The interrelationship between ligand binding and self-association of the folate binding protein. The role of detergent–tryptophan interaction

Background

The folate binding protein (FBP) regulates homeostasis and intracellular trafficking of folic acid, a vitamin of decisive importance in cell division and growth. We analyzed whether interrelationship between ligand binding and self-association of FBP plays a significant role in the physiology of folate binding.

Methods

Self-association behavior of apo- and holo-FBP was addressed through size exclusion chromatography, SDS-PAGE, mass spectrometry, surface plasmon resonance and fluorescence spectroscopy.

Results

Especially holo-FBP exhibits concentration-dependent self-association at pH 7.4 (pI), and is more prone to associate into stable complexes than apo-FBP. Even more pronounced was the tendency to complexation between apo-FBP and holo-FBP in accord with a model predicting association between apo and holo monomers [19]. This will lead to removal of apo monomers from the reaction scheme resulting in a weak incomplete ligand binding similar to that observed at FBP concentrations < 10 nM. The presence of synthetic and natural detergents normalized folate binding kinetics and resulted in appearance of monomeric holo-FBP. Fluorescence spectroscopy indicated molecular interactions between detergent and tryptophan residues located in hydrophobic structures of apo-FBP which may participate in protein associations.

General significance

Self-association into multimers may protect binding sites, and in case of holo-FBP even folate from biological degradation. High-affinity folate binding in body secretions, typically containing 1–10 nM FBP, requires the presence of natural detergents, i.e. cholesterol and phospholipids, to avoid complexation between apo- and holo-FBP.     

The effect of nanoparticle size on endocytosis dynamics depends on membrane–nanoparticle interaction

Molecular simulation studies on the interaction between nanoparticles (NPs) and cell membranes have been limited by small NP size of several nanometres. In this work, by using a simplified lipid model, we study the endocytosis of large NPs with a size being enlarged to 37.5 nm. It is found that the effect of NP size on endocytosis dynamics depends on the membrane–NP interaction. As the interaction strength between NP and lipid changes, different wrapping modes are observed. For the system with weak membrane–NP attraction, the wrapping process is controlled by the membrane bending, and thus large size of NPs (within the range of NP size we studied) would promote the wrapping dynamics. While for the case with strong membrane–NP adhesion, the wrapping process is dominated by lipid diffusion and small NPs show a larger wrapping rate. In this wrapping mode, the membrane–NP adhesion drives small NPs to move towards the membrane as the wrapping process proceeds. For relatively larger NPs, however, the membrane moves towards the NPs instead. We also find that for the second wrapping mode, the rapid wrapping rate, especially with the hydrophobic ligands on the hydrophilic NP would impose significant perturbations on membrane stability, and consequently, membrane pores may be induced during the process of NP endocytosis.     

Quercetin suppresses insulin receptor signaling through inhibition of the insulin ligand–receptor binding and therefore impairs cancer cell proliferation

Although the flavonoid quercetin is known to inhibit activation of insulin receptor signaling, the inhibitory mechanism is largely unknown. In this study, we demonstrate that quercetin suppresses insulin induced dimerization of the insulin receptor (IR) through interfering with ligand–receptor interactions, which reduces the phosphorylation of IR and Akt. This inhibitory effect further inhibits insulin stimulated glucose uptake due to decreased cell membrane translocation of glucose transporter 4 (GLUT4), resulting in impaired cancer cell proliferation. The effect of quercetin in inhibiting tumor growth was also evident in an in vivo model, indicating a potential future application for quercetin in the treatment of cancers.     

Evaluation of small ligand–protein interaction by ligation reaction with DNA-modified ligand

A method for the evaluation of interactions between protein and ligand using DNA-modified ligands, including signal enhancement of the DNA ligation reactions, is described. For proof of principle, a DNA probe modified by biotin was used. Two DNA probes were prepared with complementary sticky-ends. While one DNA probe was modified at the 5′-end of the sticky-end, the other was not modified. The probes could be ligated together by T4 DNA ligase along the strand without biotin modification. However, in the presence of streptavidin or anti-biotin Fab, the ligation reaction joining the two probes could not occur on either strand.     

Variation of protein binding cavity volume and ligand volume in protein–ligand complexes

We have systematically analyzed the variation of protein binding cavity volume of 200 protein–ligand complexes belonging to eight protein families. Wide variation in protein binding cavity volume for the same protein is observed on binding different ligands. Analysis of individual protein families shows high correlation between atom–atom interactions in binding site and ligand volume. This study implies the significance of protein flexibility in docking small molecule inhibitors on the basis of protein binding cavity volume with respect to ligand volume.     

Surface-bound selectin–ligand binding is regulated by carrier diffusion

Two-dimensional (2D) kinetics of receptor–ligand interactions governs cell adhesion in many biological processes. While the dissociation kinetics of receptor–ligand bond is extensively investigated, the association kinetics has much less been quantified. Recently receptor–ligand interactions between two surfaces were investigated using a thermal fluctuation assay upon biomembrane force probe technique (Chen et al. in Biophys J 94:694–701, 2008). The regulating factors on association kinetics, however, are not well characterized. Here we developed an alternative thermal fluctuation assay using optical trap technique, which enables to visualize consecutive binding–unbinding transition and to quantify the impact of microbead diffusion on receptor–ligand binding. Three selectin constructs (sLs, sPs, and PLE) and their ligand P-selectin glycoprotein ligand 1 were used to conduct the measurements. It was indicated that bond formation was reduced by enhancing the diffusivity of selectin-coupled carrier, suggesting that carrier diffusion is crucial to determine receptor–ligand binding. It was also found that 2D forward rate predicted upon first-order kinetics was in the order of sPs > sLs > PLE and bond formation was history-dependent. These results further the understandings in regulating association kinetics of surface-bound receptor–ligand interactions.     

Investigations of Takeout proteins’ ligand binding and release mechanism using molecular dynamics simulation

Takeout (To) proteins exist in a diverse range of insect species. They are involved in many important processes of insect physiology and behaviors. As the ligand carriers, To proteins can transport the small molecule to the target tissues. However, ligand release mechanism of To proteins is unclear so far. In this contribution, the process and pathway of the ligand binding and release are revealed by conventional molecular dynamics simulation, steered molecular dynamics simulation and umbrella sampling methods. Our results show that the α4-side of the protein is the unique gate for the ligand binding and release. The structural analysis confirms that the internal cavity of the protein has high rigidity, which is in accordance with the recent experimental results. By using the potential of mean force calculations in combination with residue cross correlation calculation, we concluded that the binding between the ligand and To proteins is a process of conformational selection. Furthermore, the conformational changes of To proteins and the hydrophobic interactions both are the key factors for ligand binding and release.     

Prospects of nanoparticle–DNA binding and its implications in medical biotechnology

Bio-nanotechnology is a new interdisciplinary R&D area that integrates engineering and physical science with biology through the development of multifunctional devices and systems, focusing biology inspired processes or their applications, in particular in medical biotechnology. DNA based nanotechnology, in many ways, has been one of the most intensively studied fields in recent years that involves the use and the creation of bio-inspired materials and their technologies for highly selective biosensing, nanoarchitecture engineering and nanoelectronics. Increasing researches have been offered to a fundamental understanding how the interactions between the nanoparticles and DNA molecules could alter DNA molecular structure and its biochemical activities. This minor review describes the mechanisms of the nanoparticle–DNA binding and molecular interactions. We present recent discoveries and research progresses how the nanoparticle–DNA binding could vary DNA molecular structure, DNA detection, and gene therapy. We report a few case studies associated with the application of the nanoparticle–DNA binding devices in medical detection and biotechnology. The potential impacts of the nanoparticles via DNA binding on toxicity of the microorganisms are briefly discussed. The nanoparticle–DNA interactions and their impact on molecular and microbial functionalities have only drown attention in recent a few years. The information presented in this review can provide useful references for further studies on biomedical science and technology.     

Species-specific interaction of seminal plasma on sperm–neutrophil binding

Bovine semen is naturally deposited in the vagina and spermatozoa migrate through the cervix into the uterus leaving the bulk of seminal plasma (SP) behind. In equine, both spermatozoa and SP are deposited directly in the uterus and SP reduces sperm binding to neutrophils and prevents the formation of DNA-based neutrophil extracellular traps (NETs). We investigated the role of bovine SP on sperm–neutrophil binding using the four most common bovine semen extenders. Contrary to equine, bovine spermatozoa removed from SP had low binding to neutrophils for up to 3 h, but as little as 10% SP increased sperm–neutrophil binding and NETs formation over time. Similar results were obtained with neutrophils isolated from peripheral blood or from the uterus. Scanning electron microscopy showed that the binding can be mediated by NETs or by direct attachment of the cell membranes for both species. The increased binding with SP reduced the number of free spermatozoa indicating that sperm transport to the site of fertilization (and thus fertility) may be hindered. Surprisingly, egg yolk negated the role of bovine SP on sperm–neutrophil binding compared to all the other semen extenders, but did not alter equine sperm binding to neutrophils. Current artificial insemination in bovine relies heavily on egg yolk extender and introduces variable amounts of SP into the uterus, which naturally remains in the vagina. Our results indicate a need to re-evaluate the composition of semen extenders and the semen processing procedures in relation to sperm transport, longevity and fertilizing ability.     

Steered molecular dynamics simulations of ligand–receptor interaction in lipocalins

Retinol binding protein (RBP) and an engineered lipocalin, DigA16, have been studied using molecular dynamics simulations. Special emphasis has been placed on explaining the ligand–receptor interaction in RBP–retinol and DigA16–digoxigenin complexes, and steered molecular dynamics simulations of 10–20 ns have been carried out for the ligand expulsion process. Digoxigenin is bound deep inside the cavity of DigA16 and forms several stable hydrogen bonds in addition to the hydrophobic van der Waals interaction with the aromatic side-chains. Four crystalline water molecules inside the ligand-binding cavity remain trapped during the simulations. The strongly hydrophobic receptor site of RBP differs considerably from DigA16, and the main source of ligand attraction comes from the phenyl side-chains. The hydrogen bonds between digoxigenin and DigA16 cause the rupture forces on ligand removal in DigA16 and RBP to differ. The mutated DigA16 residues contribute approximately one-half of the digoxigenin interaction energy with DigA16 and, of these, the energetically most important are residues His35, Arg58, Ser87, Tyr88, and Phe114. Potential “sensor loops” were found for both receptors. These are the outlier loops between residues 114–121 and 63–67 for DigA16 and RBP, respectively, and they are located near the entrance of the ligand-binding cavity. Especially, the residues Glu119 (DigA16) and Leu64 (RBP) are critical for sensing. The ligand binding energies have been estimated based on the linear response approximation of binding affinity by using a previous parametrization for retinoids and RBP.     

The RNA-binding protein Staufen1 impairs myogenic differentiation via a c-myc–dependent mechanism

Recent work has shown that Staufen1 plays key roles in skeletal muscle, yet little is known about its pattern of expression during embryonic and postnatal development. Here we first show that Staufen1 levels are abundant in mouse embryonic muscles and that its expression decreases thereafter, reaching low levels in mature muscles. A similar pattern of expression is seen as cultured myoblasts differentiate into myotubes. Muscle degeneration/regeneration experiments revealed that Staufen1 increases after cardiotoxin injection before returning to the low levels seen in mature muscles. We next prevented the decrease in Staufen1 during differentiation by generating stable C2C12 muscle cell lines overexpressing Staufen1. Cells overexpressing Staufen1 differentiated poorly, as evidenced by reductions in the differentiation and fusion indices and decreases in MyoD, myogenin, MEF2A, and MEF2C, independently of Staufen-mediated mRNA decay. However, levels of c-myc, a factor known to inhibit differentiation, were increased in C2C12 cells overexpressing Staufen1 through enhanced translation. By contrast, the knockdown of Staufen1 decreased c-myc levels in myoblasts. Collectively our results show that Staufen1 is highly expressed during early stages of differentiation/development and that it can impair differentiation by regulating c-myc, thereby highlighting the multifunctional role of Staufen1 in skeletal muscle cells.     

The fine-tuning of TRAF2–GSTP1-1 interaction: effect of ligand binding and in situ detection of the complex

We provide the first biochemical evidence of a direct interaction between the glutathione transferase P1-1 (GSTP1-1) and the TRAF domain of TNF receptor-associated factor 2 (TRAF2), and describe how ligand binding modulates such an equilibrium. The dissociation constant of the heterocomplex is K_d=0.3 μM; however the binding affinity strongly decreases when the active site of GSTP1-1 is occupied by the substrate GSH (K_d≥2.6 μM) or is inactivated by oxidation (K_d=1.7 μM). This indicates that GSTP1-1''s TRAF2-binding region involves the GSH-binding site. The GSTP1-1 inhibitor NBDHEX further decreases the complex''s binding affinity, as compared with when GSH is the only ligand; this suggests that the hydrophobic portion of the GSTP1-1 active site also contributes to the interaction. We therefore hypothesize that TRAF2 binding inactivates GSTP1-1; however, analysis of the data, using a model taking into account the dimeric nature of GSTP1-1, suggests that GSTP1-1 engages only one subunit in the complex, whereas the second subunit maintains the catalytic activity or binds to other proteins. We also analyzed GSTP1-1''s association with TRAF2 at the cellular level. The TRAF2–GSTP1-1 complex was constitutively present in U-2OS cells, but strongly decreased in S, G2 and M phases. Thus the interaction appears regulated in a cell cycle-dependent manner. The variations in the levels of individual proteins seem too limited to explain the complex''s drastic decline observed in cells progressing from the G0/G1 to the S–G2–M phases. Moreover, GSH''s intracellular content was so high that it always saturated GSTP1-1. Interestingly, the addition of NBDHEX maintains the TRAF2–GSTP1-1 complex at low levels, thus causing a prolonged cell cycle arrest in the G2/M phase. Overall, these findings suggest that a reversible sequestration of TRAF2 into the complex may be crucial for cell cycle progression and that multiple factors are involved in the fine-tuning of this interaction.     

Homogeneous time-resolved G protein-coupled receptor–ligand binding assay based on fluorescence cross-correlation spectroscopy

G protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic target classes for a wide spectrum of diseases. Drug discovery projects generally benefit from a broad range of experimental approaches for screening compound libraries and for the characterization of binding modes of drug candidates. Owing to the difficulties in solubilizing and purifying GPCRs, assay formats have been so far mainly limited to cell-based functional assays and radioligand binding assays. In this study, we used fluorescence cross-correlation spectroscopy (FCCS) to analyze the interaction of detergent-solubilized receptors to various types of GPCR ligands: endogenous peptides, small molecules, and a large surrogate antagonist represented by a blocking monoclonal antibody. Our work demonstrates the suitability of the homogeneous and time-resolved FCCS assay format for a robust, high-throughput determination of receptor–ligand binding affinities and kinetic rate constants for various therapeutically relevant GPCRs.     

Is the GehD lipase from Staphylococcus epidermidis a collagen binding adhesin?

The opportunistic human pathogen Staphylococcus epidermidis is the major cause of nosocomial biomaterial infections. S. epidermidis has the ability to attach to indwelling materials coated with extracellular matrix proteins such as fibrinogen, fibronectin, vitronectin, and collagen. To identify the proteins necessary for S. epidermidis attachment to collagen, we screened an expression library using digoxigenin-labeled collagen as well as two monoclonal antibodies generated against the Staphylococcus aureus collagen-adhesin, Cna, as probes. These monoclonal antibodies recognize collagen binding epitopes on the surface of S. aureus and S. epidermidis cells. Using this approach, we identified GehD, the extracellular lipase originally found in S. epidermidis 9, as a collagen-binding protein. Despite the monoclonal antibody cross-reactivity, the GehD amino acid sequence and predicted structure are radically different from those of Cna. The mature GehD circular dichroism spectra differs from that of Cna but strongly resembles that of a mammalian cell-surface collagen binding receptor, known as the alpha(1) integrin I domain, suggesting that they have similar secondary structures. The GehD protein is translated as a preproenzyme, secreted, and post-translationally processed into mature lipase. GehD does not have the conserved LPXTG C-terminal motif present in cell wall-anchored proteins, but it can be detected in lysostaphin cell wall extracts. A recombinant version of mature GehD binds to collagens type I, II, and IV adsorbed onto microtiter plates in a dose-dependent saturable manner. Recombinant, mature GehD protein and anti-GehD antibodies can inhibit the attachment of S. epidermidis to immobilized collagen. These results provide evidence that GehD may be a bi-functional molecule, acting not only as a lipase but also as a cell surface-associated collagen adhesin.     

Thermodynamics for base binding to four atropisomers cobalt(II) picket fence porphyrin. Intramolecular ligand–ligand interactions

Thermodynamic parameters for base binding to four atropisomers of meso-tetrakis(o-pivalamidophenyl)porphyrinatocobalt(II) were determined by spectrophotometry in toluene. The order of the affinities of the four isomers with 1-methylimidazole and pyridine is α⁴<α³<cis-α²<trans-α². The higher base affinities of the trans-α² complex compared with the α⁴ complex are due to an increase in the binding energies of the bases, although a substantial decrease of entropy changes also occurs; the differences of thermodynamic values on both complexes are −ΔΔG = 1.49 and 1.36 kcal/mol, −ΔΔH = 3.4 and 3.1 kcal/mol and −ΔΔS = 6.4 and 6.1 eu, with 1-methylimidazole and pyridine, respectively. With saturated bases pyrrolidine and piperidine, the affinities of the trans-α² complex are comparable to those of the α⁴ complex, and those of the cis-α² complex are the lowest. The increased steric repulsion between the pickets and ligated pyrrolidine or piperidine may cancel out the stabilizing effect on the base binding to the α² complexes. Proton NMR study suggests the preferential solvation of the four-coordinate species of the trans-α² complex to that of the α⁴ complex. It could be concluded that the stabilization of the base binding by the pickets is attributed to an intramolecular ligand–ligand interaction between the ligated base and the pickets rather than to the inhibition of the undesirable solvation on the active sites.     

Multivalent binding oligomers inhibit HIV Tat–TAR interaction critical for viral replication

We describe the development of a new type of scaffold to target RNA structures. Multivalent binding oligomers (MBOs) are molecules in which multiple sidechains extend from a polyamine backbone such that favorable RNA binding occurs. We have used this strategy to develop MBO-based inhibitors to prevent the association of a protein–RNA complex, Tat–TAR, that is essential for HIV replication. In vitro binding assays combined with model cell-based assays demonstrate that the optimal MBOs inhibit Tat–TAR binding at low micromolar concentrations. Antiviral studies are also consistent with the in vitro and cell-based assays. MBOs provide a framework for the development of future RNA-targeting molecules.     

Accuracy and precision of protein–ligand interaction kinetics determined from chemical shift titrations

NMR-monitored chemical shift titrations for the study of weak protein?Cligand interactions represent a rich source of information regarding thermodynamic parameters such as dissociation constants (K _D ) in the micro- to millimolar range, populations for the free and ligand-bound states, and the kinetics of interconversion between states, which are typically within the fast exchange regime on the NMR timescale. We recently developed two chemical shift titration methods wherein co-variation of the total protein and ligand concentrations gives increased precision for the K _D value of a 1:1 protein?Cligand interaction (Markin and Spyracopoulos in J Biomol NMR 53: 125?C138, 2012). In this study, we demonstrate that classical line shape analysis applied to a single set of ¹H?C¹⁵N 2D HSQC NMR spectra acquired using precise protein?Cligand chemical shift titration methods we developed, produces accurate and precise kinetic parameters such as the off-rate (k _off ). For experimentally determined kinetics in the fast exchange regime on the NMR timescale, k _off ?~?3,000?s^?1 in this work, the accuracy of classical line shape analysis was determined to be better than 5?% by conducting quantum mechanical NMR simulations of the chemical shift titration methods with the magnetic resonance toolkit GAMMA. Using Monte Carlo simulations, the experimental precision for k _off from line shape analysis of NMR spectra was determined to be 13?%, in agreement with the theoretical precision of 12?% from line shape analysis of the GAMMA simulations in the presence of noise and protein concentration errors. In addition, GAMMA simulations were employed to demonstrate that line shape analysis has the potential to provide reasonably accurate and precise k _off values over a wide range, from 100 to 15,000?s^?1. The validity of line shape analysis for k _off values approaching intermediate exchange (~100?s^?1), may be facilitated by more accurate K _D measurements from NMR-monitored chemical shift titrations, for which the dependence of K _D on the chemical shift difference (????) between free and bound states is extrapolated to ?????=?0. The demonstrated accuracy and precision for k _off will be valuable for the interpretation of biological kinetics in weakly interacting protein?Cprotein networks, where a small change in the magnitude of the underlying kinetics of a given pathway may lead to large changes in the associated downstream signaling cascade.