Restoration of structural stability and ligand binding after removal of the conserved disulfide bond in tear lipocalin

Disulfide bonds play diverse structural and functional roles in proteins. In tear lipocalin (TL), the conserved sole disulfide bond regulates stability and ligand binding. Probing protein structure often involves thiol selective labeling for which removal of the disulfide bonds may be necessary. Loss of the disulfide bond may destabilize the protein so strategies to retain the native state are needed. Several approaches were tested to regain the native conformational state in the disulfide-less protein. These included the addition of trimethylamine N-oxide (TMAO) and the substitution of the Cys residues of disulfide bond with residues that can either form a potential salt bridge or others that can create a hydrophobic interaction. TMAO stabilized the protein relaxed by removal of the disulfide bond. In the disulfide-less mutants of TL, 1.0 M TMAO increased the free energy change (ΔG⁰) significantly from 2.1 to 3.8 kcal/mol. Moderate recovery was observed for the ligand binding tested with NBD-cholesterol. Because the disulfide bond of TL is solvent exposed, the substitution of the disulfide bond with a potential salt bridge or hydrophobic interaction did not stabilize the protein. This approach should work for buried disulfide bonds. However, for proteins with solvent exposed disulfide bonds, the use of TMAO may be an excellent strategy to restore the native conformational states in disulfide-less analogs of the proteins.     

The conserved disulfide bond of human tear lipocalin modulates conformation and lipid binding in a ligand selective manner

The primary aim of this study is the elucidation of the mechanism of disulfide induced alteration of ligand binding in human tear lipocalin (TL). Disulfide bonds may act as dynamic scaffolds to regulate conformational changes that alter protein function including receptor-ligand interactions. A single disulfide bond, (Cys61-Cys153), exists in TL that is highly conserved in the lipocalin superfamily. Circular dichroism and fluorescence spectroscopies were applied to investigate the mechanism by which disulfide bond removal effects protein stability, dynamics and ligand binding properties. Although the secondary structure is not altered by disulfide elimination, TL shows decreased stability against urea denaturation. Free energy change (ΔG(0)) decreases from 4.9±0.2 to 2.1±0.3kcal/mol with removal of the disulfide bond. Furthermore, ligand binding properties of TL without the disulfide vary according to the type of ligand. The binding of a bulky ligand, NBD-cholesterol, has a decreased time constant (from 11.8±0.2 to 3.3s). In contrast, the NBD-labeled phospholipid shows a moderate decrease in the time constant for binding, from 33.2±0.2 to 22.2±0.4s. FRET experiments indicate that the hairpin CD is directly involved in modulation of both ligand binding and flexibility of TL. In TL complexed with palmitic acid (PA-TL), the distance between the residues 62 of strand D and 81 of loop EF is decreased by disulfide bond reduction. Consequently, removal of the disulfide bond boosts flexibility of the protein to reach a CD-EF loop distance (24.3?, between residues 62 and 81), which is not accessible for the protein with an intact disulfide bond (26.2?). The results suggest that enhanced flexibility of the protein promotes a faster accommodation of the ligand inside the cavity and an energetically favorable ligand-protein complex.     

Phage display reveals a novel interaction of human tear lipocalin and thioredoxin which is relevant for ligand binding

Human tear lipocalin (TL) is an unusual member of the lipocalin protein family, since it is known to bind a large variety of lipophilic ligands in vivo and acts as a cysteine proteinase inhibitor in vitro. It is suggested to function as a physiological protection factor by scavenging lipophilic potentially harmful compounds. Since protein-protein interaction or macromolecular complexation is a common feature of many lipocalins, we applied phage display technology to identify TL interacting proteins. By panning of a human prostate cDNA phagemid library against purified TL we isolated a thioredoxin (Trx) encoding phage clone. Biochemical analysis revealed that TL indeed interacts with Trx and is reduced by this redox protein. Reduction of the TL-specific disulfide bond is of functional relevance, since the reduced protein shows a nine-fold increase in ligand affinity when tested with retinoic acid as ligand.     

Natural ligand binding and transfer from liver fatty acid binding protein (LFABP) to membranes

Liver fatty acid-binding protein (LFABP) is distinctive among fatty acid-binding proteins because it binds more than one molecule of long-chain fatty acid and a variety of diverse ligands. Also, the transfer of fluorescent fatty acid analogues to model membranes under physiological ionic strength follows a different mechanism compared to most of the members of this family of intracellular lipid binding proteins. Tryptophan insertion mutants sensitive to ligand binding have allowed us to directly measure the binding affinity, ligand partitioning and transfer to model membranes of natural ligands. Binding of fatty acids shows a cooperative mechanism, while acyl-CoAs binding presents a hyperbolic behavior. Saturated fatty acids seem to have a stronger partition to protein vs. membranes, compared to unsaturated fatty acids. Natural ligand transfer rates are more than 200-fold higher compared to fluorescently-labeled analogues. Interestingly, oleoyl-CoA presents a markedly different transfer behavior compared to the rest of the ligands tested, probably indicating the possibility of specific targeting of ligands to different metabolic fates.     

Ex-FABP: a fatty acid binding lipocalin developmentally regulated in chicken endochondral bone formation and myogenesis

Extracellular fatty acid binding protein (Ex-FABP) is a 21 kDa lipocalin specifically binding fatty acids, expressed during chicken embryo development in hypertrophic cartilage, in muscle fibers and in blood granulocytes. In chondrocyte and myoblast cultures Ex-FABP expression is increased by inflammatory agents and repressed by anti-inflammatory agents. In adult cartilage Ex-FABP is expressed only in pathological conditions such as in dyschondroplastic and osteoarthritic chickens. The possible mammalian counterpart is the Neu-related lipocalin (NRL), a lipocalin overexpressed in rat mammary cancer; NRL is homologous to the human neutrophil gelatinase associated lipocalin (NGAL) expressed in granulocytes and in epithelial cells in inflammation and malignancy and to the Sip24 (super-inducible protein 24), an acute phase lipocalin expressed in mouse after turpentine injection. Immunolocalization and in situ hybridization showed that NRL/NGAL is expressed in hypertrophic cartilage, in forming skeletal muscle fibers and in developing heart. In adult cartilage NRL/NGAL was expressed in articular cartilage from osteoarthritic patients and in chondrosarcoma. Moreover, NRL was induced in chondrocyte and myoblast cultures by an inflammatory agent. We propose that these lipocalins (Ex-FABP, NRL/NGAL, Sip24) represent stress proteins physiologically expressed in tissues where active remodeling is taking place during development and also present in tissues characterized by an acute phase response due to pathological conditions.     

Adipogenic differentiating agents regulate expression of fatty acid binding protein and CD36 in the J744 macrophage cell line

Adipocyte fatty acid binding protein (aP2) is a key mediator of intracellular transport and metabolism of fatty acids. Its expression during adipocyte differentiation is regulated through the actions of peroxisome proliferator-activated receptor gamma (PPARgamma) and CCAAT/enhancer binding protein alpha (C/EBPalpha). Macrophages also express aP2, and the lack of macrophage aP2 significantly reduces atherosclerotic lesion size in hypercholesterolemic mice. We investigated the regulation of expression of macrophage aP2 and CD36, a fatty acid membrane binding protein and scavenger receptor, in response to the adipogenic agents isobutylmethylxanthine (IBMX), insulin, and dexamethasone, a combination of agents shown to induce fibroblast-to-adipocyte differentiation. Treatment of J774 macrophages with adipogenic agents significantly induced aP2 mRNA expression, while CD36 expression was inhibited. Dexamethasone was essential and sufficient to induce aP2 expression, and insulin had a synergistic effect. However, IBMX antagonized induced-aP2 expression. aP2 protein expression and [14C]oleic acid uptake by macrophages were also increased by dexamethasone. Unlike what occurs in adipocytes, adipogenic agents had mixed effects on the expression of PPARgamma and C/EBPalpha in macrophages. Our data demonstrate differences in the regulation of aP2 in adipocytes and macrophages and show that macrophage aP2 expression by adipogenic agents is independent of the PPARgamma and/or C/EBPalpha signaling pathway.     

Integrins: alternative splicing as a mechanism to regulate ligand binding and integrin signaling events

Integrins are a family of transmembrane proteins composed of heterodimers of α and β subunits. With their extracellular domain they bind extracellular matrix proteins or other cell surface molecules, and their cytoplasmic domain binds to cytoskeletal and signaling proteins. Thus, they are in an ideal position to transfer information from the extracellular environment to the interior of the cell and vice versa. For several integrin subunits, alternative splicing of mRNA leads to variations in the sequence of both extracellular and cytoplasmic domains. Many integrin splice variants have specific expression patterns, but for some time, functional differences between these variants were not evident. Recent experiments using transfected cell lines and gene targeting of specific splice variants have contributed significantly to our understanding of the function of these splice variants. The results indicate that alternative splicing is a mechanism to subtly regulate the ligand binding and signaling activity of integrins. Bio Essays 21:499–509, 1999. © 1999 John Wiley & Sons, Inc.     

Site-directed tryptophan fluorescence reveals the solution structure of tear lipocalin: evidence for features that confer promiscuity in ligand binding.

The solution structure of human TL was deduced from the position of the emission peaks after site-directed tryptophan fluorescence (SDTF). The fluorescent amino acid tryptophan was sequentially substituted for each native amino acid in the sequence. Characteristic periodicities for eight beta-strands that comprise the beta-barrel and three alpha-helices were identified. The putative beta-strand I was relatively exposed to solvent, suggesting it does not participate in the formation of the beta-barrel. The beta-strands A and F contain beta-bulges. The average lambda(max) of emission maxima reveals that strand D is at the edge of the barrel and beta-strand H interacts with the main alpha-helical domain. On the basis of the SDTF data, a 3D homology model was constructed for TL and compared to the known crystallographic structures of RBP and beta-lactoglobulin. The small size and splayed open configuration of the E-F hairpin facilitate access of ligands into the cavity mouth of TL as compared to that of RBP with a long overhanging loop that restricts access. In the model of TL, four alanine residues are positioned in the binding site as compared to bulkier residues in the corresponding positions of beta-lactoglobulin. Substitution of A51, A66, A86 to Trp results in a 3-4-fold decrease in binding affinity. The data suggest that the smaller side chains of Ala provide more capacity in the cavity of TL than the bulkier side chains (I56, I71, V92) in the cavity of beta-lactoglobulin. The structural features provide an explanation for the promiscuous binding characteristics exhibited by TL. SDTF provides a general approach for determining the solution structure of many proteins and enhances homology modeling in the absence of high sequence identity.     

Effects of ligand binding on dynamics of fatty acid binding protein and interactions with membranes

Intracellular transport of fatty acids involves binding of ligands to their carrier fatty acid binding proteins (FABPs) and interactions of ligand-free and -bound FABPs with membranes. Previous studies focused on ligand-free FABPs. Here, our amide hydrogen exchange data showed that oleic acid binding to human intestinal FABP (hIFABP) stabilizes the protein, most likely through enhancing the hydrogen-bonding network, and induces rearrangement of sidechains even far away from the ligand binding site. Using NMR relaxation techniques, we found that the ligand binding affects not only conformational exchanges between major and minor states but also the affinity of hIFABP to nanodiscs. Analyses of the relaxation and amide exchange data suggested that two minor native-like states existing in both ligand-free and -bound hIFABPs originate from global “breathing” motions, while one minor native-like state comes from local motions. The amide hydrogen exchange data also indicated that helix αII undergoes local unfolding through which ligands can exit from the binding cavity.     

Urinary neutrophil gelatinase-associated lipocalin and L-type fatty acid binding protein as diagnostic markers of early acute kidney injury after liver transplantation

Objective: We examined the value of two potential novel urinary biomarkers, neutrophil gelatinase-associated lipocalin (NGAL) and L-type fatty acid binding protein (L-FABP), in diagnosing acute kidney injury (AKI) in liver transplant recipients.

Methods: NGAL and L-FABP in urinary sample from Twenty-five patients before surgery and at 2, 4, 6, 12, 24, 48, 72 and 120 h after the anhepatic phase were tested. Standard statistics were used along with receiver-operating characteristic (ROC) analysis to evaluate the diagnostic value of selected markers.

Results: Urinary NGAL was only slightly elevated at 2 h in the non-AKI group while rose and stayed high from 2–6 h in the AKI group. However, urinary L-FABP rose transiently in both groups 2–120 h following surgery. The level of urinary NGAL presented differences at 2–6 h (p < 0.05) and urinary L-FABP at 4 h (p < 0.05) between AKI and non-AKI groups. ROC analysis showed that area under the curves (AUCs) of NGAL were 0.766, 0.773, and 0.773 at 2, 4 and 6 h respectively while 0.760 of L-FABP at 4 h.

Conclusion: Urinary NGAL rather than L-FABP appeared to be a sensitive and specific marker of AKI in liver transplant recipients.     

Crystal structure of the HNF4 alpha ligand binding domain in complex with endogenous fatty acid ligand

HNF4 alpha is an orphan member of the nuclear receptor family with prominent functions in liver, gut, kidney and pancreatic beta cells. We have solved the x-ray crystal structure of the HNF4 alpha ligand binding domain, which adopts a canonical fold. Two conformational states are present within each homodimer: an open form with alpha helix 12 (alpha 12) extended and collinear with alpha 10 and a closed form with alpha 12 folded against the body of the domain. Although the protein was crystallized without added ligands, the ligand binding pockets of both closed and open forms contain fatty acids. The carboxylic acid headgroup of the fatty acid ion pairs with the guanidinium group of Arg(226) at one end of the ligand binding pocket, while the aliphatic chain fills a long, narrow channel that is lined with hydrophobic residues. These findings suggest that fatty acids are endogenous ligands for HNF4 alpha and establish a framework for understanding how HNF4 alpha activity is enhanced by ligand binding and diminished by MODY1 mutations.     

Dehydroabietic acid, a phytochemical, acts as ligand for PPARs in macrophages and adipocytes to regulate inflammation

Obesity is characterized by an enhanced infiltration of macrophages to adipose tissues, which is closely associated with the low-grade inflammatory state and obesity-related pathologies such as type 2 diabetes and cardiovascular diseases. We showed here that dehydroabietic acid (DAA) is a potent PPARα/γ dual activator. Furthermore, we examined the anti-inflammatory effects of DAA in stimulated macrophages and in the coculture of macrophages and adipocytes. DAA significantly suppressed the production of proinflammatory mediators such as MCP-1, TNF-α, and NO in stimulated RAW 264 macrophages and in the coculture of RAW 264 macrophages and 3T3-L1 adipocytes. These results suggest that DAA is a valuable medicinal and food component for improving inflammatory changes associated with obesity-related diabetes.     

Antibacterial activity of rifamycins for M. smegmatis with comparison of oxidation and binding to tear lipocalin

A mutant of Mycobacterium smegmatis is a potential class I model substitute for Mycobacterium tuberculosis. Because not all of the rifamycins have been tested in this organism, we determined bactericidal profiles for the 6 major rifamycin derivatives. The profiles closely mirrored those established for M. tuberculosis. Rifalazil was confirmed to be the most potent rifamycin. Because the tuberculous granuloma presents a harshly oxidizing environment we explored the effects of oxidation on rifamycins. Mass spectrometry confirmed that three of the six major rifamycins showed autoxidation in the presence of trace metals. Oxidation could be monitored by distinctive changes including isosbestic points in the ultraviolet–visible spectrum. Oxidation of rifamycins abrogated anti-mycobacterial activity in M. smegmatis. Protection from autoxidation was conferred by binding susceptible rifamycins to tear lipocalin, a promiscuous lipophilic protein. Rifalazil was not susceptible to autoxidation but was insoluble in aqueous solution. Solubility was enhanced when complexed to tear lipocalin and was accompanied by a spectral red shift. The positive solvatochromism was consistent with robust molecular interaction and binding. Other rifamycins also formed a complex with lipocalin, albeit to a lesser extent. Protection from oxidation and enhancement of solubility with protein binding may have implications for delivery of select rifamycin derivatives.     

Amino acid sequence and some ligand binding properties of fatty acid-binding protein from bovine brain

Summary A fatty acid-binding protein (FABP) from the cytosol of bovine brain was purified by Sephadex G-75 filtration and electrofocusing. The purified protein migrated as a single protein band in 15% polyacrylamide gel electrophoresis with an apparent molecular mass of 14.7 kDa. To ascertain that the purified protein was a FABP, it was submitted to fatty acid-binding tests. Oleic and palmitic acids bound to brain FABP but this was not the case for palmitoyl CoA. By Scatchard analysis the ligand binding values were: Kd = 0.28 µM, Bmax (mol/mol) = 0.6 for oleic acid and Kd = 0.8 µM, Bmax (mol/mol) = 2.1 for palmitic acid. The complete amino acid sequence of the brain FABP was determined and a microheterogeneity was observed. Sequence comparison with other FABPs of known sequence and the observed microheterogeneity demonstrated the presence in brain of several homologous FABPs closely related to heart FABP.This paper corresponds to a communication at the first international workshop on fatty acid binding proteins (Maastricht, the Netherlands, September 4–5, 1989).     

Binding of the lipocalin C8gamma to human complement protein C8alpha is mediated by loops located at the entrance to the C8gamma ligand binding site

Human C8 is one of five complement components (C5b, C6, C7, C8 and C9) that interact to form the membrane attack complex (MAC). C8 is composed of a disulfide-linked C8alpha-gamma heterodimer and a noncovalently associated C8beta chain. C8alpha and C8beta are homologous to C6, C7 and C9, whereas C8gamma is the only lipocalin in the complement system. Lipocalins have a core beta-barrel structure forming a calyx with a binding site for a small molecule. In C8gamma, the calyx opening is surrounded by four loops that connect beta-strands. Loop 1 is the largest and contains Cys40 that links to Cys164 in C8alpha. To determine if these loops mediate binding of C8alpha prior to interchain disulfide bond formation in C8alpha-gamma, the loops were substituted separately and in combination for the corresponding loops in siderocalin (NGAL, Lcn2), a lipocalin that is structurally similar to C8gamma. The siderocalin-C8gamma chimeric constructs were expressed in E. coli, purified, and assayed for their ability to bind C8alpha. Results indicate at least three of the four loops surrounding the entrance to the C8gamma calyx are involved in binding C8alpha. Binding near the calyx entrance suggests C8alpha may restrict and possibly regulate access to the C8gamma ligand binding site.     

Temperature-induced conformational switch in intestinal fatty acid binding protein (IFABP) revealing an alternative mode for ligand binding

IFABP is a small beta-barrel protein with a short helix-turn-helix motif near the N-terminus that is thought to participate in the regulation of the uptake and delivery of fatty acids. In a previous work, we detected by near UV circular dichroism a reversible conformational transition of this protein occurring between 35 and 50 degrees C in the absence of fatty acids. The addition of the natural ligand oleic acid prevents this phenomenon. In both cases, the overall structure of the beta-barrel is maintained. This thermal transition is also detected by the fluorescent probe bis-anilino naphthalene sulfonic acid (bisANS) but not by its monomer ANS. In the present work, we studied in detail the interaction of each compound with IFABP as a function of temperature and in the absence or in the presence of oleic acid. A contrasting behavior was observed for these probes: (i) IFABP is able to bind two molecules of bisANS but only one molecule of ANS and (ii) oleic acid can fully displace ANS but only partially bisANS. Three independent lines of evidence, namely, fluorescence spectroscopy, circular dichroism, and limited proteolysis, indicate that there is an equilibrium among different conformations of IFABP, which differ in the extent of flexibility of the helical domain. This equilibrium can be shifted by raising temperature. bisANS is able to probe a population of IFABP in an altered state, which is more susceptible to cleavage by clostripain as compared to the apo-form, whereas the conformation of IFABP bound to oleic acid is characteristically more ordered. These results highlight the idea of an enhanced flexibility exhibited by IFABP that bears importance on its transport function, supporting the role of a dynamic entry portal region for the fatty acid ligand.     

Solution structure of human intestinal fatty acid binding protein: Implications for ligand entry and exit

The human intestinal fatty acid binding protein (I-FABP) is a small (131 amino acids) proteinwhich binds dietary long-chain fatty acids in the cytosol of enterocytes. Recently, an alanineto threonine substitution at position 54 in I-FABP has been identified which affects fatty acidbinding and transport, and is associated with the development of insulin resistance in severalpopulations including Mexican-Americans and Pima Indians. To investigate the molecularbasis of the binding properties of I-FABP, the 3D solution structure of the more commonform of human I-FABP (Ala54) was studied by multidimensional NMR spectroscopy.Recombinant I-FABP was expressed from E. coli in the presence and absence of 15N-enriched media. The sequential assignments for non-delipidated I-FABP were completed byusing 2D homonuclear spectra (COSY, TOCSY and NOESY) and 3D heteronuclear spectra(NOESY-HMQC and TOCSY-HMQC). The tertiary structure of human I-FABP wascalculated by using the distance geometry program DIANA based on 2519 distance constraintsobtained from the NMR data. Subsequent energy minimization was carried out by using theprogram SYBYL in the presence of distance constraints. The conformation of human I-FABPconsists of 10 antiparallel -strands which form two nearly orthogonal -sheets offive strands each, and two short -helices that connect the -strands A and B. Theinterior of the protein consists of a water-filled cavity between the two -sheets. TheNMR solution structure of human I-FABP is similar to the crystal structure of rat I-FABP.The NMR results show significant conformational variability of certain backbone segmentsaround the postulated portal region for the entry and exit of fatty acid ligand.     

Binding studies of tear lipocalin: the role of the conserved tryptophan in maintaining structure, stability and ligand affinity.

The principal lipid binding protein in tears, tear lipocalin (TL), binds acid and the fluorescent fatty acid analogs, DAUDA and 16-AP at one site TL compete for this binding site. A fluorescent competitive binding assay revealed that apo-TL has a high affinity for phospholipids and stearic acid (Ki) of 1.2 microM and 1.3 microM, respectively, and much less affinity for cholesterol (Ki) of 15.9 of the hydrocarbon chain. TL binds most strongly the least soluble lipids permitting these lipids to exceed their maximum solubility in aqueous solution. These data implicate TL in solubilizing and transporting lipids in the tear film. Phenylalanine, tyrosine and cysteine+ were substituted for TRP 17, the only invariant residue throughout the lipocalin superfamily. Cysteine substitution resulted in some loss os secondary structure, relaxation of aromatic side chain rigidity, decreased binding affinity for DAUDA and destabilization of structure. Mutants of TL, W17Y, and W17F showed a higher binding affinity for DAUDA than wild-type TL. Comparison of the results of the tryptophan 17 substitution in lipocalin with those of tryptophan 19 substitution in beta-lactoglobulin revealed important differences in binding characteristics that reflect the functional heterogeneity within the lipocalin family.