Unbinding Process Research Articles

Unbinding pathways of VEGFR2 inhibitors revealed by steered molecular dynamics.

A detailed atomistic description of the unbinding process of sorafenib and sunitinib, two known VEGFR2 inhibitors clinically used to treat renal cell carcinoma, was unraveled by using steered molecular dynamics (SMD) simulations. While sunitinib is a fast-dissociating binder, sorafenib exhibits quite a long residence time at this enzyme, which might impact its duration of action in vivo. In order to gain insights into the kinetically different behaviors of the two inhibitors, an SMD study was carried out, which involved a careful optimization of the force and velocity parameters. We were able to identify two different binding pathways for the two inhibitors, as sunitinib exited the ATP binding site from the cavity entrance without a rupture point while sorafenib moved opposite to the ATP binding site entrance. Furthermore, the calculated ΔGoff values clearly reflect on a qualitative level the distinct off-rates of the two inhibitors, thus suggesting that this protocol could be tried on other VEGFR2 ligands to assess its robustness and then used to rank structural analogues of these derivatives.

Probing interactions between human lung adenocarcinoma A549 cell and its aptamers at single‐molecule resolution

Because cell-specific aptamers have high potential for biomedical applications, investigation of the interaction between cell and its aptamers may be of key importance for an improved understanding of biochemical processes. Herein, the interaction between human lung adenocarcinoma A549 cell and its four aptamers was explored using single-molecule force spectroscopy (SMFS). The values of the unbinding force varied from 117.1 to 171.0 pN at the loading rate of 1.8 × 10(5) pN/s. Based on the dependence of singe molecule force on the atomic force microscopy loading rate, the corresponding kinetic parameters were obtained. The results revealed two activation barriers and two transient states in the unbinding process of aptamer/cell interaction. More importantly, the binding sites on A549 cells with its four aptamers were defined to be different using SMFS and flow cytometry. This work demonstrated that SMFS can be used as a powerful tool for exploring the aptamer/cell binding behavior at the single-molecule level, and may provide valuable information for the design and application of aptamer probes.

Molecular dynamics simulations of polyamide membrane, calcium alginate gel, and their interactions in aqueous solution.

We perform molecular dynamics (MD) simulations to investigate the cross-linked polyamide (PA) membrane, the aggregation of alginate molecules in the presence of Ca(2+) ions, and their molecular binding mechanism in aqueous solution. We use a steered molecular dynamics (SMD) approach to simulate the unbinding process between a PA membrane and an alginate gel complex. Simulation results show that Ca(2+) ions are strongly associated with the carboxylate groups in alginate molecules, forming a web structure. The adhesion force between alginate gel and PA surface during unbinding originates from several important molecular interactions. These include the short-range hydrogen bonding and van der Waals attraction forces, and the ionic bridge binding that extends much longer pulling distances due to the significant chain deformations of alginate gel and PA membrane.

The ties to unbind: age-related differences in feature (un)binding in working memory for emotional faces.

In the present study, we investigated age-related differences in the processing of emotional stimuli. Specifically, we were interested in whether older adults would show deficits in unbinding emotional expression (i.e., either no emotion, happiness, anger, or disgust) from bound stimuli (i.e., photographs of faces expressing these emotions), as a hyper-binding account of age-related differences in working memory would predict. Younger and older adults completed different N-Back tasks (side-by-side 0-Back, 1-Back, 2-Back) under three conditions: match/mismatch judgments based on either the identity of the face (identity condition), the face’s emotional expression (expression condition), or both identity and expression of the face (both condition). The two age groups performed more slowly and with lower accuracy in the expression condition than in the both condition, indicating the presence of an unbinding process. This unbinding effect was more pronounced in older adults than in younger adults, but only in the 2-Back task. Thus, older adults seemed to have a specific deficit in unbinding in working memory. Additionally, no age-related differences were found in accuracy in the 0-Back task, but such differences emerged in the 1-Back task, and were further magnified in the 2-Back task, indicating independent age-related differences in attention/STM and working memory. Pupil dilation data confirmed that the attention/STM version of the task (1-Back) is more effortful for older adults than younger adults.

Simulation Study of Interactions Between Kinesin’s Neck Linker and Motor Domain

The docking movement of kinesin’s neck linker to the motor domain is the force generation step of kinesin. This docking movement is achieved by various non-bonding interactions between neck linker and motor domain. However, the relative strength and the structural basis for these interactions are still unclear. To elucidate these problems, we unbind the docked neck linker from motor domain using steered molecular dynamics and mutation simulations. We find that the surprisingly high strength of a structure called the “ asparagine latch” arises from exquisite cooperation between hydrogen bonds and hydrophobic interactions. A β-sheet called the “ cover-neck bundle” behaves like an elastic beam in its unbinding process. The hydrophobic interaction between ILE327 (amino acid sequence in 2KIN) and the hydrophobic pocket on the motor domain is strong and provides a fixed support for elastic bending of the cover-neck bundle.

Determining the Specificity of Monoclonal Antibody HPT-101 to Tau-Peptides with Optical Tweezers

Optical tweezers-assisted dynamic force spectroscopy is employed to investigate specific receptor-ligand interactions on the level of single binding events. In particular, we analyze binding of the phosphorylation-specific monoclonal antibody (mAb) HPT-101 to synthetic tau-peptides with two potential phosphorylation sites (Thr231 and Ser235), being the most probable markers for Alzheimer's disease. Whereas the typical interpretation of enzyme-linked immunosorbent assay (ELISA) suggests that this monoclonal antibody binds exclusively to the double-phosphorylated tau-peptide, we show here by DFS that the specificity of only mAb HPT-101 is apparent. In fact, binding occurs also to each sort of monophosphorylated peptide. Therefore, we characterize the unbinding process by analyzing the measured rupture force distributions, from which the lifetime of the bond without force τ0, its characteristic length xts, and the free energy of activation ΔG are extracted for the three mAb/peptide combinations. This information is used to build a simple theoretical model to predict features of the unbinding process for the double-phosphorylated peptide purely based on data on the monophosphorylated ones. Finally, we introduce a method to combine binding and unbinding measurements to estimate the relative affinity of the bonds. The values obtained for this quantity are in accordance with ELISA, showing how DFS can offer important insights about the dynamic binding process that are not accessible with this common and widespread assay.

Unbinding Pathways from the Glucocorticoid Receptor Shed Light on the Reduced Sensitivity of Glucocorticoid Ligands to a Naturally Occurring, Clinically Relevant Mutant Receptor

Glucocorticoids are endogenous steroid hormones that regulate essential biological functions, including metabolism, growth, and apoptosis. Glucocorticoids represent the most effective anti-inflammatory agents for the treatment of several inflammatory conditions. However, the clinical use of such drugs is hampered by severe side effects. Therefore, the development of novel glucocorticoid receptor (GR) modulators with an increased therapeutic index is impelling. Herein, using steered molecular dynamics (SMD) simulations, we provide a detailed picture of the unbinding process of three clinically relevant GR modulators from GR ligand binding domains. The SMD protocol described here can be used to prioritize the synthesis of structural analogues on the basis of their potentials of mean force and calculated unbinding energies. Moreover, our results are instrumental in explaining at atomic resolution the weakened ability of dexamethasone to activate the naturally occurring mutant I747M GR, which is implicated in rare familial glucocorticoid resistance, clinically characterized by glucocorticoid insensitivity.

Unbinding Pathways of GW4064 from Human Farnesoid X Receptor As Revealed by Molecular Dynamics Simulations

Farnesoid X receptor (FXR, NR1H4) is a member of a nuclear receptor superfamily, which plays important roles in bile acid homeostasis, lipoprotein and glucose metabolism, and hepatic regeneration. GW4064 is a potent and selective FXR agonist and has become a tool compound to probe the physiological functions of FXR. Until now, the mechanism of GW4064 entering and leaving the FXR pocket is still poorly understood. Here, we report a computational study of GW4064 unbinding pathways from FXR by using several molecular dynamics (MD) simulation techniques. Based on the crystal structure of FXR in complex with GW4064, conventional MD was first used to refine the binding and check the stability of GW4064 in the FXR pocket. Random acceleration MD simulations were then performed to explore the possible unbinding pathways of GW4064 from FXR. Four main pathway clusters were found, among which three subpathways, namely Paths 2A, 2B, and 1B, were observed most frequently. Multiple steered MD simulations were further employed to estimate the maximum rupture force and the sum of the forces and to characterize the intermediate states of the ligand unbinding process. By comparing the average force profiles and structural changes, Paths 2A and 2B were identified to be the most favorable unbinding pathways. The former is located between the H1-H2 loop and the H5-H6 loop, and the latter is located in the cleft formed by the H5-H6 loop, H6, and H7. Moreover, the residues lining the pathways were analyzed for their roles in ligand unbinding. Based on our results, the possible structural modification strategies on GW4064 were also proposed.

Molecular Dynamics Simulations Suggest Ligand’s Binding to Nicotinamidase/Pyrazinamidase

The research on the binding process of ligand to pyrazinamidase (PncA) is crucial for elucidating the inherent relationship between resistance of Mycobacterium tuberculosis and PncA’s activity. In the present study, molecular dynamics (MD) simulation methods were performed to investigate the unbinding process of nicotinamide (NAM) from two PncA enzymes, which is the reverse of the corresponding binding process. The calculated potential of mean force (PMF) based on the steered molecular dynamics (SMD) simulations sheds light on an optimal binding/unbinding pathway of the ligand. The comparative analyses between two PncAs clearly exhibit the consistency of the binding/unbinding pathway in the two enzymes, implying the universality of the pathway in all kinds of PncAs. Several important residues dominating the pathway were also determined by the calculation of interaction energies. The structural change of the proteins induced by NAM’s unbinding or binding shows the great extent interior motion in some homologous region adjacent to the active sites of the two PncAs. The structure comparison substantiates that this region should be very important for the ligand’s binding in all PncAs. Additionally, MD simulations also show that the coordination position of the ligand is displaced by one water molecule in the unliganded enzymes. These results could provide the more penetrating understanding of drug resistance of M. tuberculosis and be helpful for the development of new antituberculosis drugs.

Delineation of the Unbinding Pathway of α-Conotoxin ImI from the α7 Nicotinic Acetylcholine Receptor

α-Conotoxins potently and specifically inhibit isoforms of nicotinic acetylcholine receptors (nAChRs) and are used as molecular probes and as drugs or drug leads. Interactions occurring during binding and unbinding events are linked to binding kinetics, and knowledge of these interactions could help in the development of α-conotoxins as drugs. Here, the unbinding process for the prototypical α-conotoxin ImI/α7-nAChR system was investigated theoretically, and three exit routes were identified using random accelerated molecular dynamics simulations. The route involving the smallest conformation perturbation was further divided into three subpathways, which were studied using steered molecular dynamics simulations. Of the three subpathways, two had better experimental support and lower potential of mean force, indicating that they might be sampled more frequently. Additionally, these subpathways were supported by previous experimental studies. Several pairwise interactions, including a cation-π interaction and charge and hydrogen bond interactions, were identified as potentially playing important roles in the unbinding event.

Molecular Basis of Ligand Dissociation in β-Adrenergic Receptors

The important and diverse biological functions of β-adrenergic receptors (βARs) have promoted the search for compounds to stimulate or inhibit their activity. In this regard, unraveling the molecular basis of ligand binding/unbinding events is essential to understand the pharmacological properties of these G protein-coupled receptors. In this study, we use the steered molecular dynamics simulation method to describe, in atomic detail, the unbinding process of two inverse agonists, which have been recently co-crystallized with β1 and β2ARs subtypes, along four different channels. Our results indicate that this type of compounds likely accesses the orthosteric binding site of βARs from the extracellular water environment. Importantly, reconstruction of forces and energies from the simulations of the dissociation process suggests, for the first time, the presence of secondary binding sites located in the extracellular loops 2 and 3 and transmembrane helix 7, where ligands are transiently retained by electrostatic and Van der Waals interactions. Comparison of the residues that form these new transient allosteric binding sites in both βARs subtypes reveals the importance of non-conserved electrostatic interactions as well as conserved aromatic contacts in the early steps of the binding process.

Cooperative crosslink (un)binding in slowly driven bundles of semiflexible filaments

Combining simulations and theory I study the interplay between bundle elastic degrees of freedom and crosslink binding propensity. By slowly driving bundles into a deformed configuration, and depending on the mechanical stiffness of the crosslinking agent, the binding affinity is shown to display a sudden and discontinuous drop. This indicates a cooperative unbinding process that involves the crossing of a free-energy barrier. Choosing the proper crosslinker therefore not only allows us to change the composite elastic properties of the bundle but also the relevant time scales which can be tuned from the single crosslink binding rate to the much longer escape time over the free-energy barrier.

Influenza Virus Adhesion to Living Cells Measured by Single Virus Force Spectroscopy (SVFS) and Force Probe MD Simulation

Influenza virus belongs to a wide range of viruses that are enclosed in a lipid envelope. The major spike protein of the viral envelope hemagglutinin (HA) binds sialic acid (SA) residues of glycoproteins on the plasma membrane of the host cells. This represents the first step of infection and requires multiple simultaneous interactions since the affinity between one single HA-SA pair is very low (10-13 M-1).The binding interaction of influenza virus adhesion to living cells was probed by means of dynamic force spectroscopy and force probe molecular dynamics (MD) simulation. We applied three independent approaches to measure the unbinding force between influenza virus and a host cell membrane. Using optical tweezers and AFM based single molecule force spectroscopy we were able to characterize the binding energy on the single molecule level. Unbinding events where analysed and revealed a multimodal rupture force distribution which suggests sequential binding of multiple receptors. We determined the interacting force between hemagglutinin and its receptor sialic acid to be ∼10pN. Furthermore we used molecular dynamics simulation to gain information about the binding architecture and the sequence of the unbinding process. MD simulation allowed us a more detailed view of the energy landscape that governs the interaction between HA and its ligand.The combination of experimental and simulated force spectroscopy covers a very large force regime and provides information that could not be obtained with either one or the other method. The techniques are complementary and provide detailed insights of the molecular interactions involved in influenza virus attachment.

Steered Molecular Dynamics Simulations of the Electron Transfer Complex between Azurin and Cytochrome c551

The unbinding properties of the transient complex between the electron transfer proteins azurin and cytochrome c(551) have been investigated by steered molecular dynamics simulations by pulling four different atoms of cytochrome c(551). The atoms to be pulled have been chosen to closely match the conditions of a previous experimental study by atomic force spectroscopy. The temporal evolution of the distance between different couples of atoms of the intermolecular hydrogen bonds and of the unbinding force have been analyzed for the various setups and by applying seven different pulling speed values. The observed variability in the response of the system has been traced back to the occurrence of different pathways during the unbinding process. Furthermore, an analysis of the unbinding force as a function of the pulling speed analyzed in the framework of the thermally activated model has allowed the extraction of the dissociation rate constant at equilibrium. These results have been discussed in connection with the transient character of the system and with the available experimental data by atomic force spectroscopy.

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.

Downhill binding energy surface of the barnase–barstar complex

We have employed biased molecular dynamics simulations in explicit solvent to characterize the one-dimensional potential of mean force for the dissociation process of the barnase-barstar protein-protein complex. Unbinding of barstar from wild-type barnase was compared with dissociation from four charge-deletion mutants of barnase. Interestingly, we find in all cases that unbinding of barnase and barstar is an uphill process on a smooth, tilted energy landscape. The total free energy difference between the dissociated and bound state was similar for wild-type barnase-barstar and for the R87A mutant of barnase. The values for the three other mutant barnase mutants K27A, R59A, and R83Q were only about half as much. Besides, we have analyzed the conformational dynamics of important residues at the barnase-barstar interface. In the bound state, their conformational fluctuations are reduced relatively to the free state because of the formation of intermolecular contacts. Interestingly, we find that some residues also show decreased mobility at intermediate stages of the unbinding process suggesting that these residues may be involved in the first contacts being formed on binding. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 977-985, 2010.

Insight into the Dynamic Interaction of Different Carbohydrates with Human Surfactant Protein D: Molecular Dynamics Simulations

The unbinding process of three monosaccharides--galactose, glucose, and mannose--from human surfactant protein D (hSP-D) was investigated by the molecular docking and molecular dynamics methods to explore the cause of different dynamic interaction between these monosaccharides and the protein. The results show that the low affinity of galactose for hSP-D is attributed to the different binding conformation from the other two monosaccharides. The sugar coordinates to the calcium ion by the hydroxyl groups in the C2 and C3 atoms, so it cannot form the effective interaction with hSP-D. Glucose and mannose have similar binding conformations with hSP-D. Their difference in the affinity is induced by the interaction between the hydroxyl group in the C2 atom and the residue Asp325. The direction of the hydroxyl group in mannose results in the formation of the hydrogen bond with Asp325 and further makes mannose hydrogen-bond to the residues Glu329 and Arg343 by the hydroxyl groups in the C3, C4, and C6 atoms. As glucose only forms three hydrogen bonds with the residues Glu321, Asn323, and Glu329 by the hydroxyl groups in the C3 and C4 atoms, its interaction with hSP-D is weaker than that of mannose. Thus glucose has a lower energy barrier of dissociation. This work could provide the more penetrating understanding of hSP-D physiological functions.

Unbinding of glucose from human pulmonary surfactant protein D studied by steered molecular dynamics simulations

Steered molecular dynamics (SMD) simulations were performed to investigate the dynamic characteristics in the unbinding process of α- d-glucose from human pulmonary surfactant protein D (SP-D). The result shows that the residues Glu321, Asp325, Glu329 and Arg343 are responsible for the different dynamic strength from the affinity in the stable state of thermodynamics. Especially the hydrogen bonds between the residues Glu321, Glu329 and glucose increase their interaction and contribute the counteraction to the external force. The change of the directions of the hydroxyl groups in the C2 and C6 atoms of α- d-glucose also affects the dynamic strength in the pulling procedure.

Single molecular dynamic interactions between glycophorin A and lectin as probed by atomic force microscopy

Glycophorin A (GpA) is one of the most abundant transmembrane proteins in human erythrocytes and its interaction with lectins has been studied as model systems for erythrocyte related biological processes. We performed a force measurement study using the force mode of atomic force microscopy (AFM) to investigate the single molecular level biophysical mechanisms involved in GpA-lectin interactions. GpA was mounted on a mica surface or natively presented on the erythrocyte membrane and probed with an AFM tip coated with the monomeric but multivalent Psathyrella velutina lectin (PVL) through covalent crosslinkers. A dynamic force spectroscopy study revealed similar interaction properties in both cases, with the unbinding force centering around 60 pN with a weak loading rate dependence. Hence we identified the presence of one energy barrier in the unbinding process. Force profile analysis showed that more than 70% of GpAs are free of cytoskeletal associations in agreement with previous reports.

Single-Molecule Study on Intermolecular Interaction between C60and Porphyrin Derivatives: Toward Understanding the Strength of the Multivalency

Multivalency is important in molecular self-assembly, although it remains a challenge to correlate the single-molecule results with the multivalent interaction. As the first example, we have combined a well-defined self-assembling system with AFM-based single-molecule force spectroscopy (SMFS) to investigate the intermolecular interactions and multivalency between C(60) and porphyrin derivatives. Compared with the interaction between C(60) and a single porphyrin (29 pN), SMFS has revealed that porphyrin tweezers can provide an enhanced binding interaction with C(60), resulting in a more than 2-fold higher unbinding force (75 pN, at the same loading rate). In addition, a much lower dissociation rate and a shorter effective distance between the bound state and transition state of the interaction are indicated by dynamic force spectroscopy. These results provide new quantitative information on the divalency effect in the unbinding process of C(60) and porphyrin tweezers at the single-molecule level, which is of significance in understanding the strength of the multivalency in the molecular assembly.