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.     

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.     

Evidence for internal and external binding sites on human tear lipocalin

8-Anilino-1-naphthalenesulfonic acid (ANS) is widely used as a probe for locating binding sites of proteins. To characterize the binding sites of tear lipocalin (TL), we studied ANS binding to apoTL by steady-state and time-resolved fluorescence. Deconvolution of ANS binding revealed that two lifetime components, 16.99 ns and 2.76 ns at pH 7.3, have dissociation constants of 0.58 μM and 5.7 μM, respectively. At pH 3.0, the lifetime components show decreased affinities with dissociation constants of 2.42 μM and ∼21 μM, respectively. Selective displacement of ANS molecules from the ANS-apoTL complex by stearic acid discriminates the internal and external binding sites. Dependence of the binding affinity on ionic strength under various conditions provides strong evidence that an electrostatic interaction is involved. Time-resolved fluorescence is a promising tool to segregate multiple binding sites of proteins.     

Hydrophobic ligand binding properties of the human lipocalin apolipoprotein M

Apolipoprotein M (apoM) is a plasma protein associated mainly with HDL. ApoM is suggested to be important for the formation of prebeta-HDL, but its mechanism of action is unknown. Homology modeling has suggested apoM to be a lipocalin. Lipocalins share a structurally conserved beta-barrel, which in many lipocalins bind hydrophobic ligands. The aim of this study was to test the ability of apoM to bind different hydrophobic substances. ApoM was produced both in Escherichia coli and in HEK 293 cells. Characterization of both variants with electrophoretic and immunological methods suggested apoM from E. coli to be correctly folded. Intrinsic tryptophan fluorescence of both apoM variants revealed that retinol, all-trans-retinoic acid, and 9-cis-retinoic acid bound (dissociation constant = 2-3 microM), whereas other tested substances (e.g., cholesterol, vitamin K, and arachidonic acid) did not. The intrinsic fluorescence of two apoM mutants carrying single tryptophans was quenched by retinol and retinoic acid to the same extent as wild-type apoM, indicating that the environment of both tryptophans was affected by the binding. In conclusion, the binding of retinol and retinoic acid supports the hypothesis that apoM is a lipocalin. The physiological relevance of this binding has yet to be elucidated.     

Resolution of ligand positions by site-directed tryptophan fluorescence in tear lipocalin

The lipocalin superfamily of proteins functions in the binding and transport of a variety of important hydrophobic molecules. Tear lipocalin is a promiscuous lipid binding member of the family and serves as a paradigm to study the molecular determinants of ligand binding. Conserved regions in the lipocalins, such as the G strand and the F-G loop, may play an important role in ligand binding and delivery. We studied structural changes in the G strand of holo- and apo-tear lipocalin using spectroscopic methods including circular dichroism analysis and site-directed tryptophan fluorescence. Apo-tear lipocalin shows the same general structural characteristics as holo-tear lipocalin including alternating periodicity of a beta-strand, orientation of amino acid residues 105, 103, 101, and 99 facing the cavity, and progressive depth in the cavity from residues 105 to 99. For amino acid residues facing the internal aspect of cavity, the presence of a ligand is associated with blue shifted spectra. The collisional rate constants indicate that these residues are not less exposed to solvent in holo-tear lipocalin than in apo-tear lipocalin. Rather the spectral blue shifts may be accounted for by a ligand induced rigidity in holo-TL. Amino acid residues 94 and 95 are consistent with positions in the F-G loop and show greater exposure to solvent in the holo- than the apo-proteins. These findings are consistent with the general hypothesis that the F-G loop in the holo-proteins of the lipocalin family is available for receptor interactions and delivery of ligands to specific targets. Site-directed tryptophan fluorescence was used in combination with a nitroxide spin labeled fatty acid analog to elucidate dynamic ligand interactions with specific amino acid residues. Collisional quenching constants of the nitroxide spin label provide evidence that at least three amino acids of the G strand residues interact with the ligand. Stern-Volmer plots are inconsistent with a ligand that is held in a static position in the calyx, but rather suggest that the ligand is in motion. The combination of site-directed tryptophan fluorescence with quenching by nitroxide labeled species has broad applicability in probing specific interactions in the solution structure of proteins and provides dynamic information that is not attainable by X-ray crystallography.     

Interstrand loops CD and EF act as pH-dependent gates to regulate fatty acid ligand binding in tear lipocalin

Tear lipocalin (TL), a major component of human tears, shows pH-dependent endogenous ligand binding. The structural and conformational changes associated with ligand release in the pH range of 7.5-3.0 are monitored by circular dichroism spectroscopy and site-directed tryptophan fluorescence. In the transition from pH 7.5 to pH 5.5, the ligand affinity for 16-(9-anthroyloxy)palmitic acid (16AP) and 8-anilino-1-naphthalenesulfonic acid is reduced. At pH 4.0 these ligands no longer bind within the TL calyx. From pH 7.3 to pH 3.0, the residues on loops CD and EF, which overhang the calyx entrance, show reduced accessibility to acrylamide. In addition resonance energy transfer is enhanced between residues on the two loops; the distance between the loops narrows. These findings suggest that apposition of the loops at low pH excludes the ligand from the intracavitary binding site. The conformational changes observed in transition from pH 7.3 to pH 3.0 for loops CD and EF are quite different. The CD loop shows less population reshuffling than the EF loop with an acidic environment, probably because backbone motion is restrained by the adjacent disulfide bond. The Trp fluorescence wavelength maximum (lambda(max)) reflects internal electrostatic interactions for positions on loops CD and EF. The titration curves of lambda(max) for mutants on the EF loop fit the Hendersen-Hasselbalch equation for two apparent pK(a) values, while the CD loop positions fit satisfactorily with one pK(a) value. Midpoints of transition for the binding affinity of TL tryptophan mutants to 16AP occur at pH 5.5-6.1. Replacement of each amino acid on either loop by single tryptophan mutation does not disrupt the pH-dependent binding affinity to 16AP. Taken together the data suggest that pH-driven ligand release involves ionization changes in several titratable residues associated with CD and EF loop apposition and occlusion of the calyx.     

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.     

Substrate binding and the catalytic reactions in cbb3-type oxidases: The lipid membrane modulates ligand binding

Heme–copper oxidases (HCuOs) are the terminal components of the respiratory chain in the mitochondrial membrane or the cell membrane in many bacteria. These enzymes reduce oxygen to water and use the free energy from this reaction to maintain a proton-motive force across the membrane in which they are embedded. The heme–copper oxidases of the cbb₃-type are only found in bacteria, often pathogenic ones since they have a low K_m for O₂, enabling the bacteria to colonize semi-anoxic environments. Cbb₃-type (C) oxidases are highly divergent from the mitochondrial-like aa₃-type (A) oxidases, and within the heme–copper oxidase family, cbb₃ is the closest relative to the most divergent member, the bacterial nitric oxide reductase (NOR). Nitric oxide reductases reduce NO to N₂O without coupling the reaction to the generation of any electrochemical proton gradient. The significant structural differences between A- and C-type heme–copper oxidases are manifested in the lack in cbb₃ of most of the amino acids found to be important for proton pumping in the A-type, as well as in the different binding characteristics of ligands such as CO, O₂ and NO. Investigations of the reasons for these differences at a molecular level have provided insights into the mechanism of O₂ and NO reduction as well as the proton-pumping mechanism in all heme–copper oxidases. In this paper, we discuss results from these studies with the focus on the relationship between proton transfer and ligand binding and reduction. In addition, we present new data, which show that CO binding to one of the c-type hemes of CcoP is modulated by protein–lipid interactions in the membrane. These results show that the heme c-CO binding can be used as a probe of protein–membrane interactions in cbb₃ oxidases, and possible physiological consequences for this behavior are discussed.     

Evolution rescues folding of human immunodeficiency virus-1 envelope glycoprotein GP120 lacking a conserved disulfide bond

The majority of eukaryotic secretory and membrane proteins contain disulfide bonds, which are strongly conserved within protein families because of their crucial role in folding or function. The exact role of these disulfide bonds during folding is unclear. Using virus-driven evolution we generated a viral glycoprotein variant, which is functional despite the lack of an absolutely conserved disulfide bond that links two antiparallel β-strands in a six-stranded β-barrel. Molecular dynamics simulations revealed that improved hydrogen bonding and side chain packing led to stabilization of the β-barrel fold, implying that β-sheet preference codirects glycoprotein folding in vivo. Our results show that the interactions between two β-strands that are important for the formation and/or integrity of the β-barrel can be supported by either a disulfide bond or β-sheet favoring residues.     

Structural and thermodynamic consequences of removal of a conserved disulfide bond from equine beta-lactoglobulin

A disulfide bond between cysteine 66 and cysteine 160 of equine beta-lactoglobulin was removed by substituting cysteine residues with alanine. This disulfide bond is conserved across the lipocalin family. The conformation and stability of the disulfide-deleted mutant protein was investigated by circular dichroism. The mutant protein assumes a native-like structure under physiological conditions and assumes a helix-rich molten globule structure at acid pH or at moderate concentrations of urea as the wild-type protein does. The urea-induced unfolding experiment shows that the stability of the native conformation was reduced but that of the molten globule intermediate is not significantly changed at pH 4 by removal of the disulfide bond. On the other hand, the molten globule at acid pH was destabilized by removal of the disulfide bond. This difference in the stabilizing effect of the disulfide bond was interpreted by the effect of the disulfide in keeping the molecule compact against the electrostatic repulsion at acid pH. In contrast to the wild-type protein, the circular dichroism spectrum in the molten globule state at acid pH depends on anion concentration, suggesting that the expansion of the molecule through electrostatic repulsion induces alpha-helices as observed in the cold denatured state of the wild-type protein.     

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.     

Role of an intrachain disulfide bond in the conformation and stability of ovalbumin

Ovalbumin, which contains one intrachain disulfide bond and four cysteine sulfhydryls, was reduced with dithiothreitol under non-denaturing conditions, and its conformation and stability were compared with those of the disulfide-bonded form. The CD spectrum in the far-UV region revealed that the overall conformation of the reduced form is similar to that of the disulfide-bonded one. Likewise, the inaccessibility to trypsin and the non-reactivity of the four cysteine sulfhydryls, exhibited by the native disulfide-bonded ovalbumin, were still retained in the disulfide-reduced form. Thus, the reduced ovalbumin appeared to substantially take the native-like conformation. However, the near-UV CD spectrum slightly differed between the native and disulfide-reduced forms. Protein alkylation with a fluorescent dye and subsequent sequence analysis showed that the two sulfhydryls (Cys73 and Cys120) originating from the disulfide bond are highly reactive in the reduced form. Furthermore, upon proteolysis with subtilisin, the N-terminal side of Cys73 was cleaved in the reduced form, but not in the disulfide-bonded one. Upon heat denaturation, the transition temperature of the reduced form was lower, by 6.8 degrees C, than that of the disulfide-bonded one. Thus, we concluded that ovalbumin has a native-like conformation in its disulfide-reduced form, but that the local conformation of the reduced form fluctuates more than that of the disulfide-bonded one. Such local destabilization may be related to the decreased stability against heat denaturation.     

Heme orientation modulates histidine dissociation and ligand binding kinetics in the hexacoordinated human neuroglobin

Neuroglobin (Ngb) is a globin present in the brain and retina of mammals. This hexacoordinated hemoprotein binds small diatomic molecules, albeit with lower affinity compared with other globins. Another distinctive feature of most mammalian Ngb is their ability to form an internal disulfide bridge that increases ligand affinity. As often seen for prosthetic heme b containing proteins, human Ngb exhibits heme heterogeneity with two alternative heme orientations within the heme pocket. To date, no details are available on the impact of heme orientation on the binding properties of human Ngb and its interplay with the cysteine oxidation state. In this work, we used ¹H NMR spectroscopy to probe the cyanide binding properties of different Ngb species in solution, including wild-type Ngb and the single (C120S) and triple (C46G/C55S/C120S) mutants. We demonstrate that in the disulfide-containing wild-type protein cyanide ligation is fivefold faster for one of the two heme orientations (the A isomer) compared with the other isomer, which is attributed to the lower stability of the distal His64–iron bond and reduced steric hindrance at the bottom of the cavity for heme sliding in the A conformer. We also attribute the slower cyanide reactivity in the absence of a disulfide bridge to the tighter histidine–iron bond. More generally, enhanced internal mobility in the CD loop bearing the disulfide bridge hinders access of the ligand to heme iron by stabilizing the histidine–iron bond. The functional impact of heme disorder and cysteine oxidation state on the properties of the Ngb ligand is discussed.     

The role of interchain disulfide bond in a recombinant human interleukin-17A variant

Interleukin-17A (IL-17A) is the prototype of IL-17 family and has been implicated in the pathogenesis of a variety of autoimmune diseases. Therefore its structural and functional properties are of great medical interest. During our research on a recombinant human IL-17A (rhIL-17A) variant, four isoforms were obtained when it was refolded. While isoforms 1 and 2 represented non-covalent dimers, isoforms 3 and 4 were determined to be covalent dimers. All four isoforms were structurally similar by Circular Dichroism and fluorescence spectroscopy studies, but differential scanning calorimetry demonstrated thermal stability in the order of isoform 1 = isoform 2 < isoform 4 < isoform 3. In addition, compared to covalent dimers (isoform 3 and 4), the non-covalent dimers (isoforms 1 and 2) are slightly less active in a receptor-binding assay but at least 5-fold less active in a cell-based assay.     

Allostery mediates ligand binding to Grb2 adaptor in a mutually exclusive manner

Allostery plays a key role in dictating the stoichiometry and thermodynamics of multi‐protein complexes driving a plethora of cellular processes central to health and disease. Herein, using various biophysical tools, we demonstrate that although Sos1 nucleotide exchange factor and Gab1 docking protein recognize two non‐overlapping sites within the Grb2 adaptor, allostery promotes the formation of two distinct pools of Grb2–Sos1 and Grb2–Gab1 binary signaling complexes in concert in lieu of a composite Sos1–Grb2–Gab1 ternary complex. Of particular interest is the observation that the binding of Sos1 to the nSH3 domain within Grb2 sterically blocks the binding of Gab1 to the cSH3 domain and vice versa in a mutually exclusive manner. Importantly, the formation of both the Grb2–Sos1 and Grb2–Gab1 binary complexes is governed by a stoichiometry of 2:1, whereby the respective SH3 domains within Grb2 homodimer bind to Sos1 and Gab1 via multivalent interactions. Collectively, our study sheds new light on the role of allostery in mediating cellular signaling machinery. Copyright © 2013 John Wiley & Sons, Ltd.     

The relative ligand binding preference of the murine ileal lipid binding protein

The ileal lipid binding protein (ILBP), a member of the intracellular lipid binding protein family, is a 14-kDa protein that has bile and fatty acids as possible physiological ligands. The ligand binding specificity of this protein is not well characterized. Therefore, we studied the lipid binding activity of purified recombinant murine ILBP (mILBP) in vitro. These studies demonstrated by direct analysis the interaction of mILBP with naturally occurring bile and fatty acids. The rank order of binding preference for fatty acids, or unconjugated and conjugated bile acids, was assessed. Among fatty acids, mILBP preferred species that had longer chain length and increased saturation, similar to other members of the intracellular lipid binding protein family. Among the bile acids, mILBP showed the greatest preference for conjugated species that contained a doubly hydroxylated steroid moiety. The results demonstrate that mILBP exhibits a preference for certain species of bile and fatty acids.     

Linkage between ligand binding and control of tubulin conformation.

The effect of both antimitotic drugs and nucleotide analogues on the magnesium-induced self-association of purified tubulin into 42S double rings has been examined by sedimentation velocity. In the absence of magnesium, all complexes sedimented as the 5.8S species. The binding of colchicine to tubulin led to a small but consistent (-0.1 to -0.2 kcal/mol) enhancement in the self-association of tubulin alpha-beta dimers. In the absence of nucleotide at the exchangeable site, tubulin retained a weak ability (K2 = 7.5 x 10(3) M-1) to self-associate, which was unchanged by the addition of guanosine or GMP. Analogues with altered P-O-P bonds (GMPPCP, GMPPNP) did not support ring formation at the protein concentrations examined, although GMPPCP supported microtubule assembly. When the exchangeable site was occupied by nucleotides altered on the gamma-phosphate (GTP gamma S, GTP gamma F), rings were formed; tubulin-GTP gamma F formed rings to an extent slightly greater than did tubulin-GTP, and tubulin-GTP gamma S to about the same extent as tubulin-GDP. Both of these analogues are inhibitors of microtubule assembly. These results are consistent with a model [Melki, R., Carlier, M.-F., Pantaloni, D., & Timasheff, S. N. (1989) Biochemistry 28, 9143-9152] in which an equilibrium exists between straight (microtubule-forming) and curved (ring-forming) conformations of tubulin. Furthermore, the present results indicate that the "switch" which controls the nature of the final polymeric product via free energy linkages is the occupancy of the gamma-phosphate binding locus of the exchangeable site by a properly coordinated metal-nucleotide complex.     

Prawn lipocalin: characterization of a color shift induced by gene knockdown and ligand binding assay

The lipocalin family of proteins functions in the transport of steroids, carotenoids, retinoids, and other small hydrophobic molecules. Recently, a lipocalin (MrLC) was isolated from the prawn Macrobrachium rosenbergii and its expression varied with the molting cycle. In this study, knockdown of the MrLC gene by RNA interference (RNAi) was performed and resulted in a shift in body color from blue to orangish red over the entire carapace. By immune-gold electron microscopy, MrLC was found to co-localize with the lipid droplets in subepidermal adiose tissue that were found to be decreased dramatically in MrLC knockdown prawns, in which a reduction in relative fat content was also quantified. Furthermore, MrLC was found to specifically bind astaxanthin and molt hormone (20-hydroxyecdysone) in both in vitro ligand binding assay and in vivo native ligand detection. These results suggested that MrLC plays roles in the regulation of coloration through its association with astaxanthin and may also be involved in the regulation of molting in crustacean.