Solution structure and backbone dynamics of human liver fatty acid binding protein: fatty acid binding revisited

Liver fatty acid binding protein (L-FABP), a cytosolic protein most abundant in liver, is associated with intracellular transport of fatty acids, nuclear signaling, and regulation of intracellular lipolysis. Among the members of the intracellular lipid binding protein family, L-FABP is of particular interest as it can i), bind two fatty acid molecules simultaneously and ii), accommodate a variety of bulkier physiological ligands such as bilirubin and fatty acyl CoA. To better understand the promiscuous binding and transport properties of L-FABP, we investigated structure and dynamics of human L-FABP with and without bound ligands by means of heteronuclear NMR. The overall conformation of human L-FABP shows the typical β-clam motif. Binding of two oleic acid (OA) molecules does not alter the protein conformation substantially, but perturbs the chemical shift of certain backbone and side-chain protons that are involved in OA binding according to the structure of the human L-FABP/OA complex. Comparison of the human apo and holo L-FABP structures revealed no evidence for an "open-cap" conformation or a "swivel-back" mechanism of the K90 side chain upon ligand binding, as proposed for rat L-FABP. Instead, we postulate that the lipid binding process in L-FABP is associated with backbone dynamics.     

Catalytic activities of Werner protein are affected by adduction with 4-hydroxy-2-nonenal

4-Hydroxy-2-nonenal (HNE) is a reactive α,β-unsaturated aldehyde generated during oxidative stress and subsequent peroxidation of polyunsaturated fatty acids. Here, Werner protein (WRN) was identified as a novel target for modification by HNE. Werner syndrome arises through mutations in the WRN gene that encodes the RecQ DNA helicase which is critical for maintaining genomic stability. This hereditary disease is associated with chromosomal instability, premature aging and cancer predisposition. WRN appears to participate in the cellular response to oxidative stress and cells devoid of WRN display elevated levels of oxidative DNA damage. We demonstrated that helicase/ATPase and exonuclease activities of HNE-modified WRN protein were inhibited both in vitro and in immunocomplexes purified from the cell extracts. Sites of HNE adduction in human WRN were identified at Lys577, Cys727, His1290, Cys1367, Lys1371 and Lys1389. We applied in silico modeling of the helicase and RQC domains of WRN protein with HNE adducted to Lys577 and Cys727 and provided a potential mechanism of the observed deregulation of the protein catalytic activities. In light of the obtained results, we postulate that HNE adduction to WRN is a post-translational modification, which may affect WRN conformational stability and function, contributing to features and diseases associated with premature senescence.     

Modification of Akt2 by 4-hydroxynonenal inhibits insulin-dependent Akt signaling in HepG2 cells

The production of reactive aldehydes such as 4-hydroxy-2-nonenal (4-HNE) is a key component of the pathogenesis in a spectrum of hepatic diseases involving oxidative stress such as alcoholic liver disease (ALD). One consequence of ALD is increased insulin resistance in hepatocytes. To understand the effects of 4-HNE on insulin signaling in liver cells, we employed a model using hepatocellular carcinoma cell line HepG2. Previously, we have demonstrated an increase in the level of Akt phosphorylation is mediated by 4-HNE inhibition of PTEN, a direct regulator of Akt. In this work, we evaluated the effects of 4-HNE on insulin-dependent stimulation of the Akt2 pathway. We demonstrate that 4-HNE selectively leads to phosphorylation of Akt2. Although Akt2 is phosphorylated following 4-HNE treatment, the level of downstream phosphorylation of Akt substrates such as GSK3β and MDM2 is significantly decreased. Pretreatment with 4-HNE prevented insulin-dependent Akt signaling and decreased intracellular Akt activity by 87%. Using biotin hydrazide capture, it was confirmed that 4-HNE treatment of cells resulted in carbonylation of Akt2, which was not observed in untreated control cells. Using a synthetic GSK3α/β peptide as a substrate, treatment of recombinant human myristoylated Akt2 (rAkt2) with 20 or 40 μM 4-HNE inhibited rAkt2 activity by 30 or 85%, respectively. Matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF) identified Michael addition adducts of 4-HNE with His196, His267, and Cys311 of rAkt2. Computation-based molecular modeling analysis of 4-HNE adducted to His196 and Cys311 of Akt2 suggests inhibition of GSK3β peptide binding by 4-HNE in the Akt2 substrate binding pocket. The inhibition of Akt by 4-HNE provides a novel mechanism for increased insulin resistance in ALD. These data provide a potential mechanism of dysregulation of Akt2 during events associated with sustained hepatocellular oxidative stress.     

Posttranslational modification and regulation of glutamate-cysteine ligase by the α,β-unsaturated aldehyde 4-hydroxy-2-nonenal

4-Hydroxy-2-nonenal (4-HNE) is a lipid peroxidation product formed during oxidative stress that can alter protein function via adduction of nucleophilic amino acid residues. 4-HNE detoxification occurs mainly via glutathione (GSH) conjugation and transporter-mediated efflux. This results in a net loss of cellular GSH, and restoration of GSH homeostasis requires de novo GSH biosynthesis. The rate-limiting step in GSH biosynthesis is catalyzed by glutamate-cysteine ligase (GCL), a heterodimeric holoenzyme composed of a catalytic (GCLC) and a modulatory (GCLM) subunit. The relative levels of the GCL subunits are a major determinant of cellular GSH biosynthetic capacity and 4-HNE induces the expression of both GCL subunits. In this study, we demonstrate that 4-HNE can alter GCL holoenzyme formation and activity via direct posttranslational modification of the GCL subunits in vitro. 4-HNE directly modified Cys553 of GCLC and Cys35 of GCLM in vitro, which significantly increased monomeric GCLC enzymatic activity, but reduced GCL holoenzyme activity and formation of the GCL holoenzyme complex. In silico molecular modeling studies also indicate these residues are likely to be functionally relevant. Within a cellular context, this novel posttranslational regulation of GCL activity could significantly affect cellular GSH homeostasis and GSH-dependent detoxification during periods of oxidative stress.     

Conformational and dynamics changes induced by bile acids binding to chicken liver bile acid binding protein

The correlation between protein motions and function is a central problem in protein science. Several studies have demonstrated that ligand binding and protein dynamics are strongly correlated in intracellular lipid binding proteins (iLBPs), in which the high degree of flexibility, principally occurring at the level of helix-II, CD, and EF loops (the so-called portal area), is significantly reduced upon ligand binding. We have recently investigated by NMR the dynamic properties of a member of the iLBP family, chicken liver bile acid binding protein (cL-BABP), in its apo and holo form, as a complex with two bile salts molecules. Binding was found to be regulated by a dynamic process and a conformational rearrangement was associated with this event. We report here the results of molecular dynamics (MD) simulations performed on apo and holo cL-BABP with the aim of further characterizing the protein regions involved in motion propagation and of evaluating the main molecular interactions stabilizing bound ligands. Upon binding, the root mean square fluctuation values substantially decrease for CD and EF loops while increase for the helix-loop-helix region, thus indicating that the portal area is the region mostly affected by complex formation. These results nicely correlate with backbone dynamics data derived from NMR experiments. Essential dynamics analysis of the MD trajectories indicates that the major concerted motions involve the three contiguous structural elements of the portal area, which however are dynamically coupled in different ways whether in the presence or in the absence of the ligands. Motions of the EF loop and of the helical region are part of the essential space of both apo and holo-BABP and sample a much wider conformational space in the apo form. Together with NMR results, these data support the view that, in the apo protein, the flexible EF loop visits many conformational states including those typical of the holo state and that the ligand acts stabilizing one of these pre-existing conformations. The present results, in agreement with data reported for other iLBPs, sharpen our knowledge on the binding mechanism for this protein family.     

Dynamics of cellular retinoic acid binding protein I on multiple time scales with implications for ligand binding

Cellular retinoic acid binding protein I (CRABPI) belongs to the family of intracellular lipid binding proteins (iLBPs), all of which bind a hydrophobic ligand within an internal cavity. The structures of several iLBPs reveal minimal structural differences between the apo (ligand-free) and holo (ligand-bound) forms, suggesting that dynamics must play an important role in the ligand recognition and binding processes. Here, a variety of nuclear magnetic resonance (NMR) spectroscopy methods were used to systematically study the dynamics of both apo and holo CRABPI at various time scales. Translational and rotational diffusion constant measurements were used to study the overall motions of the proteins. Both apo and holo forms of CRABPI tend to self-associate at high (1.2 mM) concentrations, while at low concentrations (0.2 mM), they are predominantly monomeric. Rapid amide exchange rate and laboratory frame relaxation rate measurements at two spectrometer field strengths (500 and 600 MHz) were used to probe the internal motions of the individual residues. Several residues in the apo form, notably within the ligand recognition region, exhibit millisecond time scale motions that are significantly arrested in the holo form. In contrast, no significant differences in the high-frequency motions were observed between the two forms. These results provide direct experimental evidence for dynamics-induced ligand recognition and binding at a specifically defined time scale. They also exemplify the importance of dynamics in providing a more comprehensive understanding of how a protein functions.     

Glutathione-protein mixed disulfide decreases the affinity of rat liver fatty acid-binding protein for unsaturated fatty acid

.16 +/- 0.062% of the fatty acid-binding protein purified from 50 mM N-ethylmaleimide-treated rat liver (L-FABP) was determined as a form S-thiolated by glutathione (L-FABP-SSG). L-FABP-SSG, which was prepared in vitro through thiol-disulfide exchange reaction, showed more acidic pI (approximately 5.0) than the pI (approximately 7.0) of reduced L-FABP. S-thiolation of L-FABP by glutathione decreased the affinity of the protein for unsaturated fatty acids without changing the equimolar maximum binding. The changes in Kd were from 0.63 +/- 0.054 microM to 1.03 +/- 0.14 microM for oleic acid, from 0.63 +/- 0.028 microM to 0.97 +/- 0.12 microM for linoleic acid and from 0.85 +/- 0.050 microM to 1.45 +/- 0.024 microM for arachidonic acid. This modification did not alter the affinity nor the maximum binding for saturated fatty acids, which were determined to be Kd of approximately 1.0 microM for palmitic acid and approximately 0.9 microM for stearic acids, and equimolar maximum binding for both fatty acids. The binding affinity of L-FABP for unsaturated fatty acid may be regulated by redox state of the liver.     

Analysis of naphthalene adduct binding sites in model proteins by tandem mass spectrometry

The electrophilic metabolites of the polyaromatic hydrocarbon naphthalene have been shown to bind covalently to proteins and covalent adduct formation correlates with the cytotoxic effects of the chemical in the respiratory system. Although 1,2-naphthalene epoxide, naphthalene diol epoxide, 1,2-naphthoquinone, and 1,4-napthoquinone have been identified as reactive metabolites of interest, the role of each metabolite in total covalent protein adduction and subsequent cytotoxicity remains to be established. To better understand the target residues associated with the reaction of these metabolites with proteins, mass spectrometry was used to identify adducted residues following (1) incubation of metabolites with actin and protein disulfide isomerase (PDI), and (2) activation of naphthalene in microsomal incubations containing supplemental actin or PDI. All four reactive metabolites bound to Cys, Lys or His residues in actin and PDI. Cys(17) of actin was the only residue adducted by all metabolites; there was substantial metabolite selectivity for the majority of adducted residues. Modifications of actin and PDI, following microsomal incubations containing (14)C-naphthalene, were detected readily by 2D gel electrophoresis and phosphor imaging. However, target modifications on tryptic peptides from these isolated proteins could not be readily detected by MALDI/TOF/TOF and only three modified peptides were detected using high resolution-selective ion monitoring (HR-SIM). All the reactive metabolites investigated have the potential to modify several residues in a single protein, but even in tissues with very high rates of naphthalene activation, the extent of modification was too low to allow unambiguous identification of a significant number of modified residues in the isolated proteins.     

Protein function annotation by local binding site surface similarity

Hundreds of protein crystal structures exist for proteins whose function cannot be confidently determined from sequence similarity. Surflex‐PSIM, a previously reported surface‐based protein similarity algorithm, provides an alternative method for hypothesizing function for such proteins. The method now supports fully automatic binding site detection and is fast enough to screen comprehensive databases of protein binding sites. The binding site detection methodology was validated on apo/holo cognate protein pairs, correctly identifying 91% of ligand binding sites in holo structures and 88% in apo structures where corresponding sites existed. For correctly detected apo binding sites, the cognate holo site was the most similar binding site 87% of the time. PSIM was used to screen a set of proteins that had poorly characterized functions at the time of crystallization, but were later biochemically annotated. Using a fully automated protocol, this set of 8 proteins was screened against ～60,000 ligand binding sites from the PDB. PSIM correctly identified functional matches that predated query protein biochemical annotation for five out of the eight query proteins. A panel of 12 currently unannotated proteins was also screened, resulting in a large number of statistically significant binding site matches, some of which suggest likely functions for the podorly characterized proteins. Proteins 2014; 82:679–694. © 2013 Wiley Periodicals, Inc.     

Solution structure, dynamics, and hydrodynamics of the calcium-bound cross-reactive birch pollen allergen Bet v 4 reveal a canonical monomeric two EF-hand assembly with a regulatory function

Birch pollinosis is one of the prevailing allergic diseases. In all, 5-20% of birch pollinotics mount IgE antibodies against the minor birch pollen allergen Bet v 4, a Ca2+-binding polcalcin. Due to IgE cross-reactivity among the polcalcins these patients are polysensitized to various plant pollens. Determination of the high-resolution structure of holo Bet v 4 by heteronuclear NMR spectroscopy reveals a canonical two EF-hand assembly in the open conformation with interhelical angles closely resembling holo calmodulin. The polcalcin-specific amphipathic COOH-terminal alpha-helix covers only a part of the hydrophobic groove on the molecular surface. Unlike the polcalcin Phl p 7 from timothy grass, which was recently shown to form a domain-swapped dimer, the hydrodynamic parameters from NMR relaxation, NMR translational diffusion, and analytical ultracentrifugation indicate that both apo and holo Bet v 4 are predominantly monomeric, raising the question of the physiological and immunological significance of the dimeric form of these polcalcins, whose physiological function is still unknown. The reduced helicity and heat stability in the CD spectra, the poor chemical shift dispersion of the NMR spectra, and the slightly increased hydrodynamic radius of apo Bet v 4 indicate a reversible structural transition upon Ca2+ binding, which explains the reduced IgE binding capacity of apo Bet v 4. The remarkable structural similarity of holo Bet v 4 and holo Phl p 7 in spite of different oligomerization states explains the IgE cross-reactivity and indicates that canonical monomers and domain-swapped dimers may be of similar allergenicity. Together with the close structural homology to calmodulin and the hydrophobic ligand binding groove this transition suggests a regulatory function for Bet v 4.     

Selective binding of cholesterol by recombinant fatty acid binding proteins

The sterol binding specificity of rat recombinant liver fatty acid binding protein (L-FABP) and intestinal fatty acid binding protein (I-FABP) was characterized with [3H]cholesterol and a fluorescent sterol analog dehydroergosterol. Ligand binding analysis, fluorescence spectroscopy, and activation of microsomal acyl-CoA:cholesterol acyltransferase activity showed that L-FABP-bound sterols. 1) Lipidex-1000 assay showed a dissociation constant Kd = 0.78 +/- 0.18 microM and stoichiometry of 0.47 +/- 0.16 mol/mol for [3H]cholesterol binding to L-PABP. 2) With [3H]cholesterol/phosphatidylcholine liposomes, the cholesterol binding parameters for L-FABP were Kd = 1.53 +/- 0.28 microM and stoichiometry 0.83 +/- 0.07 mol/mol. 3) L-FABP interaction with dehydroergosterol altered the fluorescence intensity and polarization of dehydroergosterol. Dehydroergosterol bound to L-FABP with Kd = 0.37 microM and a stoichiometry of 0.83 mol/mol. 4) Cholesterol and dehydroergosterol decreased L-FABP tyrosine lifetime. Dehydroergosterol binding produced sensitized emission of bound dehydroergosterol with longer lifetime.5) L-FABP bound two cis-parinaric acid molecules/molecule of protein. Cholesterol displaced one of these bound cis-parinaric acids. 6) L-FABP enhanced acyl-CoA:cholesterol acyltransferase in a concentration-dependent manner. In contrast, these assays indicated that I-FABP did not bind sterols. Thus, L-FABP appears able to bind 1 mol of cholesterol/mol of L-FABP, the L-FABP sterol binding site is equivalent to one of the two fatty acid binding sites, and L-FABP stimulates acyl-CoA:cholesterol acyltransferase by transfer of cholesterol.     

Oxidative stress-mediated aldehyde adduction of GRP78 in a mouse model of alcoholic liver disease: functional independence of ATPase activity and chaperone function

Pathogenesis in alcoholic liver disease (ALD) is complicated and multifactorial but clearly involves oxidative stress and inflammation. Currently, conflicting reports exist regarding the role of endoplasmic reticulum (ER) stress in the etiology of ALD. The glucose-regulated protein 78 (GRP78) is the ER homolog of HSP70 and plays a critical role in the cellular response to ER stress by serving as a chaperone assisting protein folding and by regulating the signaling of the unfolded protein response (UPR). Comprising three functional domains, an ATPase, a peptide-binding, and a lid domain, GRP78 folds nascent polypeptides via the substrate-binding domain. Earlier work has indicated that the ATPase function of GRP78 is intrinsically linked and essential to its chaperone activity. Previous work in our laboratory has indicated that GRP78 and the UPR are not induced in a mouse model of ALD but that GRP78 is adducted by the lipid electrophiles 4-hydroxynonenal (4-HNE) and 4-oxononenal (4-ONE) in vivo. As impairment of GRP78 has the potential to contribute to pathogenesis in ALD, we investigated the functional consequences of aldehyde adduction on GRP78 function. Identification of 4-HNE and 4-ONE target residues in purified human GRP78 revealed a marked propensity for Lys and His adduction within the ATPase domain and a relative paucity of adduct formation within the peptide-binding domain. Consistent with these findings, we observed a concomitant dose-dependent decrease in ATP-binding and ATPase activity without any discernible impairment of chaperone function. Collectively, our data indicate that ATPase activity is not essential for GRP78-mediated chaperone activity and is consistent with the hypothesis that ER stress does not play a primary initiating role in the early stages of ALD.     

4-Hydroxynonenal regulates 26S proteasomal degradation of alcohol dehydrogenase

The lipid peroxidation product 4-hydroxynonenal (4-HNE) has been shown to interfere with protein function. The goal of this study was to determine the effects of substrate modification by 4-HNE on protein degradation. Equine liver alcohol dehydrogenase (ADH, EC 1.1.1.1) treated with 2-fold molar excess 4-HNE was degraded by a rabbit reticulocyte lysate (RRL) system approximately 1.5-fold faster than control, while treatment with concentrations up to 100-fold molar excess aldehyde were inhibitory to degradation. Involvement of the 26S proteasome (EC 3.4.99.46) was demonstrated through the use of specific proteasome and ATPase inhibitors, and confirmed by measuring the extent of ADH polyubiquitination. Tryptic digestion and LC/MS analysis of 4-HNE-treated ADH identified modification of two zinc chelating Cys residues. Through molecular modeling experiments a conformational shift in both zinc-containing regions was predicted, with an approximate doubling of the distance between the structural zinc and its respective chelating residues. Modification of residues in the active site zinc binding motif resulted in less pronounced alteration in protein structure. The data presented here demonstrate accelerated ubiquitination and proteasomal degradation of ADH modified with 4-HNE, and suggest a conformational change after 4-HNE docking as a mechanism behind these observations.     

4-Hydroxynonenal induces Cx46 hemichannel inhibition through its carbonylation

Hemichannels formed by connexins mediate the exchange of ions and signaling molecules between the cytoplasm and the extracellular milieu. Under physiological conditions hemichannels have a low open probability, but in certain pathologies their open probability increases, which can result in cell damage. Pathological conditions are characterized by the production of a number of proinflammatory molecules, including 4-hydroxynonenal (4-HNE), one of the most common lipid peroxides produced in response to inflammation and oxidative stress. The aim of this work was to evaluate whether 4-HNE modulates the activity of Cx46 hemichannels. We found that 4-HNE (100 μM) reduced the rate of 4′,6-diamino-2-fenilindol (DAPI) uptake through hemichannels formed by recombinant human Cx46 fused to green fluorescent protein, an inhibition that was reversed partially by 10 mM dithiothreitol. Immunoblot analysis showed that the recombinant Cx46 expressed in HeLa cells becomes carbonylated after exposure to 4-HNE, and that 10 mM dithiothreitol reduced its carbonylation. We also found that Cx46 was carbonylated by 4-HNE in the lens of a selenite-induced cataract animal model. The exposure to 100 μM 4-HNE decreased hemichannel currents formed by recombinant rat Cx46 in Xenopus laevis oocytes. This inhibition also occurred in a mutant expressing only the extracellular loop cysteines, suggesting that other Cys are not responsible for the hemichannel inhibition by carbonylation. This work demonstrates for the first time that Cx46 is post-translationally modified by a lipid peroxide and that this modification reduces Cx46 hemichannel activity.     

Crystallographic and biochemical analyses of the metal-free Haemophilus influenzae Fe3+-binding protein.

The crystal structure of the iron-free (apo) form of the Haemophilus influenzae Fe(3+)-binding protein (hFbp) has been determined to 1.75 A resolution. Information from this structure complements that derived from the holo structure with respect to the delineation of the process of iron binding and release. A 21 degrees rotation separates the two structural domains when the apo form is compared with the holo conformer, indicating that upon release of iron, the protein undergoes a change in conformation by bending about the central beta-sheet hinge. A surprising finding in the apo-hFbp structure was that the ternary binding site anion, observed in the crystals as phosphate, remained bound. In solution, apo-hFbp bound phosphate with an affinity K(d) of 2.3 x 10(-3) M. The presence of this ternary binding site anion appears to arrange the C-terminal iron-binding residues conducive to complementary binding to Fe(3+), while residues in the N-terminal binding domain must undergo induced fit to accommodate the Fe(3+) ligand. These observations suggest a binding process, the first step of which is the binding of a synergistic anion such as phosphate to the C-terminal domain. Next, iron binds to the preordered half-site on the C-terminal domain. Finally, the presence of iron organizes the N-terminal half-site and closes the interdomain hinge. The use of the synergistic anion and this iron binding process results in an extremely high affinity of the Fe(3+)-binding proteins for Fe(3+) (nFbp K'(eff) = 2.4 x 10(18) M(-1)). This high-affinity ligand binding process is unique among the family of bacterial periplasmic binding proteins and has interesting implications in the mechanism of iron removal from the Fe(3+)-binding proteins during FbpABC-mediated iron transport across the cytoplasmic membrane.     

Conformational Transitions upon Ligand Binding: Holo-Structure Prediction from Apo Conformations

Biological function of proteins is frequently associated with the formation of complexes with small-molecule ligands. Experimental structure determination of such complexes at atomic resolution, however, can be time-consuming and costly. Computational methods for structure prediction of protein/ligand complexes, particularly docking, are as yet restricted by their limited consideration of receptor flexibility, rendering them not applicable for predicting protein/ligand complexes if large conformational changes of the receptor upon ligand binding are involved. Accurate receptor models in the ligand-bound state (holo structures), however, are a prerequisite for successful structure-based drug design. Hence, if only an unbound (apo) structure is available distinct from the ligand-bound conformation, structure-based drug design is severely limited. We present a method to predict the structure of protein/ligand complexes based solely on the apo structure, the ligand and the radius of gyration of the holo structure. The method is applied to ten cases in which proteins undergo structural rearrangements of up to 7.1 Å backbone RMSD upon ligand binding. In all cases, receptor models within 1.6 Å backbone RMSD to the target were predicted and close-to-native ligand binding poses were obtained for 8 of 10 cases in the top-ranked complex models. A protocol is presented that is expected to enable structure modeling of protein/ligand complexes and structure-based drug design for cases where crystal structures of ligand-bound conformations are not available.     

Proposed mechanisms for binding of apo[a] kringle type 9 to apo B-100 in human lipoprotein[a].

The protein component of human lipoprotein[a] consists primarily of two apolipoproteins, apo[a] and apo B-100, linked through a cystine disulfide(s). In the amino acid sequence of apo bd, Cys4057 located within a plasminogen kringle 4-like repeat sequence (3991-4068) is believed to form a disulfide bond with a specific cysteine residue in apo B-100. Our fluorescence-labeling experiments and molecular modeling studies have provided evidence for possible interactions between this apo[a] kringle type and apo B-100. The fluorescent probe, fluorescein-5-maleimide, was used in parallel experiments to label free sulfhydryl moieties in lipoprotein[a] and low-density lipoprotein (LDL). In apo B-100 of LDL, Cys3734 was labeled with the probe, but this site was not labeled in autologous lipoprotein[a]. The result strongly implicates Cys3734 of apo B-100 as the residue forming the disulfide linkage with Cys4057 of apo[a]. To explore possible noncovalent interactions between apo B-100 and apo[a], the crystallographic coordinates for plasminogen kringle 4 were used to generate molecular models of the apo[a] kringle-repeat sequence (3991-4068, LPaK9), the only plasminogen kringle 4 type repeat in apo[a] having an extra cysteine residue not involved in an intramolecular disulfide bond. The Cys4057 residue (henceforth designated as Cys67 in the LPaK9 sequence) is believed to form an intermolecular disulfide bond with a cysteine of apo B-100. In computer graphics molecular models of LPaK9, Cys67 is located on the surface of the kringle near the lysine ligand binding site. Selected segments of the LDL apo B-100 sequence that contain free sulfhydryl cysteines were subjected to energy minimization and docking with the ligand binding site and adjacent regions of the LPaK9 model. In the docking experiments, apo B-100 segment 3732-3745 (PSCKLDFREIQIYK) displayed the best fit and the largest number of van der Waals contacts with models of LPaK9. Other apo B-100 peptides with sulfhydryl cysteine were found to be less compatible when minimized with this kringle. These results support and extend previously suggested mechanisms for a complex interaction between apo[a] and apo B-100 that involve more than a simple covalent disulfide bond.     

Effects of heme on the structure of the denatured state and folding kinetics of cytochrome b562

Heme-linked proteins, such as cytochromes, are popular subjects for protein folding studies. There is the underlying question of whether the heme affects the structure of the denatured state by cross-linking it and forming other interactions, which would perturb the folding pathway. We have studied wild-type and mutant cytochrome b562 from Escherichia coli, a 106 residue four-alpha-helical bundle. The holo protein apparently refolds with a half-life of 4 micros in its ferrous state. We have analysed the folding of the apo protein using continuous-flow fluorescence as well as stopped-flow fluorescence and CD. The apo protein folded much more slowly with a half-life of 270 micros that was unaffected by the presence of exogenous heme. We examined the nature of the denatured states of both holo and apo proteins by NMR methods over a range of concentrations of guanidine hydrochloride. The starting point for folding of the holo protein in concentrations of denaturant around the denaturation transition was a highly ordered native-like species with heme bound. Fully denatured holo protein at higher concentrations of denaturant consisted of denatured apo protein and free heme. Our results suggest that the very fast folding species of denatured holo protein is in a compact state, whereas the normal folding pathway from fully denatured holo protein consists of the slower folding of the apo protein followed by the binding of heme. These data should be considered in the analysis of folding of heme proteins.     

Determination of apo and holo retinoic acid-binding protein levels in retinoid-responsive transformed cells by high-performance size-exclusion chromatography

A method to measure the endogenous levels of apo and holo cellular retinoic acid-binding proteins was developed using calf testis cytosol as the source of retinoic acid-binding protein. [3H]Retinoic acid-retinoic acid-binding protein complexes were assayed by high-performance size-exclusion chromatography. Preincubation of cytosol with 10 mM p-hydroxymercuribenzoate at 4 degrees C resulted in complete inhibition of retinoic acid binding to apo retinoic acid-binding protein. In addition, total dissociation of preformed holo retinoic acid-binding protein complexes was noted within 20 min after mercurial addition. Thus, p-hydroxymercuribenzoate converted the total pool of cellular retinoic acid-binding protein (apo plus holo) to mercurial-protein complexes unable to bind retinoic acid in vitro. Mercurial inhibition of retinoic acid-retinoic acid-binding protein complex formation was totally reversed upon the addition of 50 mM dithiothreitol. Total cytosolic retinoic acid-binding protein was determined from specific retinoic acid binding after treatment with p-hydroxymercuribenzoate and dithiothreitol. Apo cellular retinoic acid-binding protein concentration was measured by determining specific radioligand binding prior to p-hydroxymercuribenzoate treatment, and correcting for exchange of endogenously bound retinoid with exogenous tritiated retinoic acid. Holo cellular retinoic acid-binding protein concentration was derived from the difference between total and apo retinoic acid-binding protein concentrations. Using this method, we have demonstrated that retinoid-responsive EJ and T24 human bladder carcinoma cell lines and AT3A and AT3B rat pancreatic acinar carcinoma cell lines lack detectable levels of either apo or holo cellular retinoic acid-binding protein. These results established that retinoid inhibition of transformed bladder and acinar cell proliferation in culture was mediated by a cellular retinoic acid-binding protein-independent mechanism.     

Ligand-induced conformational change of a protein reproduced by a linear combination of displacement vectors obtained from normal mode analysis

The conformational change of a protein upon ligand binding was examined by normal mode analysis (NMA) based on an elastic-network model (ENM) for a full-atom system using dihedral angles as independent variables. Specifically, we investigated the extent to which conformational change vectors of atoms from an apo form to a holo form of a protein can be represented by a linear combination of the displacement vectors of atoms in the apo form calculated for the lowest-frequency m normal modes (m=1, 2,…, 20). In this analysis, the latter vectors were best fitted to the former ones by the least-squares method. Twenty-two paired proteins in the holo and apo forms, including three dimer pairs, were examined. The results showed that, in most cases, the conformational change vectors were reproduced well by a linear combination of the displacement vectors of a small number of low-frequency normal modes. The conformational change around an active site was reproduced as well as the entire conformational change, except for some proteins that only undergo significant conformational changes around active sites. The weighting factors for 20 normal modes optimized by the least-squares fitting characterize the conformational changes upon ligand binding for these proteins. The conformational changes sampled around the apo form of a protein by the linear combination of the displacement vectors obtained by ENM-based NMA may help solve the flexible-docking problem of a protein with another molecule because the results presented herein suggest that they have a relatively high probability of being involved in an actual conformational change.