Ligand Binding Mechanics of Maltose Binding Protein

In the past decade, single-molecule force spectroscopy has provided new insights into the key interactions stabilizing folded proteins. A few recent studies probing the effects of ligand binding on mechanical protein stability have come to quite different conclusions. While some proteins seem to be stabilized considerably by a bound ligand, others appear to be unaffected. Since force acts as a vector in space, it is conceivable that mechanical stabilization by ligand binding is dependent on the direction of force application. In this study, we vary the direction of the force to investigate the effect of ligand binding on the stability of maltose binding protein (MBP). MBP consists of two lobes connected by a hinge region that move from an open to a closed conformation when the ligand maltose binds. Previous mechanical experiments, where load was applied to the N and C termini, have demonstrated that MBP is built up of four building blocks (unfoldons) that sequentially detach from the folded structure. In this study, we design the pulling direction so that force application moves the two MBP lobes apart along the hinge axis. Mechanical unfolding in this geometry proceeds via an intermediate state whose boundaries coincide with previously reported MBP unfoldons. We find that in contrast to N-C-terminal pulling experiments, the mechanical stability of MBP is increased by ligand binding when load is applied to the two lobes and force breaks the protein-ligand interactions directly. Contour length measurements indicate that MBP is forced into an open conformation before unfolding even if ligand is bound. Using mutagenesis experiments, we demonstrate that the mechanical stabilization effect is due to only a few key interactions of the protein with its ligand. This work illustrates how varying the direction of the applied force allows revealing important details about the ligand binding mechanics of a large protein.     

Improved Ligand-Protein Binding Affinity Predictions Using Multiple Binding Modes

Accurate ligand-protein binding affinity prediction, for a set of similar binders, is a major challenge in the lead optimization stage in drug development. In general, docking and scoring functions perform unsatisfactorily in this application. Docking calculations, followed by molecular dynamics simulations and free energy calculations can be applied to improve the predictions. However, for targets with large, flexible binding sites, with no experimentally determined binding modes for a set of ligands, insufficient sampling can decrease the accuracy of the free energy calculations. Cytochrome P450s, a protein family of major importance for drug metabolism, is an example of a challenging target for binding affinity predictions. As a result, the choice of starting structure from the docking solutions becomes crucial. In this study, an iterative scheme is introduced that includes multiple independent molecular dynamics simulations to obtain weighted ensemble averages to be used in the linear interaction energy method. The proposed scheme makes the initial pose selection less crucial for further simulation, as it automatically calculates the relative weights of the various poses. It also properly takes into account the possibility that multiple binding modes contribute similarly to the overall affinity, or of similar compounds occupying very different poses. The method was applied to a set of 12 compounds binding to cytochrome P450 2C9 and it displayed a root mean-square error of 2.9 kJ/mol.     

Mixed Mode of Ligand-DNA Binding Results in S-Shaped Binding Curves

Abstract

S-shaped binding curves often characterize interactions of ligands with nucleic acid molecules as analyzed by different physicochemical and biophysical techniques. S-shaped experimental binding curves are usually interpreted as indicative of the positive cooperative interactions between the bound ligand molecules. This paper demonstrates that S-shaped binding curves may occur as a result of the “mixed mode” of DNA binding by the same ligand molecule. Mixed mode of the ligand-DNA binding can occur, for example, due to 1) isomerization or dimerization of the ligands in solution or on the DNA lattice, 2) their ability to intercalate the DNA and to bind it within the minor groove in different orientations. DNA- ligand complexes are characterized by the length of the ligand binding site on the DNA lattice (so-called “multiple-contact” model). We show here that if two or more complexes with different lengths of the ligand binding sites could be produced by the same ligand, the dependence of the concentration of the complex with the shorter length of binding site on the total concentration of ligand should be S-shaped. Our theoretical model is confirmed by comparison of the calculated and experimental CD binding curves for bis-netropsin binding to poly(dA-dT) poly(dA-dT). Bis-netropsin forms two types of DNA complexes due to its ability to interact with the DNA as monomers and trimers. Experimental S-shaped bis-netropsin-DNA binding curve is shown to be in good correlation with those calculated on the basis of our theoretical model. The present work provides new insight into the analysis of ligand-DNA binding curves.     

Melatonin Binding Sites

The distribution and characterization of specific melatonin binding sites were studied using 125I-melatonin. Autoradiography revealed only three sites of specific melatonin binding in brain: the suprachiasmatic nuclei, the median eminence, and the small part of choroid plexus at the caudal end of the fourth ventricle. Two other sites were detected outside the CNS: the anterior pituitary and the retina. The specific binding of 125I-melatonin was saturable and reversible. The dissociation constant (KD) of the binding sites was 60 pM. The concentration of the binding sites (Bmax) in the median eminence was 26 fmol/mg protein, and in the pituitary 3 fmol/mg protein. Specificity of the binding sites was tested by displacement of 125I-melatonin. The order of potency--melatonin much less than N-acetyl-5-hydroxytryptamine less than 5-methoxytryptamine much less than 5-hydroxytryptamine = 3,4-dihydroxyphenylethylamine = noradrenaline--shows high specificity of the binding sites for melatonin.     

肌动蛋白结合蛋白

肌动蛋白结合蛋白是一类调节肌动蛋白聚合、成束或交联的蛋白质,迄今已经发现160多种。通过与肌动蛋白相互作用,直接或间接参与肌动蛋白纤丝的聚合及解聚、纤丝成束与交联,从而介导细胞形态的维持、细胞运动等众多生物学功能。     

Equilibrium Binding Analysis of Neural Cell Adhesion Molecule Binding to Heparin

The kinetics of neural cell adhesion molecule (NCAM) binding to heparin were studied in a heparin-Sepharose-based solid-phase binding assay. The observed binding is time dependent and saturable. A binding constant of 5.2 +/- 1.4 X 10(-8) M is observed for binding of newborn rat NCAM to heparin. This is approximately 25 times lower than the binding constant determined for newborn rat NCAM homophilic binding. Both Scatchard and Hill plot analyses suggest the presence of only one binding site. Fab' fragments of antibodies to rat NCAM significantly inhibit binding, a result indicating that a specific site on NCAM is involved in binding to heparin. The binding is inhibited by heparin (IC50, approximately 5 micrograms/ml), whereas chondroitin sulfate is a less potent inhibitor (IC50, approximately 15 micrograms/ml).     

Binding properties of diethylcarbamazine

Diethylcarbamazine (DEC) reacted with liver cell plasma membrane of rodent hosts-cotton rat, albino rat and Mastomys natalensis exhibiting the presence of both saturable and unsaturable components. The presence of lectins or sugar derivatives did not affect the binding significantly. The drug showed similar binding pattern with serum but the saturation was reached at a much lower concentration of the ligand. Data obtained with a variety of macromolecules, particularly with the homopolymers of amino acids indicate that DEC does not require any specific constituent of the membrane for binding. The nonspecific nature of DEC binding does not provide any convincing clue for the accumulation of microfilariae specifically in the liver following the drug treatment.     

Binding protein-dependent transport systems

Bacterial binding protein-dependent transport systems are the best characterized members of a superfamily of transporters which are structurally, functionally, and evolutionary related to each other. These transporters are not only found in bacteria but also in yeasts, plants, and animals including man, and include both import and export systems. Although any single system is relatively specific, different systems handle very different substrates which can be inorganic ions, amino acids, sugars, large polysaccharides, or even proteins. Some are of considerable medical importance, including Mdr, the protein responsible for multidrug resistance in human tumors, and the product of the cystic fibrosis locus. In this article we review the current state of knowledge on the structure and function of the protein components of these transporters, the mechanism by which transport is mediated, and the role of ATP in the transport process.     

Enhancement by Gangliosides of the Binding of Serotonin to Serotonin Binding Protein

Abstract: Several gangliosides, especially G_D3 (disialosyllactosyl ceramide) in the presence of another lipid (lecithin) were found to enhance the binding of serotonin to serotonin binding protein (SBP) severalfold. In our conditions, this enhancement was linear to a concentration of 2.7 × 10⁻⁶I G_D3 and a three- to fivefold increase in binding capacity of SBP was obtained with 8.8 × 10⁻⁶ M. The addition of this ganglioside led to an increase of serotonin binding sites, but not to an increase in the affinity of SBP to serotonin. Optimal binding capacity was found with a ratio of lecithin to ganglioside of 6: 1 (w/w). No binding was found in the absence of either SBP or Fe²⁺ (binding of serotonin to SBP is dependent on Fe²⁺). Other glycosphingolipids (sulfatide, G_D1a, G_D1b, G_M1) showed lesser effects at low concentration, whereas asialo-G_M1, cytolipin H, galactocerebroside and G_M3 had insignificant effects. Since earlier studies suggested a storage role for serotonin binding protein, the interaction of gangliosides with this protein may regulate the concentration of the biogenic amine in the synapse.