Ligand binding characteristics and aggregation behavior of purified cow's milk folate binding protein depends on the presence of amphiphatic substances including cholesterol,phospholipids, and synthetic detergents

Folate binding protein was purified from cow's milk by a combination of cation exchange chromatography and methotrexate-AH-sepharose affinity chromatography. Dilution of the preparation to concentrations of protein less than 10 nM resulted in drastic changes of radioligand (folate) binding characteristics, i.e., a decrease in binding affinity with a change from upward to downward convex Scatchard plots and increased ligand dissociation combined with appearance of weak-affinity aggregated forms of the binding protein on gel filtration. These findings, consistent with a model predicting dimerization between unliganded and liganded monomers, were reversed in the presence of material eluted from the affinity column after adsorption of the protein(cofactor) or cholesterol, phospholipids, and synthetic detergents. The latter amphiphatic substances form micelles and lipid bilayers which could separate hydrophobic unliganded monomers from hydrophilic liganded monomers in the surrounding aqueous medium and thereby prevent association between these monomeric forms prevailing at low concentrations of the protein. Our data have some bearings on studies which show that cholesterol and phospholipids are necessary for the clustering of folate receptors in the cell membrane; a process required for optimum receptor function and internalization of folate.     

Purification, Biochemical Characterization, Binding Activity, and Selectivity of a Glutamate Binding Protein from Bovine Brain

A glutamate binding protein was purified from bovine brain to apparent homogeneity. The procedure used for the purification of this protein involved extraction of a crude synaptic membrane fraction with Na-cholate, followed by solubilization of the binding protein from the membranes by Triton X-100, and, finally, affinity batch separation of the protein on L-glutamate-loaded glass fiber. The molecular characteristics of the purified protein were similar to those previously described for the glutamate binding protein from rat brain synaptic membranes and included the following: small Mr (14,000), acidic (pI = 4.7) protein with a single NH2-terminal amino acid (tyrosine), and significant absorption at wave-lengths greater than 300 nm. Complete amino acid analysis of the protein was not achieved, either because of destruction of some amino acids or of incomplete hydrolysis of the protein. The protein bound L-glutamate with high affinity (KD = 0.87 microM), exhibited one class of L-glutamate binding sites, and bound glutamate with a stoichiometry of 0.7 mol ligand/mol protein. The displacement of protein-bound L-glutamic acid by other neuroactive amino acids had characteristics similar to those observed for the displacement of L-glutamate from rat brain synaptic membrane or purified protein binding sites. Finally, the metal ligand formers KCN and NaN3 inhibited the activity of this protein just as they have been shown to do in rat brain synaptic membranes or the purified protein.     

A high-throughput differential filtration assay to screen and select detergents for membrane proteins

Structural studies on integral membrane proteins are routinely performed on protein-detergent complexes (PDCs) consisting of purified protein solubilized in a particular detergent. Of all the membrane protein crystal structures solved to date, a subset of only four detergents has been used in more than half of these structures. Unfortunately, many membrane proteins are not well behaved in these four detergents and/or fail to yield well-diffracting crystals. Identification of detergents that maintain the solubility and stability of a membrane protein is a critical step and can be a lengthy and “protein-expensive” process. We have developed an assay that characterizes the stability and size of membrane proteins exchanged into a panel of 94 commercially available and chemically diverse detergents. This differential filtration assay (DFA), using a set of filtered microplates, requires sub-milligram quantities of purified protein and small quantities of detergents and other reagents and is performed in its entirety in several hours.     

Crystal Structure of the Mp1p Ligand Binding Domain 2 Reveals Its Function as a Fatty Acid-binding Protein

Penicillium marneffei is a dimorphic, pathogenic fungus in Southeast Asia that mostly afflicts immunocompromised individuals. As the only dimorphic member of the genus, it goes through a phase transition from a mold to yeast form, which is believed to be a requisite for its pathogenicity. Mp1p, a cell wall antigenic mannoprotein existing widely in yeast, hyphae, and conidia of the fungus, plays a vital role in host immune response during infection. To understand the function of Mp1p, we have determined the x-ray crystal structure of its ligand binding domain 2 (LBD2) to 1.3 Å. The structure reveals a dimer between the two molecules. The dimer interface forms a ligand binding cavity, in which electron density was observed for a palmitic acid molecule interacting with LBD2 indirectly through hydrogen bonding networks via two structural water molecules. Isothermal titration calorimetry experiments measured the ligand binding affinity (K_d) of Mp1p at the micromolar level. Mutations of ligand-binding residues, namely S313A and S332A, resulted in a 9-fold suppression of ligand binding affinity. Analytical ultracentrifugation assays demonstrated that both LBD2 and Mp1p are mostly monomeric in vitro, no matter with or without ligand, and our dimeric crystal structure of LBD2 might be the result of crystal packing. Based on the conformation of the ligand-binding pocket in the dimer structure, a model for the closed, monomeric form of LBD2 is proposed. Further structural analysis indicated the biological importance of fatty acid binding of Mp1p for the survival and pathogenicity of the conditional pathogen.     

Ligand binding characteristics of two molecular forms,one equipped with a hydrophobic glycosyl phosphatidylinositol tail,of the folate binding protein purified from human milk

Two molecular forms of the folate binding protein were isolated and purified from human milk by a combination of cation exchange- and affinity chromatography. One protein (27 kDa) was a cleavage product of the other 100 kDa protein as evidenced by N-terminal amino acid sequence homology and a reduction in the molecular size of the latter protein to 27 kDa after cleavage of its hydrophobic glycosylphosphatidylinositol tail by phosphatidylinositol-specific phospholipase C. High-affinity binding of [³H]folate was characterized by upward convex Scatchard plots and increasing ligand binding affinity with decreasing concentrations of both proteins. Downward convex Scatchard plots and binding affinities showing no dependence on the protein concentration were, however, observed in highly diluted solutions of both proteins. Radioligand binding was inhibited by folate analogs, and dissociation of radioligand was slow at pH 7.4 but rapid and complete at pH 5.0 and 3.5. Ligand binding quenched the tryptophan fluorescence of the 27 kDa protein suggesting that tryptophan is present at the binding site and/or ligand binding induces a conformation change that affects tryptophan environment in the protein. The 27 kDa protein representing soluble folate binding protein exhibited a greater affinity for ligand binding than the 100 kDa protein which possesses a hydrophobic tail identical to the one that anchors the folate receptor to the cell membrane.     

Interactions of the intact FsrC membrane histidine kinase with its pheromone ligand GBAP revealed through synchrotron radiation circular dichroism

FsrC is the membrane-bound histidine kinase component of the Fsr two-component signal transduction system involved in quorum sensing in the hospital-acquired infection agent Enterococcus faecalis. Synchrotron radiation circular dichroism spectroscopy was used here to study the intact purified protein solubilised in detergent micelles. Conditions required for FsrC stability in detergent were firstly determined and tested by prolonged exposure of stabilised protein to far-ultraviolet radiation. Using stabilised purified protein, far-ultraviolet synchrotron radiation circular dichroism revealed that FsrC is 61% α-helical and that it is relatively thermostable, retaining at least 57% secondary structural integrity at 90°C in the presence or absence of gelatinase biosynthesis-activating pheromone (GBAP). Whilst binding of the quorum pheromone ligand GBAP did not significantly affect FsrC secondary structure, near-ultraviolet spectra revealed that the tertiary structure in the regions of the Tyr and Trp residues was significantly affected. Titration experiments revealed a calculated k(d) value of 2μM indicative of relatively loose binding of gelatinase biosynthesis-activating pheromone to FsrC. Although use of synchrotron radiation circular dichroism has been applied to membrane proteins previously, to our knowledge this is the first report of its use to determine a k(d) value for an intact membrane protein. Based on our findings, we suggest that synchrotron radiation circular dichroism will be a valuable technique for characterising ligand binding by other membrane sensor kinases and indeed other membrane proteins in general. It further provides a valuable screening tool for membrane protein stability under a range of detergent conditions prior to downstream structural methods such as crystallisation and NMR experiments particularly when lower detergent concentrations are used.     

Endocytosis of oxidized low density lipoprotein through scavenger receptor CD36 utilizes a lipid raft pathway that does not require caveolin-1

The scavenger receptor CD36 binds a diverse array of ligands, including thrombospondin-1, oxidized low density lipoprotein (OxLDL), fatty acids, anionic phospholipids, and apoptotic cells. CD36 has been reported to be present in lipid rafts/caveolae, but little is known about the membrane trafficking of this protein at baseline or following ligand binding. Here, we determined that expression of CD36 in Chinese hamster ovary (CHO) cells and endogenous expression of CD36 in C32 cells led to a homogeneous distribution of the protein on the plasma membrane, as judged by confocal fluorescence microscopy. This homogeneous pattern was observed both by anti-CD36 antibody staining and by live cell imaging of CHO cells expressing a chimeric CD36-green fluorescent protein construct. In contrast, caveolin-1 displayed its usual punctate surface distribution. Correspondingly, dual labeling of CD36 and caveolin-1 showed essentially no overlap, neither by immunofluorescence light microscopy nor by immunogold electron microscopy. Furthermore, isolation of lipid rafts by sucrose gradient ultracentrifugation of cold Triton X-100 cell lysates yielded both CD36 and caveolin-1, but immunoprecipitates of caveolin-1 did not contain CD36. Binding of Ox-LDL led to internalization of CD36 and OxLDL into endosomal structures that did not contain caveolin-1 or transferrin but that co-internalized the glycosyl-phosphatidylinositol-anchored protein decay accelerating factor, a lipid raft protein. Furthermore, expression of CD36 in the caveolin-1-negative KB cell line is sufficient for OxLDL-induced internalization of CD36, indicating that caveolin-1 is not required for this endocytic process. Taken together, these data demonstrate that at steady state, CD36 is localized in lipid rafts but not in caveolae, and that binding of OxLDL to CD36 leads to endocytosis through a lipid raft pathway that is distinct from the clathrin-mediated or caveolin internalization pathways.     

The complex of the insect LDL receptor homolog, lipophorin receptor, LpR, and its lipoprotein ligand does not dissociate under endosomal conditions

The insect low-density lipoprotein (LDL) receptor (LDLR) homolog, lipophorin receptor (LpR), mediates endocytic uptake of the single insect lipoprotein, high-density lipophorin (HDLp), which is structurally related to LDL. However, in contrast to the fate of LDL, which is endocytosed by LDLR, we previously demonstrated that after endocytosis, HDLp is sorted to the endocytic recycling compartment and recycled for re-secretion in a transferrin-like manner. This means that the integrity of the complex between HDLp and LpR is retained under endosomal conditions. Therefore, in this study, the ligand-binding and ligand-dissociation capacities of LpR were investigated by employing a new flow cytometric assay, using LDLR as a control. At pH 5.4, the LpR-HDLp complex remained stable, whereas that of LDLR and LDL dissociated. Hybrid HDLp-binding receptors, containing either the beta-propeller or both the beta-propeller and the hinge region of LDLR, appeared to be unable to release ligand at endosomal pH, revealing that the stability of the complex is imparted by the ligand-binding domain of LpR. The LpR-HDLp complex additionally appeared to be EDTA-resistant, excluding a low Ca(2+) concentration in the endosome as an alternative trigger for complex dissociation. From binding of HDLp to the above hybrid receptors, it was inferred that the stability upon EDTA treatment is confined to LDLR type A (LA) ligand-binding repeats 1-7. Additional (competition) binding experiments indicated that the binding site of LpR for HDLp most likely involves LA-2-7. It is therefore proposed that the remarkable stability of the LpR-HDLp complex is attributable to this binding site. Together, these data indicate that LpR and HDLp travel in complex to the endocytic recycling compartment, which constitutes a key determinant for ligand recycling by LpR.     

Consensus design of a NOD receptor leucine rich repeat domain with binding affinity for a muramyl dipeptide,a bacterial cell wall fragment

Repeat proteins have recently emerged as especially well‐suited alternative binding scaffolds due to their modular architecture and biophysical properties. Here we present the design of a scaffold based on the consensus sequence of the leucine rich repeat (LRR) domain of the NOD family of cytoplasmic innate immune system receptors. Consensus sequence design has emerged as a protein design tool to create de novo proteins that capture sequence‐structure relationships and interactions present in nature. The multiple sequence alignment of 311 individual LRRs, which are the putative ligand‐recognition domain in NOD proteins, resulted in a consensus sequence protein containing two internal and N‐ and C‐capping repeats named CLRR2. CLRR2 protein is a stable, monomeric, and cysteine free scaffold that without any affinity maturation displays micromolar binding to muramyl dipeptide, a bacterial cell wall fragment. To our knowledge, this is the first report of direct interaction of a NOD LRR with a physiologically relevant ligand.     

Purification and characterization of a copper-binding protein from Asian periwinkle Littorina brevicula

The Asian periwinkle, Littorina brevicula, is highly resistant to a wide range of heavy metal concentrations and its metal-binding protein(s) are induced in the presence of cadmium (Cd) and zinc (Zn). In this study, we isolated and characterized a novel copper-binding protein (Cu-BP). Following purification by Sephacryl S-100 chromatography, Cu-BP contained an equal amount of Zn in non-exposed physiological conditions. However, Zn is replaced by Cu at the binding site upon addition of excess Cu (100 microM CuCl(2)) to the cytosol or after a long period (60 days) of exposure of the periwinkles to the metal ion (150 microg/l CuCl(2)). The ligand was further purified by DEAE-Sepharose anion-exchange chromatography and C(18) reverse-phase HPLC. The molecular weight of the purified protein was determined as 11.38 kDa by MALDI-TOF MS analyses. This Cu-BP is distinct from common mollusk metallothionein (MT) in that it contains significantly lower number of Cys (8 residues) and high levels of aromatic amino acids, Tyr and Phe. The protein additionally contains His and Met, which are absent in the MT-like Cd-BP of L. brevicula. The finding that Cu-BP in the Asian periwinkle is distinct from MT-like Cd-BP suggests that the timely expression of specific metal-binding proteins allows added protection against each heavy metal in severely polluted conditions.     

The structural basis of receptor-binding by Escherichia coli associated with diarrhea and septicemia

GafD in Escherichia coli G (F17) fimbriae is associated with diarrheal disease, and the structure of the ligand-binding domain, GafD1-178, has been determined at 1.7A resolution in the presence of the receptor sugar N-acetyl-D-glucosamine. The overall fold is a beta-barrel jelly-roll fold. The ligand-binding site was identified and localized to the side of the molecule. Receptor binding is mediated by side-chain as well main-chain interactions. Ala43-Asn44, Ser116-Thr117 form the sugar acetamide specificity pocket, while Asp88 confers tight binding and Trp109 appears to position the ligand. There is a disulfide bond that rigidifies the acetamide specificity pocket. The three fimbrial lectins, GafD, FimH and PapG share similar beta-barrel folds but display different ligand-binding regions and disulfide-bond patterns. We suggest an evolutionary path for the evolution of the very diverse fimbrial lectins from a common ancestral fold.     

Expression, purification, stability optimization and characterization of human Aurora B kinase domain from E. coli

Aurora B kinase plays a critical role in regulating mitotic progression, and its dysregulation has been linked to tumorigenesis. The structure of the kinase domain of human Aurora B and the complementary information of binding thermodynamics of known Aurora inhibitors is lacking. Towards that effort, we sought to identify a human Aurora B construct that would be amenable for large-scale protein production for biophysical and structural studies. Although the designed AurB_69-333 construct expressed at high levels in Escherichia coli, the purified protein was largely unstable and prone to aggregation. We employed thermal-shift assay for high-throughput screening of 192 conditions to identify optimal pH and salt conditions that increased the stability and minimized aggregation of AurB_69-333. Direct ligand binding analyses using temperature-dependent circular dichroism (TdCD) and TR-FRET-based Lanthascreen™ binding assay showed that the purified protein was folded and functional. The affinity rank-order obtained using TdCD and Lanthascreen™ binding assay correlated with enzymatic IC50 values measured using full-length Aurora B protein for all the inhibitors tested except for AZD1152. The direct binding results support the hypothesis that the purified human AurB_69-333 fragment is a good surrogate for its full-length counterpart for biophysical and structural analyses.     

Rabbit antibodies against the folate binding protein from cow's milk. Production,characterization and use for development of an enzyme-linked immunosorbent assay (ELISA)

Rabbit antibodies to purified folate binding protein from cow's milk whey were used for development of a two-site enzyme-linked immunosorbent assay (ELISA) for quantitation of bovine folate binding proteins, The folate binding proteins in human milk and serum showed no cross-reactivity. A partial saturation of purified bovine folate binder with folate gave rise to an increased antigenicity probably due to a ligand (folate)-induced exposure of antigenic sites on the protein.     

Tuning ligand affinity,specificity, and folding stability of an engineered lipocalin variant -- a so-called 'anticalin' -- using a molecular random approach

Anticalins are prepared by reshaping the ligand pocket of a natural lipocalin via protein engineering in order to recognize a prescribed ligand. In this manner, the anticalin DigA with specificity for digoxigenin was previously derived from the bilin-binding protein (BBP), a natural lipocalin from Pieris brassicae. The four peptide loops that form its ligand-binding site were randomized and a cognate variant was selected from the resulting library. Here, we propose a concept for improving the ligand-binding properties of this anticalin in an in vitro affinity maturation process by step-wise randomization of restricted areas of the loop region. Following selection on digoxigenin-binding activity via phage display and colony screening, several DigA variants were thus obtained. The recombinant proteins were thoroughly characterized in terms of ligand affinity and specificity, secondary structure and thermal stability against unfolding. The variant DigA16/19, which carries several new mutations, exhibits clearly improved affinity for digoxigenin, with K(D)=12.4 nM. Hence, it is suitable as a sensitive reagent in biochemical detection experiments, especially when produced as a functional fusion protein with alkaline phosphatase as reporter enzyme. In addition, DigA16/19 possesses enhanced ligand specificity and recognizes part of the linker that was used for fixing the steroid group to a carrier protein. Finally, the digoxigenin-binding anticalins appear to have high physico-chemical stability, with T(m) values in the 70 degrees C range. Our present findings support the notion that anticalins provide a useful class of compact and robust ligand-receptor proteins that can be tailored for practical demands.     

Expression, purification, and characterization of multiple, multifunctional human glucocorticoid receptor proteins

The glucocorticoid receptor (GR) is a nuclear receptor protein that plays a central role in glucose homeostasis, the stress response, control of the hypothalamic-pituitary-adrenal axis, and immuno-inflammatory processes via binding of the natural steroid, cortisol. GR is a well-validated drug target and continues to be an important target for new drug discovery efforts. Here, we describe a basic and simple method for Escherichia coli expression and purification of a variety of human GR proteins that contain all three of the functional domains of the protein: the activation function-1 domain, the DNA-binding domain, and the ligand-binding domain. We present characterization data to show that these purified, multifunctional GR proteins are active for ligand, coactivator, and DNA-binding. The work presented here should serve as a reference for future mechanistic, structural and drug discovery efforts that require purified, full or near full length, GR protein.     

Ligand displaces heat shock protein 90 from overlapping binding sites within the aryl hydrocarbon receptor ligand-binding domain

Hsp90 (heat shock protein of 90 kDa) is often found associated with functional domains of client proteins, including those for ligand binding, dimerization, DNA binding, and enzymatic activity. Although Hsp90 can maintain the conformation of functionally important domains prior to activation of the client protein, its specific binding site and the mechanism(s) of Hsp90 dissociation during activation are unknown. Here, we have identified and characterized residues involved in Hsp90 binding within the aryl hydrocarbon receptor (AhR) ligand-binding domain and demonstrate that they overlap with those involved in ligand binding. In agreement with this spatial model, ligand binding results in Hsp90 dissociation from the AhR Per-ARNT-Sim B fragment. Interestingly, whereas Hsp90-binding residues within the ligand-binding domain were not involved in Hsp90-dependent AhR protein stability, several of these residues are important for ligand-dependent AhR activation, and their mutation resulted in conversion of two AhR antagonists/partial agonists into full AhR agonists. These studies reveal co-localization of a tentative Hsp90-binding site with that for AhR ligand binding and provide the first molecular mechanism for Hsp90 dissociation in the activation of a client protein.     

Identification of PSF as a protein kinase Calpha-binding protein in the cell nucleus

Protein kinase C (PKC) isoforms are present in the cell nucleus in diverse cell lines and tissues. Since little is known about proteins interacting with PKC inside the cell nucleus, we used Neuro-2a neuroblastoma cells, in which PKCalpha is present in the nucleus, to screen for nuclear binding partners for PKC. Applying overlay assays, we detected several nuclear proteins which bind to PKCalpha. Specificity of binding was shown by its dependence on PKC activation by phorbol ester, calcium, and phosphatidylserine. The PKC-binding proteins were partially purified and analyzed by microsequencing and mass spectrometry. Four proteins could be identified: PTB-associated splicing factor (PSF), p68 RNA helicase, and the heterogeneous nuclear ribonucleoprotein (hnRNP) proteins A3 and L. In the case of PSF, binding to PKC could also be demonstrated in a GST-pull-down assay using GST-PKCalpha, expressed in insect cells. Phosphorylation experiments revealed that PSF is a weak in vitro substrate for PKCalpha.     

Screening, identification, and characterization of mechanistically diverse inhibitors of the Mycobacterium tuberculosis enzyme, pantothenate kinase (CoaA)

The authors describe the discovery of anti-mycobacterial compounds through identifying mechanistically diverse inhibitors of the essential Mycobacterium tuberculosis (Mtb) enzyme, pantothenate kinase (CoaA). Target-driven drug discovery technologies often work with purified enzymes, and inhibitors thus discovered may not optimally inhibit the form of the target enzyme predominant in the bacterial cell or may not be available at the desired concentration. Therefore, in addition to addressing entry or efflux issues, inhibitors with diverse mechanisms of inhibition (MoI) could be prioritized before hit-to-lead optimization. The authors describe a high-throughput assay based on protein thermal melting to screen large numbers of compounds for hits with diverse MoI. Following high-throughput screening for Mtb CoaA enzyme inhibitors, a concentration-dependent increase in protein thermal stability was used to identify true binders, and the degree of enhancement or reduction in thermal stability in the presence of substrate was used to classify inhibitors as competitive or non/uncompetitive. The thermal shift-based MoI assay could be adapted to screen hundreds of compounds in a single experiment as compared to traditional biochemical approaches for MoI determination. This MoI was confirmed through mechanistic studies that estimated K(ie) and K(ies) for representative compounds and through nuclear magnetic resonance-based ligand displacement assays.     

Purification of the major outer membrane protein of Azospirillum brasilense, its affinity to plant roots, and its involvement in cell aggregation

The major outer membrane protein (MOMP) of the nitrogen-fixing rhizobacterium Azospirillum brasilense strain Cd was purified and isolated by gel filtration, and antiserum against this protein was obtained. A screening of the binding of outer membrane proteins (OMPs) of A. brasilense to membrane-immobilized root extracts of various plant species revealed different affinities for the MOMP, with a stronger adhesion to extracts of cereals in comparison with legumes and tomatoes. Moreover, this protein was shown to bind to roots of different cereal seedlings in an in vitro adhesion assay. Incubation of A. brasilense cells with MOMP-antiserum led to fast agglutination, indicating that the MOMP is a surface-exposed protein. Cells incubated with Fab fragments obtained from purified MOMP-antiserum immunoglobulin G exhibited significant inhibition of bacterial aggregation as compared with controls. Bacteria preincubated with Fab fragments showed weaker adhesion to corn roots in comparison to controls without Fab fragments. These findings suggest that the A. brasilense MOMP acts as an adhesin involved in root adsorption and cell aggregation of this bacterium.