Unbinding Processes Research Articles

Kinetics of pH-dependent interactions between PD-1 and PD-L1 immune checkpoint proteins from molecular dynamics.

Immune checkpoint blockade of signaling pathways such as PD-1/PD-L1 has recently opened up a new avenue for highly efficient immunotherapeutic strategies to treat cancer. Since tumor microenvironments are characterized by lower pH (5.5-7.0), pH-dependent protein-ligand interactions can be exploited as efficient means to regulate drug affinity and specificity for a variety of malignancies. In this article, we investigate the mechanism and kinetics of pH-dependent binding and unbinding processes for the PD-1/PD-L1 checkpoint pair employing classical molecular dynamics simulations. Two representative pH levels corresponding to circumneutral physiological conditions of blood (pH 7.4) and acidic tumor microenvironment (pH 5.5) are considered. Our calculations demonstrate that pH plays a key role in protein-ligand interactions with small pH changes leading to several orders of magnitude increase in binding affinity. By identifying the binding pocket in the PD-1/PD-L1 complex, we show a pivotal role of the His68 protonation state of PD-1in the complex stabilization at low pH. The results on the reaction rate constants are in qualitative agreement with available experimental data. The obtained molecular details are important for further engineering of binding/unbinding kinetics to formulate more efficient immune checkpoint blockade strategies.

Simulation of Spontaneous G Protein Activation Reveals a New Intermediate Driving GDP Unbinding

Activation of heterotrimeric G proteins is a key step in many signaling cascades. However, a complete mechanism for this process, which requires allosteric communication between binding sites that are ∼30 Å apart, remains elusive. We construct an atomically detailed model of G protein activation by combining three powerful computational methods: metadynamics, Markov state models (MSMs), and CARDS analysis of correlated motions. We uncover a mechanism that is consistent with a wide variety of structural and biochemical data. Surprisingly, the rate-limiting step for activation, GDP dissociation, correlates with tilting rather than translation of the GPCRbinding helix 5. β-Strands 1-3 and helix 1 emerge as hubs in the allosteric network that links conformational changes in the GPCR-binding site to disordering of the distal nucleotidebinding site and consequent GDP release. Our approach and insights provide foundations for understanding disease-implicated G protein mutants, illuminating slow events in allosteric networks, and examining unbinding processes with slow off-rates. Published in: Sun, X.∗, & Singh, S.∗, et al. Simulation of spontaneous G protein activation reveals a new intermediate driving GDP unbinding. eLife, 7, (2018).

Enhanced Sampling Simulations of Ligand Unbinding Kinetics Controlled by Protein Conformational Changes.

Understanding unbinding kinetics of protein-ligand systems is of great importance for the design of ligands with desired specificity and safety. In recent years, enhanced sampling techniques have emerged as effective tools for studying unbinding kinetics of protein-ligand systems at the atomistic level. However, in many protein-ligand systems, the ligand unbinding processes are strongly coupled to protein conformational changes and the disclosure of the hidden degrees of freedom closely related to the protein conformational changes so that sampling is enhanced over these degrees of freedom remains a great challenge. Here, we show how potential-scaled molecular dynamics (sMD) and infrequent metadynamics (InMetaD) simulation techniques can be combined to successfully reveal the unbinding mechanism of 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-[18F]fluo-rodibenzo[b,d]thiophene 5,5-dioxide ([18F]ASEM) from a chimera structure of the α7-nicotinic acetylcholine receptor. By using sMD simulations, we disclosed that the "close" to "open" conformational change of loop C plays a key role in the ASEM unbinding process. By carrying out InMetaD simulations with this conformational change taken into account as an additional collective variable, we further captured the key states in the unbinding process and clarified the unbinding mechanism of ASEM from the protein. Our work indicates that combining sMD and InMetaD simulation techniques can be an effective approach for revealing the unbinding mechanism of a protein-ligand system where protein conformational changes control the unbinding process.

Solvent effects on molecular encapsulation of Toussantine-A by chitosan nanoparticle: A metadynamics study

We report a metadynamics study of toussantine-A with chitosan nanoparticle in dimethyl sulfoxide and water in order to assess the solvent related effects on free energy and interaction stability between the two compounds. We use well-tempered metadynamics to characterize the binding and unbinding processes of toussantine-A from chitosan. The calculated binding free energies support unbinding process deduced from metadynamics simulation. Toussantine-A interacts poorly with chitosan in the presence of dimethyl sulfoxide when compared to water; the dimethyl sulfoxide gives rising a reverse effect on drug binding that favours to the unbinding of toussantine-A.

How Complex Is the Concanavalin A-Carboxypeptidase Y Interaction?

Lectin-carbohydrate interactions can be exploited in ultrasensitive biochemical recognition or medical diagnosis. For this purpose, besides the high specificity of the interactions, an appropriate methodology for their quantitative and detailed characterization is demanded. In this work, we determine the unbinding properties of the concanavalin A-carboxypeptidase Y complex, which is important for characterization of glycoproteins on the surface of biological cells. To achieve the goal, we have developed a methodology based on dynamic force spectroscopy measurements and two advanced theoretical models of force-induced unbinding. Our final results allowed excluding both, rebinding processes and the multibarrier character of the interaction potential, as possible explanations of the concanavalin A-carboxypeptidase Y unbinding mechanisms. Such characteristics as the position and height of the activation barrier and the force-free dissociation rate were determined. We hope our paper contributes to a better understanding of the unbinding processes in receptor-ligand complexes.

CaverDock: a molecular docking-based tool to analyse ligand transport through protein tunnels and channels.

Protein tunnels and channels are key transport pathways that allow ligands to pass between proteins' external and internal environments. These functionally important structural features warrant detailed attention. It is difficult to study the ligand binding and unbinding processes experimentally, while molecular dynamics simulations can be time-consuming and computationally demanding. CaverDock is a new software tool for analysing the ligand passage through the biomolecules. The method uses the optimized docking algorithm of AutoDock Vina for ligand placement docking and implements a parallel heuristic algorithm to search the space of possible trajectories. The duration of the simulations takes from minutes to a few hours. Here we describe the implementation of the method and demonstrate CaverDock's usability by: (i) comparison of the results with other available tools, (ii) determination of the robustness with large ensembles of ligands and (iii) the analysis and comparison of the ligand trajectories in engineered tunnels. Thorough testing confirms that CaverDock is applicable for the fast analysis of ligand binding and unbinding in fundamental enzymology and protein engineering. User guide and binaries for Ubuntu are freely available for non-commercial use at https://loschmidt.chemi.muni.cz/caverdock/. The web implementation is available at https://loschmidt.chemi.muni.cz/caverweb/. The source code is available upon request. Supplementary data are available at Bioinformatics online.

Understanding Corona Exchange Dynamics on Single-Walled Carbon Nanotubes with Multiplexed Fluorescence Monitoring

Adsorption of polymers on single-walled carbon nanotubes (SWCNTs) has enabled developments in molecular sensing, in vivo imaging, gene and drug delivery, and chirality sorting applications. Noncovalent functionalization offers a modification route that preserves the pristine atomic structure, thus retaining the intrinsic near-infrared fluorescence of the SWCNTs for sensing or imaging functions, and offers a reversible binding mode for cargo delivery or chirality separation processes. However, noncovalent adsorption is an inherently dynamic process, where exchange occurs between molecules in the bulk solution and molecules on the surface, into what is known as the ‘corona phase’. In the case of polymers on SWCNTs, the nature, strength, and kinetics of both the polymer binding and unbinding processes are important contributors to the success of polymer-SWCNT based technologies. Understanding this binding process is especially important for intended uses of functionalized SWCNTs in biological environments, where native biomolecules compete with the original polymer to occupy the SWCNT surface. Binding of proteins and other biomolecules to the SWCNT not only disrupts the intended functionality of the nanoparticle, but further leads to potentially adverse biocompatibility outcomes. We present an assay to study the process of corona exchange dynamics between solution-phase and corona-phase polymers on SWCNTs. Real-time tracking of polymer and protein adsorption and desorption events are conducted with systematic variation of the molecular entities and solution conditions. This assay exploits the quenching property of fluorophores in close proximity to the SWCNT surface to monitor ligand binding and unbinding events. Binding profiles are thus extracted from the experimental assay and used to inform a kinetic model of the system. This study encompasses exchange of SWCNT-adsorbed ssDNA and either ssDNA, surfactant, or protein added to the bulk solution with multiplexed fluorescence tracking of all entities. Moreover, the kinetics of these binding profiles are validated against an orthogonal and more ubiquitously-used platform monitoring solvatochromic shifting of the near-infrared SWCNT spectrum as a proxy for SWCNT corona perturbations (Beyene et al., Nano Letters, 2018; Heller et al., PNAS, 2011; Jena et al., ACS Appl. Mater. Interfaces, 2017). The work presented herein develops an understanding of the fundamental corona exchange mechanism, contextualizes the nature of the ligand exchange process versus SWCNT solvatochromic shifting, and provides insight into performance of these designed SWCNT-based systems in biologically relevant, protein-rich conditions.

Stimulus-response recoding during inhibitory control is associated with superior frontal and parahippocampal processes

Inhibitory control is affected by perceptual processes, but the mechanisms how perceptual features affect response inhibition are poorly understood. Theoretical frameworks detailing how variations in stimulus features that create overlaps between response categories can affect action control, like the Theory of Event Coding (TEC), have not been transferred to inhibitory control. We present a novel Go/Nogo paradigm in which we varied stimulus feature overlap between Go and Nogo trials. To examine what cognitive-neurophysiological subprocesses and functional neuroanatomical structures are modulated by stimulus-response feature overlap and recoding during inhibitory control, we combine event-related potential (ERP) recordings with source localization analyses. We show that response inhibition was compromised when stimulus features overlapped between Go and Nogo trials. The EEG data show that the recoding of stimulus-response mappings induced by such a stimulus feature overlaps affects subprocesses from perceptual gating/categorization (P1 ERP-component) to pre-motor inhibition (Nogo-N2 ERP-component) and motor inhibition (Nogo-P3 ERP-component). Although these are distinct processes, overlapping neuronal structures are associated with these modulations. The cascade of processes starts in the superior frontal cortex and is associated with perceptual categorization mechanisms. Subsequently, pre-motor inhibition or stimulus-response unbinding processes are modulated in parahippocampal structures before stimulus-response rebinding and motor inhibition is accomplished in parahippocampal and superior frontal structures. The study shows how perceptual processes can affect response inhibition using a theoretical framework, which has, until now, not been brought into connection with inhibitory control and establishes links between neurophysiology and functional neuroanatomy of inhibitory control with the TEC framework.

Free Energy of Specific Cholesterol-GPCR Interactions

G-protein coupled receptors, the largest class of membrane proteins, are the targets for 30-50% of drugs. Many membrane proteins have been shown to be functionally dependent on cholesterol, several of which have also been shown to bind cholesterol at well-defined locations on their membrane-facing surface. In this work, all-atom simulation together with well-tempered metadynamics is used to study the free energy landscape of cholesterol binding sites on the A2A adenosine receptor, a G-protein coupled receptor that is a target for the treatment of Parkinson's disease. Characterizing cholesterol binding and unbinding processes, and its corresponding response of the receptor, is of great importance towards better drug designs targeting G-protein coupled receptors. The protocol is generic, and readily applicable to other GPCRs and membrane proteins.

Multi-dimensional spectral gap optimization of order parameters (SGOOP) through conditional probability factorization.

Spectral gap optimization of order parameters (SGOOP) [P. Tiwary and B. J. Berne, Proc. Natl. Acad. Sci. U. S. A. 113, 2839 (2016)] is a method for constructing the reaction coordinate (RC) in molecular systems, especially when they are plagued with hard to sample rare events, given a larger dictionary of order parameters or basis functions and limited static and dynamic information about the system. In its original formulation, SGOOP is designed to construct a 1-dimensional RC. Here we extend its scope by introducing a simple but powerful extension based on the notion of conditional probability factorization where known features are effectively washed out to learn additional and possibly hidden features of the energy landscape. We show how SGOOP can be used to proceed in a sequential and bottom-up manner to (i) systematically probe the need for extending the dimensionality of the RC and (ii) if such a need is identified, learn additional coordinates of the RC in a computationally efficient manner. We formulate the method and demonstrate its utility through three illustrative examples, including the challenging and important problem of calculating the kinetics of benzene unbinding from the protein T4L99A lysozyme, where we obtain excellent agreement in terms of dissociation pathway and kinetics with other sampling methods and experiments. In this last case, starting from a larger dictionary of 11 order parameters that are generic for ligand unbinding processes, we demonstrate how to automatically learn a 2-dimensional RC, which we then use in the infrequent metadynamics protocol to obtain 16 independent unbinding trajectories. We believe our method will be a big step in increasing the utility of SGOOP in performing intuition-free sampling of complex systems. Finally, we believe that the utility of our protocol is amplified by its applicability to not just SGOOP but also other generic methods for constructing the RC.

Simulation of spontaneous G protein activation reveals a new intermediate driving GDP unbinding.

Activation of heterotrimeric G proteins is a key step in many signaling cascades. However, a complete mechanism for this process, which requires allosteric communication between binding sites that are ~30 Å apart, remains elusive. We construct an atomically detailed model of G protein activation by combining three powerful computational methods: metadynamics, Markov state models (MSMs), and CARDS analysis of correlated motions. We uncover a mechanism that is consistent with a wide variety of structural and biochemical data. Surprisingly, the rate-limiting step for GDP release correlates with tilting rather than translation of the GPCR-binding helix 5. β-Strands 1 - 3 and helix 1 emerge as hubs in the allosteric network that links conformational changes in the GPCR-binding site to disordering of the distal nucleotide-binding site and consequent GDP release. Our approach and insights provide foundations for understanding disease-implicated G protein mutants, illuminating slow events in allosteric networks, and examining unbinding processes with slow off-rates.

Structure-Based Virtual Screening of High-Affinity ATP-Competitive Inhibitors Against Human Lemur Tyrosine Kinase-3 (LMTK3) Domain: A Novel Therapeutic Target for Breast Cancer.

Human lemur tyrosine kinase-3 (LMTK3) is an oncogenic kinase known to regulate ER-α through phosphorylation and is considered to be a novel therapeutic target for breast cancer. In this work, we have studied the ATP-binding mechanism with LMTK3 domain and also carried out virtual screening on LMTK3 to identify lead compounds using Dock blaster server. The top scored compounds obtained from Dock blaster were then narrowed down further to six lead compounds (ZINC37996511, ZINC83363046, ZINC3745998, ZINC50456700, ZINC83351792 and ZINC83364581) based on high-binding affinity and non-bonding interactions with LMTK3 using Autodock 4.2 program. We found in comparison to ATP, the lead compounds bind relatively stronger to LMTK3. The relative binding free energy results from MM-PBSA/GBSA method further indicate the strong binding affinity of lead compounds over ATP to LMTK3 in the dynamic system. Further, potential of mean force (PMF) study for ATP and lead compounds with LMTK3 have been performed to explore the unbinding processes and the free energy barrier. From the PMF results, we observed that the lead compounds have higher dissociation energy barriers than the ATP. Our findings suggest that these lead compounds may compete with ATP, and could act as probable potential inhibitors for LMTK3.

Investigating Small-Molecule Ligand Binding to G Protein-Coupled Receptors with Biased or Unbiased Molecular Dynamics Simulations.

An increasing number of G protein-coupled receptor (GPCR) crystal structures provide important-albeit static-pictures of how small molecules or peptides interact with their receptors. These high-resolution structures represent a tremendous opportunity to apply molecular dynamics (MD) simulations to capture atomic-level dynamical information that is not easy to obtain experimentally. Understanding ligand binding and unbinding processes, as well as the related responses of the receptor, is crucial to the design of better drugs targeting GPCRs. Here, we discuss possible ways to study the dynamics involved in the binding of small molecules to GPCRs, using long timescale MD simulations or metadynamics-based approaches.

Protein-ligand (un)binding kinetics as a new paradigm for drug discovery at the crossroad between experiments and modelling.

In the last three decades, protein and nucleic acid structure determination and comprehension of the mechanisms, leading to their physiological and pathological functions, have become a cornerstone of biomedical sciences. A deep understanding of the principles governing the fates of cells and tissue at the molecular level has been gained over the years, offering a solid basis for the rational design of drugs aimed at the pharmacological treatment of numerous diseases. Historically, affinity indicators (i.e. Kd and IC50/EC50) have been assumed to be valid indicators of the in vivo efficacy of a drug. However, recent studies pointed out that the kinetics of the drug-receptor binding process could be as important or even more important than affinity in determining the drug efficacy. This eventually led to a growing interest in the characterisation and prediction of the rate constants of protein-ligand association and dissociation. For instance, a drug with a longer residence time can kinetically select a given receptor over another, even if the affinity for both receptors is comparable, thus increasing its therapeutic index. Therefore, understanding the molecular features underlying binding and unbinding processes is of central interest towards the rational control of drug binding kinetics. In this review, we report the theoretical framework behind protein-ligand association and highlight the latest advances in the experimental and computational approaches exploited to investigate the binding kinetics.

Impact of human galectin-1 binding to saccharide ligands on dimer dissociation kinetics and structure.

Endogenous lectins can control critical biological responses, including cell communication, signaling, angiogenesis and immunity by decoding glycan-containing information on a variety of cellular receptors and the extracellular matrix. Galectin-1 (Gal-1), a prototype member of the galectin family, displays only one carbohydrate recognition domain and occurs in a subtle homodimerization equilibrium at physiologic concentrations. Such equilibrium critically governs the function of this lectin signaling by allowing tunable interactions with a preferential set of glycosylated receptors. Here, we used a combination of experimental and computational approaches to analyze the kinetics and mechanisms connecting Gal-1 ligand unbinding and dimer dissociation processes. Kinetic constants of both processes were found to differ by an order of magnitude. By means of steered molecular dynamics simulations, the ligand unbinding process was followed monitoring water occupancy changes. By determining the water sites in a carbohydrate binding place during the unbinding process, we found that rupture of ligand-protein interactions induces an increase in energy barrier while ligand unbinding process takes place, whereas the entry of water molecules to the binding groove and further occupation of their corresponding water sites contributes to lowering of the energy barrier. Moreover, our findings suggested local asymmetries between the two subunits in the dimer structure detected at a nanosecond timescale. Thus, integration of experimental and computational data allowed a more complete understanding of lectin ligand binding and dimerization processes, suggesting new insights into the relationship between Gal-1 structure and function and renewing the discussion on the biophysics and biochemistry of lectin-ligand lattices.

Insight into Crizotinib Resistance Mechanisms Caused by Three Mutations in ALK Tyrosine Kinase using Free Energy Calculation Approaches

As a safe and efficacious drug, crizotinib was approved by the U.S. Food and Drug Administration (FDA) in 2011 for the treatment of advanced fusion-type nonsmall-cell lung cancer. Although high response ratio was detected from the patients treated with crizotinib, the cancer has eventually conferred resistance to crizotinib. Several drug resistance mutations have been found in the anaplastic lymphoma kinase (ALK) tyrosine kinase domain as the target for crizotinib, but the drug resistance mechanisms remain unclear. Therefore, in this study, the adaptive biasing force (ABF) method and two-end-state free energy calculation approaches were employed to elucidate the resistance mechanisms of crizotinib induced by the mutations L1152R, G1202R, and S1206Y. The ABF simulation results suggest that the reaction coordinates for the unbinding processes of crizotinib from the binding pockets of the mutated ALKs is different from that of the wild type ALK. The potentials of mean force for the crizotinib unbinding and the binding free energies predicted by the two-end-state free energy calculations are consistent with the experimental data. Our results indicate that the three mutations weaken the binding affinity of crizotinib obviously and lead to drug resistance. The free energy decomposition analysis illustrates the importance of the loss of two important H-bonds in the L1152R and S1206Y mutants on drug resistance. The entropy analysis shows that the entropy term plays a critical role in the substantial change of the conformational entropies of G1202R and L1152R. Our results reveal the mechanisms of drug resistance and provide vital clues for the development of new inhibitors to combat drug resistance.

Mechanism and Energetics of Ligand Release in the Aspartate Transporter GltPh

The bacterial aspartate transporter GltPh cotransports three Na(+) ions with the substrate. The mechanism and energetics of ligand binding have previously been studied using molecular dynamics simulations on the crystal structure of GltPh captured in the outward-facing state. Here we use the recent crystal structure of the inward-facing state of GltPh to study the reverse process of unbinding of ligands. Gating behavior is studied in the presence of different ligands. A detailed characterization of the intracellular gate is given, pointing out the differences from the extracellular gate. We then perform free energy simulations to calculate the binding affinities of all the ligands in different combinations, from which the unbinding order is determined as Na2, (gate opens), Asp, Na1, and Na3. The strong coupling between Asp and Na1 is quantified from several free energy calculations. Na3 has the largest affinity to GltPh, and therefore, its unbinding is proposed as the rate-limiting step in the transport cycle. The release time of Na3, estimated from Kramers' rate theory, is shown to be consistent with the experimental turnover rate of the transporter.

Stress relaxation through crosslink unbinding in cytoskeletal networks

The mechanical properties of cells are dominated by the cytoskeleton, an interconnected network of long elastic filaments. The connections between the filaments are provided by crosslinking proteins, which constitute, next to the filaments, the second important mechanical element of the network. An important aspect of cytoskeletal assemblies is their dynamic nature, which allows remodeling in response to external cues. The reversible nature of crosslink binding is an important mechanism that underlies these dynamical processes. Here, we develop a theoretical model that provides us insight into how the mechanical properties of cytoskeletal networks may depend on their underlying constituting elements. We incorporate three important ingredients: non-affine filament deformations in response to network strain; the interplay between filament and crosslink mechanical properties; and reversible crosslink (un)binding in response to the imposed stress. With this we are able to self-consistently calculate the nonlinear modulus of the network as a function of deformation amplitude and crosslink as well as filament stiffnesses. During loading, crosslink unbinding processes lead to a relaxation of stress and therefore to a reduction of the network modulus and eventually to network failure, when all crosslinks are unbound. This softening due to crosslink unbinding generically competes with an inherent stiffening response, which may be due to either filament or crosslink nonlinear elasticity.

Unbinding Process of Amelogenin and Fibrinogen Adsorbed on Different Solid Surfaces Using AFM

The interaction of proteins with solid surfaces is a fundamental phenomenon in the biomaterials field. We investigated, using atomic force microscopy (AFM), the interactions of a recombinant amelogenin with titanium, a biphasic calcium phosphate (BCP) and mica. The unbinding processes were compared to those of an earlier studied protein, namely fibrinogen. Force spectroscopy (AFM) experiments were carried out at 0 ms, 102 ms, 103 ms and 104 ms of contact time. In general, the rupture forces increased as a function of interaction time. The unbinding forces of amelogenin interacting with the BCP surface were always stronger than those of the amelogenin-titanium system. The unbinding forces of fibrinogen interacting with the BCP surface were always much stronger than those of the fibrinogen-titanium system. For the most part, this study provides direct evidence that recombinant amelogenin binds more strongly than fibrinogen on the studied substrates.

Application of an Inter-Protein Coarse-Grained Force Field to Binding Process of Actomyosin

Protein-protein interaction is important for many biological processes. Especially, when molecular motors fulfill their functions, they sometimes form multi-subunit complex or move through alternate binding and unbinding processes. Therefore, it is important to model inter-protein interaction appropriately in order to reveal working mechanism of molecular motors. Coarse-grained model is often used to analyze working mechanism of molecular motors for computational efficiency, but modeling of inter-protein interaction sometimes becomes difficult problem. In this study, we apply a sequence dependent inter-protein coarse-grained force field (each amino acid is coarse-grained to one bead) that considers electrostatic interaction between charged residues and sequence dependent contact interaction [Kim and Hummer, JMB (2008)] to a molecular motor, actomyosin. Myosin is known to take detached, weakly bound and strongly bound state to its rail protein, actin, during ATP hydrolysis cycle, and the force is thought to be generated during the weakly-to-strongly transition. These binding states should be coupled to nucleotide dependent conformational changes of myosin such as open-close motion of the actin-binding cleft and rearrangement of surface loops like loop II. We will discuss the effect of the myosin conformational changes on the binding process.