Binding Of Deoxycholate Research Articles

Structural and functional characterization of the IpaD π-helix reveals critical roles in DOC interaction, T3SS apparatus maturation, and Shigella virulence

Shigella spp. are highly pathogenic members of the Enterobacteriaceae family, causing ∼269 million cases of bacillary dysentery and >200,000 deaths each year. Like many Gram-negative pathogens, Shigella rely on their type three secretion system (T3SS) to inject effector proteins into eukaryotic host cells, driving both cellular invasion and evasion of host immune responses. Exposure to the bile salt deoxycholate (DOC) significantly enhances Shigella virulence and is proposed to serve as a critical environmental signal present in the small intestine that prepares Shigella’s T3SS for efficient infection of the colonic epithelium. Here, we uncover critical mechanistic details of the Shigella-specific DOC signaling process by describing the role of a π-helix secondary structure element within the T3SS tip protein IpaD. Biophysical characterization and high-resolution structures of IpaD mutants lacking the π-helix show that it is not required for global protein structure, but that it defines the native DOC binding site and prevents off target interactions. Additionally, Shigella strains expressing the π-helix deletion mutants illustrate the pathogenic importance of its role in guiding DOC interaction as flow cytometry and gentamycin protection assays show that the IpaD π-helix is essential for DOC-mediated apparatus maturation and enhanced invasion of eukaryotic cells. Together, these findings add to our understanding of the complex Shigella pathogenesis pathway and its evolution to respond to environmental bile salts by identifying the π-helix in IpaD as a critical structural element required for translating DOC exposure to virulence enhancement.

Towards the design of hypolipidaemic peptides: Deoxycholate binding affinity of hydrophobic peptide aggregates of casein plastein

Plastein is a thixotropic product of protease-induced peptide aggregation. This study evaluated the effects of the modified peptide structure of plastein on its affinity for a secondary bile acid. Casein plastein formation resulted in decreased free amino nitrogen and increased mean particle size, surface hydrophobicity and hydrophobic-to-hydrophilic amino acid ratio of the peptides. The net negative surface charge of casein hydrolysate (CH) was decreased (P < 0.05) in crude plastein (CPc) and completely lost in the isolated plastein (CPi) resulting in the formation of insoluble aggregates. The surface and structural changes in CPi were associated with 38.5 ± 0.0% binding of physiological level of sodium deoxycholate, higher (P < 0.05) than the activities of CPc (23.1 ± 0.0%) and CH (12.8 ± 4.4%). Maximum specific ligand binding (Bmax) was higher (P < 0.05) for CPc (0.391 ± 0.021 mM/mg protein) than CH (0.246 ± 0.043 mM/mg protein) and CPi (0.234 ± 0.005 mM/mg protein). However, CPi had the strongest affinity for sodium deoxycholate with the lowest dissociation constant (Kd) of 2.719 ± 0.146 mM, which was 2.5- and 3-folds lower (P < 0.05) than those of CH and CPc, respectively. Understanding deoxycholate-binding affinity of peptides can facilitate the design of potent food-based BA sequestrants for managing hypercholesterolaemia (dyslipidaemia) and associated health conditions.

Bet v 1 – a Trojan horse for small ligands boosting allergic sensitization?

Birch pollen allergy represents the main cause of winter and spring pollinosis in the temperate climate zone of the northern hemisphere and sensitization towards Bet v 1, the major birch pollen allergen, affects over 100 million allergic patients. The major birch pollen allergen Bet v 1 has been described as promiscuous acceptor for a wide variety of hydrophobic ligands. In search of intrinsic properties of Bet v 1, which account responsible for the high allergenic potential of the protein, we thought to investigate the effects of ligand-binding on immunogenic as well as allergenic properties. As surrogate ligand of Bet v 1 sodium deoxycholate (DOC) was selected. Recombinant and natural Bet v 1 were characterised physico-chemically as well as immunologically in the presence or absence of DOC, and an animal model of allergic sensitization was established. Moreover, human IgE binding to Bet v 1 was analysed by nuclear magnetic resonance (NMR) spectroscopy. Ligand-binding had an overall stabilizing effect on Bet v 1. This translated in a Th2 skewing of the immune response in a mouse model. Analyses of human IgE binding on Bet v 1 in mediator release assays revealed that ligand-bound allergen-induced degranulation at lower concentrations; however, in basophil activation tests with human basophils ligand-binding did not show this effect. For the first time, human IgE epitopes on Bet v 1 were determined using antibodies isolated from patients' sera. The IgE epitope mapping of Bet v 1 demonstrated the presence of multiple binding regions. Deoxycholate binding stabilizes conformational IgE epitopes on Bet v 1; however, the epitopes themselves remain unaltered. Therefore, we speculate that humans are exposed to both ligand-bound and free Bet v 1 during sensitization, disclosing the ligand-binding cavity of the allergen as key structural element.

Identification of the bile salt binding site on ipad from Shigella flexneri and the influence of ligand binding on IpaD structure

Type III secretion (TTS) is an essential virulence factor for Shigella flexneri, the causative agent of shigellosis. The Shigella TTS apparatus (TTSA) is an elegant nanomachine that is composed of a basal body, an external needle to deliver effectors into human cells, and a needle tip complex that controls secretion activation. IpaD is at the tip of the nascent TTSA needle where it controls the first step of TTS activation. The bile salt deoxycholate (DOC) binds to IpaD to induce recruitment of the translocator protein IpaB into the maturing tip complex. We recently used spectroscopic analyses to show that IpaD undergoes a structural rearrangement that accompanies binding to DOC. Here, we report a crystal structure of IpaD with DOC bound and test the importance of the residues that make up the DOC binding pocket on IpaD function. IpaD binds DOC at the interface between helices α3 and α7, with concomitant movement in the orientation of helix α7 relative to its position in unbound IpaD. When the IpaD residues involved in DOC binding are mutated, some are found to lead to altered invasion and secretion phenotypes. These findings suggest that adoption of a DOC bound structural state for IpaD primes the Shigella TTSA for contact with host cells. The data presented here and in the studies leading up to this work provide the foundation for developing a model of the first step in Shigella TTS activation.

Conformational Changes in IpaD from Shigella flexneri upon Binding Bile Salts Provide Insight into the Second Step of Type III Secretion

Shigella flexneri uses its type III secretion apparatus (TTSA) to inject host-altering proteins into targeted eukaryotic cells. The TTSA is composed of a basal body and an exposed needle with invasion plasmid antigen D (IpaD) forming a tip complex that controls secretion. The bile salt deoxycholate (DOC) stimulates recruitment of the translocator protein IpaB into the maturing TTSA needle tip complex. This process appears to be triggered by a direct interaction between DOC and IpaD. Fluorescence spectroscopy and NMR spectroscopy are used here to confirm the DOC-IpaD interaction and to reveal that IpaD conformational changes upon DOC binding trigger the appearance of IpaB at the needle tip. Förster resonance energy transfer between specific sites on IpaD was used here to identify changes in distances between IpaD domains as a result of DOC binding. To further explore the effects of DOC binding on IpaD structure, NMR chemical shift mapping was employed. The environments of residues within the proposed DOC binding site and additional residues within the "distal" globular domain were perturbed upon DOC binding, further indicating that conformational changes occur within IpaD upon DOC binding. These events are proposed to be responsible for the recruitment of IpaB at the TTSA needle tip. Mutation analyses combined with additional spectroscopic analyses confirm that conformational changes in IpaD induced by DOC binding contribute to the recruitment of IpaB to the S. flexneri TTSA needle tip. These findings lay the foundation for determining how environmental factors promote TTSA needle tip maturation prior to host cell contact.

Deoxycholate Interacts with IpaD of Shigella flexneri in Inducing the Recruitment of IpaB to the Type III Secretion Apparatus Needle Tip

Type III secretion (TTS) is an essential virulence function for Shigella flexneri that delivers effector proteins that are responsible for bacterial invasion of intestinal epithelial cells. The Shigella TTS apparatus (TTSA) consists of a basal body that spans the bacterial inner and outer membranes and a needle exposed at the pathogen surface. At the distal end of the needle is a "tip complex" composed of invasion plasmid antigen D (IpaD). IpaD not only regulates TTS, but is required for the recruitment and stable association of the translocator protein IpaB at the TTSA needle tip in the presence of deoxycholate or other bile salts. This phenomenon is not accompanied by induction of TTS or the recruitment of IpaC to the Shigella surface. We now show that IpaD specifically binds fluorescein-labeled deoxycholate and, based on energy transfer measurements and docking simulations, this interaction appears to occur where the N-terminal domain of IpaD meets its central coiled-coil, a region that may also be involved in needle-tip interactions. TTS is initiated as a series of distinct steps and that small molecules present in the bacterial milieu are capable of inducing the first step of TSS through interactions with the needle tip protein IpaD. Furthermore, the amino acids proposed to be important for deoxycholate binding by IpaD appear to have significant roles in regulating tip complex composition and pathogen entry into host cells.

Dietary fibre and the mouse colon: Its influence on luminal pH, reducing activity and bile acid binding

Female, Swiss mice were fed semi-synthetic diets for 33 days. The diets were fibre-free (FF) or supplemented with corn bran (CB) 12%, wheat bran (WB) 12%, alfalfa (AL) 12%, pectin (P) 6%, cellulose (CL) 6%, or lignin (LG) 6%. Fibre caused little hyperplasia of the colon mucosa. The number of cells per crypt was increased 9–13% and the crypt column length by 14–19% in the CL, AL and LG groups. CB caused rather less hyperplasia, WB less again and P caused none. The colon mucosal DNA content was approximately 5–10% lower in mice given supplemental fibre. The pH of the contents of the distal colon was apparently unaffected by fibre. Measurement of a non-specific, non-enzymic reducing activity indicated that activity was doubled by AL and LG, lowered 41% by CB but little changed by WB, CL and P. The deoxycholate binding capacity of the colon contents was increased 3–4-fold by LG, whereas the other fibre sources were without appreciable effect. This high binding capacity by LG was also seen in the material used for diet preparation. Analyses of the contents of the caecum and of the remaining colon indicated that as food residue passes from the caecum to the remaining colon little change in binding capacity occurs.

Molecular weight of poly(C9). 12 to 18 C9 molecules form the transmembrane channel of complement.

Poly(C9), the tubular 27 S complex forming the transmembrane channel of the membrane attack complex of complement, was purified to homogeneity by gel filtration and sucrose density gradient ultracentrifugation. The molecular weight of poly(C9) was determined by two independent methods in addition to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. First, sedimentation equilibrium analysis using 0.2% SDS or 1% deoxycholate containing buffer as solvents yielded a point weight average molecular weight exclusive of bound detergent of 0.9 to 1.3 X 10(6) and a weight average molecular weight of all poly(C9) complexes of 1,050,000 +/- 40,000 (S.D.). SDS and deoxycholate binding to poly(C9) was measured in an air-driven ultracentrifuge and was determined to be 0.53 +/- 0.065 (S.D.) g of SDS and 0.26 +/- 0.015 (S.D.) g of deoxycholate/g of poly(C9), respectively. Second, the mass of 27 S poly(C9) devoid of bound detergent was determined by electron scattering of unstained specimens in the scanning transmission electron microscope. The molecular weight obtained by this method was 1,078,000 +/- 194,000. The inner diameter of poly(C9) tubules imaged in top view projections by negative staining electron microscopy varied between 9 and 12 mm. The accumulated data suggest a true heterogeneity of the molecular weight of poly(C9) due to polymers with varying protomer numbers. Using a mean value of 73,500 for the molecular weight of monomeric C9, the protomer number of poly(C9) tubules appear to vary between 12 and 18 C9 subunits. Approximately 50-75% of the tubules have 14 to 16 subunits as deduced from the mass distribution determined by electron scattering and from ring size measurements. It is suggested that poly(C9) tubules with various protomer numbers may arise due to limited flexibility in the C9-C9 interaction.

Interaction of the mouse and bovine myelin basic proteins and two cleavage fragments with anionic detergents

The binding of deoxycholate and dodecyl sulfate to the mouse and bovine myelin basic proteins and two peptide fragments, obtained by cleavage of the bovine basic protein at its single tryptophan residue, was examined. Complete equilibrium binding isotherms for both detergents were obtained by examining their binding to each of the polypeptides immobilized on agarose. The bulk of the binding of dodecyl sulfate was found to be highly cooperative, and at saturation all four polypeptides bound far more detergent than globular, water-soluble proteins. The sum of the dodecyl sulfate bound by each of the two bovine basic protein cleavage fragments was almost twice that bound by the intact protein at saturation, suggesting that cleavage of the bovine basic protein exposes sites for additional binding of dodecyl sulfate. At pH values below pH 8.0, an additional cooperative transition was observed below the critical micelle concentration of sodium dodecyl sulfate in the binding isotherms of all four polypeptides. The midpoint of this transition corresponded to an apparent p K of approximately 5.5; however, the destruction of 90% of the histidine residues in the bovine basic protein had no effect on this transition. At pH 9.2 and moderate ionic strength ( I = 0.1), the bulk of the binding of deoxycholate to the mouse and bovine basic proteins occurred at and above the critical micelle concentration of the detergent; and saturation values of deoxycholate binding to these two proteins were considerably higher than that reported for globular, water-soluble proteins. In marked contrast to the results with dodecyl sulfate, neither cleavage fragment was observed to bind deoxycholate. The results suggest that the higher ordered structure of the bovine basic protein may play an important role in the binding of anionic amphiphiles to the protein.

Purification and characterization of a cholesterol-binding protein from human pancreas.

The protein composition of human intestinal lavage fluids was analysed by electroimmunoassay. In addition to secretory immunoglobulin A and other components that were antigenically related to serum proteins, a number of gut-specific proteins were detected. One of these was found to exhibit the capacity of binding sodium deoxycholate and cholesterol. After isolation of this cholesterol-binding protein from intestinal fluids, immunohistochemical studies utilizing a specific antiserum indicated the pancreas to be the organ of its synthesis. The protein was subsequently purified from necrobiotic pancreas tissues and was found to be composed of a single polypeptide chain with a mol. wt. of 28 000 and an isoelectric point of pH4.9. The deoxycholate binding capacity determined by gel chromatography in the presence of [3H]deoxycholate was calculated to be approx. 24 mol of deoxycholate/mol of protein. In the intestinal fluids the protein appeared to be present in firm association with cholesterol, phospholipids, triacylglycerols and bile salts as a macromolecular protein-lipid complex. The possibility is raised that the pancreas-derived, cholesterol-binding protein may fulfil a function as an intestinal 'lipoprotein'.

Interaction of tubulin with non-denaturing amphiphiles.

Soluble purified calf brain tubulin contains extensive and easily accessible regions capable of hydrophobic interactions. The binding of non-ionic and mild anionic detergents to this protein has been characterized by difference absorption spectroscopy and equilibrium gel chromatography with labelled ligands. Tubulin bound reversibly and co-operatively 0.42 +/- 0.05 g deoxycholate per g protein and bound octyl glucoside at a minimal stoichiometry of 0.26 g per g protein. Binding of deoxycholate and octyl glucoside perturbed the protein absorption, quenched the fluorescence, and produced a moderate change in the far u.v. circular dichroism of tubulin. These changes have been interpreted as the result of detergent binding near aromatic amino acids and the production of a structural change different from detergent-induced denaturation. Deoxycholate and octyl glucoside inhibited colchicine binding. Octyl glucoside and Triton X-100 inhibited the in vitro self-assembly of tubulin into microtubules, whereas small concentrations of deoxycholate were found to enhance microtubule formation.

The dispersion of gram-negative lipopolysaccharide by deoxycholate. Subunit molecular weight.

The lipopolysaccharide (LPS) was isolated from three strains of Salmonella typhimurium, a "smooth" strain, STM 7, the Ra mutant, TV 119, and the Re mutant, SL 1102. The effect of depletion of divalent cations on structure and the effect of deoxycholate on hydrodynamic behavior were studied. The results confirmed previous work by others that divalent cations and hydrophobic forces are important factors influencing LPS size and morphology. The binding of deoxycholate to LPS was measured. When the weight average molecular weights of the deoxycholate-dissociated LPS were determined by sedimentation equilibrium and corrected for bound deoxycholate, the values 5,555, 10,607, and 15,592, respectively, for Re, Ra, and "smooth" LPS were in good agreement with calculated formula weights. Although others have suggested that the basic LPS subunit is a trimer, our results suggest that it exists in the dimeric form.

Binding of deoxycholate to lipoprotein lipase

Binding of deoxycholate to lipoprotein lipase from bovine milk was demonstrated by equilibrium dialysis and by charge shift electrophoresis. The detergent increased the solubility and stability of the enzyme in aqueous buffers, but did not change its elution position on gel filtration. It is concluded that each molecule of enzyme binds several molecules of deoxycholate without denaturation. This signifies the presence of a lipid-binding region in the native structure of the enzyme.

Selective inactivation of lactate dehydrogenase of rat tissues by sodium deoxycholate

Soluble lactate dehydrogenase (EC 1.1.1.27) extracted from brain, skeletal and cardiac muscle and liver of rats, and purified isoenzymes LDH-1 and LDH-5, were incubated with sodium deoxycholate. Deoxycholate almost totally inactivated isoenzyme LDH-5 (A4), whereas it left isoenzyme LDH-1 (B4) unaffected. Tissue lactate dehydrogenase was inactivated to different degrees depending on the origin of the enzyme. Electrophoretic isoenzyme studies of tissue lactate dehydrogenase showed the loss of activity to be quantitatively related to the overall percentage of subunit A distributed among the homotetramer LDH-5 and the heterotetramers LDH-2, LDH-3 and LDH-4. It was concluded that subunit A of lactate dehydrogenase interacts selectively with deoxycholate, irrespective of its association with subunit B. Distinct changes in electrophoretic mobilities of deoxycholate-treated isoenzymes strongly indicated an indiscriminate binding of deoxycholate by all LDH isoenzymes, probably through hydrophobic interactions. The results suggest that the inactivation of the enzyme is non-competitive, but the basis of the selectivity of deoxycholate towards subunit A is not known at present.

SC5b-9: Regeneration of the Ability to Interact with Lipid by Selective Removal of the S-Protein

Abstract The various complexes were prepared by mixing isolated C5b-6 in the presence of detergents or phospholipids with equimolar amounts of C7, C7 and C8, or with C7, C8 and C9 in molar ratios of 1:1:1:3. C5b-9 was also prepared by treating the SC5b-9 complex purified from inulin activated serum with 6 mM Na deoxycholate (DOC) at pH 8.1 and subjecting it to gel filtration on Sepharose 6B in the presence of DOC. This treatment resulted in selective dissociation of the S-protein, in exchange for binding of DOC by C5b-9. Detergent-binding studies showed that C5b-6, C5b-7 and C5b-8 bind 26, 28 and 65 mole 14C-DOC per mole of complex, respectively, as measured by equilibrium dialysis. C5b-9 (prepared from SC5b-9) binds 86 mole DOC per mole of complex, measured by gel filtration in the presence of 3H-DOC. DOC binding as well as cetyl trimethyl ammonium bromide (CTAB) binding of all complexes could also be demonstrated by charge shift electrophoresis.

Activity and structural changes of purified kallikrein with detergent treatment

The esterase activity of rat urinary kallikrein is increased up to fourfold by the anionic detergent, deoxycholate and the nonionic detergents, Triton X-100, Lubrol PX, and Brij 58. The cationic detergents, benzyltriphenylphosphonium chloride and cetyltri-methylammonium bromide, inhibit kallikrein activity. Certain trypsin inhibitors stimulate kallikrein activity but this stimulation is not observed when kallikrein is preincubated with deoxycholate. In addition, deoxycholate weakens the inhibition of kallikrein activity by Trasylol. Deoxycholate-induced conformational changes of kallikrein are noted by a change in circular dichroism spectra in the far and near ultraviolet region. A maximal change of ellipticity at 275 nm suggests binding of deoxycholate to kallikrein at or around the tyrosine residue(s) or changes in the microenvironment of these residue(s).

Spin-Labeling of Adenosine Triphosphatase in Sarcoplasmic Reticulum Membrane and Change in the State of the Spin Labels Induced by Deoxycholate

Fragmented sarcoplasmic reticulum (SR) was reacted with a thiol-directed spin label, N-(1-oxyl-2,2,6,6,-tetramethyl-4-piperidinyl)maleimide, under various conditions. It was found that ATP inhibited the binding of the label to SR protein in the initial phase of the reaction, but as the incubation time was extended up to 18 h, the amount of label bound to SR protein in the control and ATP-containing samples became almost identical. The Ca2+-dependent ATPase control and ATP-containing samples became almost identical The Ca2+-dependent ATPase (ATP phosphohydrolase [EC 3.6.1.3]) of SR was protected by the presence of ATP during incubation with relatively low concentrations of spin label, irrespective of the total amount of label bound, although with increasing concentration of bound label the ATPase activity decreased. Deoxycholate slightly reduced the rotational freedom of the label bound to SR protein and decreased the initial rate of quenching of protein-bound nitroxide by ascorbate. From an analysis of these results, it was concluded that the binding of deoxycholate to protein decreases the accessibility of ascorbate to the protein-bound label.

The binding of deoxycholate, Triton X-100, sodium dodecyl sulfate, and phosphatidylcholine vesicles to cytochrome b5.

Cytochrome b5 is composed of two domains that can be isolated after tryptic cleavage as two polypeptide fragments. One fragment is globular and hydrophilic and contains the heme; the other fragment is rich in hydrophobic amino acids and is essential for recombination of cytochrome b5 with microsomal membranes (Ito, A., and Sato, R. (1968), J. Biol. Chem. 243, 4922; Spatz, L., and Strittmatter, P. (1971), Proc. Nat. Acad. Sci. U.S. 68, 1042). Equilibrium dialysis and sedimentation equilibrium measurements of the binding of deoxycholate, Triton X-100 and dodecyl sulfate show that neither intact cytochrome b5 nor its proteolytic fragments possess high affinity binding sites for any of these amphiphiles. However, each detergent binds to the protein in a highly cooperative manner at concentrations near the critical micelle concentration. Binding measurements using the separated tryptic fragments show that deoxycholate and Triton X-100 (both nondenaturing detergents) bind to the hydrophobic fragment to the same extent as to intact cytochrome b5, and not at all to the polar fragment. Sodium dodecyl sulfate (a denaturing detergent) is bound to both tryptic fragments, but 70% of the detergent is bound to the hydrophobic fragment although it comprises only 30% of the protein mass. Less detailed measurements were made with synthetic and natural phosphatidylcholines, and show that the intact protein is quantitatively incorporated into phosphatidylcholine vesicles, but that no interaction with the polar fragment occurs. These results are interpreted in terms of the hydrophobic domain of cytochrome b5 having a diffuse hydrophobic surface that can act as a nonspecific nucleus for the formation of a micelle with a variety of amphiphilic substances. This domain of the molecule will insert into any available hydrophobic environment, whether it be detergent micelles, synthetic phospholipid vesicles, or the microsomal membrane. The incorporation of cytochrome b5 into the microsomal membrane is only a specialized case of the general property.

The Binding of Deoxycholate and Triton X-100 to Proteins

Deoxycholate and Triton X-100 resemble other anionic and neutral amphiphiles in binding to the high affinity sites of native bovine serum albumin. There are four principal sites for each ligand and about 14 weaker sites for deoxycholate; more than half the strong sites are common to each ligand, or octyl and dodecyl sulfates, or both, on the basis of competitive binding studies. On the other hand, cooperative binding of large amounts of ligand with accompanying denaturation, such as is observed for dodecyl sulfate and many other ionic detergents for serum albumin and most other proteins, does not take place with deoxycholate and Triton X-100. The probable reason is that these substances have a relatively low critical micelle concentration, so that a monomeric concentration sufficient for the cooperative mode of binding cannot be attained. The utility of deoxycholate and Triton X-100 for the solubilization of membrane proteins in native or near-native form is consistent with these results.