memory and hysteresis effects for binding vs. unbinding. Our study thus provides a significant step forward toward efficient, physics-based interpretation and predictions of the complex kinetics in realistic ligand-receptor systems.