Dynamics of internal water in fatty acid binding protein: computer simulations and comparison with experiments

Multiple molecular dynamics (MD) simulations of fully solvated rat intestinal fatty acid binding protein (I-FABP) were conducted to investigate the dynamics of internal water molecules. Although the long time average of the number of internal water molecules in I-FABP is 22 as shown by the X-ray crystal structure, MD simulations predict large variations in the instantaneous number of internal water molecules on the nanosecond time scale. The computational model employed predicts that w135 (internal) and w217 (located on the protein surface) may be the water molecules with long residence times observed in previously reported magnetic relaxation dispersion studies. The average residence time of approximately 20 internal water molecules occupying the fatty acid binding cavity is estimated to be between 0.6 and 2.0 nanoseconds. Exchange of internal water in I-FABP appears to occur almost exclusively through the interface of beta-strands EF with the rest of the protein, which has significant implications for the pathways of the fatty acid entry and exit from the binding cavity. Proteins 2001;43:65-72.     

Refinement of the structure of Escherichia coli-derived rat intestinal fatty acid binding protein with bound oleate to 1.75-A resolution. Correlation with the structures of the apoprotein and the protein with bound palmitate.

The structure of rat intestinal fatty acid binding protein (I-FABP) with bound oleate (C18:1) has been refined with x-ray diffraction data to a resolution of 1.75 A. The protein contains 10 anti-parallel beta strands composed of 99 residues and 2 short helices of 14 residues. Oleate is located in the interior of the protein in a bent conformation with C1-C12 more ordered than C13-C18. Two of the eight ordered waters in I-FABP:oleate are part of a hydrogen bond network that includes the carboxylate of oleate, the guanidinium group of Arg106, the nitrogen of the indole group of Trp82, and the side chain of Gln115. Most of the methylenes of bound oleate reside in a crevice formed by hydrophobic and aromatic side chains. Tyr70 and Tyr117 envelop the acyl chain from C3 to C8 forming contacts with both the convex and concave faces of its van der Waals surface. The hydroxyls of each phenolic side chain hydrogen bond to ordered water molecules. Two ordered waters make van der Waals contact with the concave face of the bound fatty acid. The omega-terminal methyl of oleate is oriented so that it points toward the center of the benzene of Phe55 allowing it to form van der Waals interactions with its component methylenes. Comparison of the structure of I-FABP:oleate with a recently refined 1.19-A model of apoI-FABP and an earlier 2.0-A model of I-FABP:palmitate revealed a remarkable degree of similarity in the positions of their main chain and side chain atoms and in the conformations of the bound oleate and palmitate. The principal differences were confined to a few discrete regions of the protein. The helical domain, the type I turn between beta strands C and D, and the ring of Phe55 together form a solvent-accessible portal to the interior of the protein. They are repositioned in I-FABP:oleate (and I-FABP:palmitate) so that the binding cavity is even more accessible to solvent and its volume is increased. The side chain of Phe55 which shows discrete disorder in the apoprotein functions as an omega-terminal "sensing device": moving progressively outward toward the surface as the chain length of the bound fatty acid increases by 2 methylenes. Tyr70 and Tyr117 which also show discrete disorder in the apoprotein structure due to rotation around their C alpha-C beta bonds, are stabilized in a single, well ordered position in the holoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

A "structural" water molecule in the family of fatty acid binding proteins

A single water molecule (w135), buried within the structure of rat intestinal fatty acid binding protein (I-FABP), is investigated by NMR, molecular dynamics simulations, and analysis of known crystal structures. An ordered water molecule was found in structurally analogous position in 24 crystal structures of nine different members of the family of fatty acid binding proteins. There is a remarkable conservation of the local structure near the w135 binding site among different proteins from this family. NMR cross-relaxation measurements imply that w135 is present in the I-FABP:ANS (1-sulfonato-8-(1')anilinonaphthalene) complex in solution with the residence time of >300 ps. Mean-square positional fluctuations of w135 oxygen observed in MD simulations (0.18 and 0.13 A2) are comparable in magnitude to fluctuations exhibited by the backbone atoms and result from highly constrained binding pocket as revealed by Voronoi volumes (averages of 27.0 +/- 1.8 A3 and 24.7 +/- 2.2 A3 for the two simulations). Escape of w135 from its binding pocket was observed only in one MD simulation. The escape process was initiated by interactions with external water molecules and was accompanied by large deformations in beta-strands D and E. Immediately before the release, w135 assumed three distinct states that differ in hydrogen bonding topology and persisted for about 15 ps each. Computer simulations suggest that escape of w135 from the I-FABP matrix is primarily determined by conformational fluctuations of the protein backbone and interactions with external water molecules.     

Solution structure of human intestinal fatty acid binding protein with a naturally-occurring single amino acid substitution (A54T) that is associated with altered lipid metabolism

The human intestinal fatty acid binding protein (I-FABP) belongs to a family of intracellular lipid binding proteins. This 15 kDa protein binds dietary long-chain fatty acids in the cytosol of enterocytes. A naturally-occurring nucleotide polymorphism at codon 54, which produces either an alanine-containing (A54) or a threonine-containing (T54) protein, has been identified. These two I-FABP forms display differential binding and transport of fatty acids across cells, and their alleles are associated with in vivo insulin resistance and/or altered lipid metabolism in several human populations. The three-dimensional solution structure of the more common A54 form was previously determined in our lab. A direct comparison between human A54 and T54 I-FABP has now been performed to help elucidate the structural origins of their physiological distinctions. The solution structure of T54 I-FABP is highly homologous to that of A54 I-FABP, with the same overall three-dimensional fold that includes an antiparallel beta-clam motif. Chemical shift differences between the two proteins suggest only minor local structural changes within the "portal region" and no significant alterations elsewhere. Hence, the slightly stronger binding of fatty acids to T54 I-FABP does not originate from residues in direct contact with the bound fatty acid. Instead, it appears that the larger Thr(54) side chain affects the passage of the ligand through the entry portal. Structural details of this portal region will be discussed in view of the influence residue 54 exerts on the functional properties of human I-FABP.     

Molecular simulations of β‐lactoglobulin complexed with fatty acids reveal the structural basis of ligand affinity to internal and possible external binding sites

The interaction of saturated fatty acids of different length (C8:0 to C18:0) with β‐lactoglobulin (βLG) was investigated by molecular dynamics simulation and docking approaches. The results show that the presence of such ligands in the hydrophobic central cavity of βLG, known as the protein calyx, determines an enhancement of atomic fluctuations compared with the unliganded form, especially for loops at the entrance of the binding site. Concerted motions are evidenced for protein regions that could favor the binding of ligands. The mechanism of anchoring of fatty acids of different length is similar for the carboxylate head‐group, through electrostatic interactions with the side chains of Lys60/Lys69. The key protein residues to secure the hydrocarbon chain are Phe105/Met107, which adapt their conformation upon ligand binding. In particular, Phe105 provides an additional hydrophobic clamp only for the tail of the two fatty acids with the longest chains, palmitic, and stearic acid, which are known to bind βLG with a high affinity. The search of additional external binding sites for fatty acids, distinct from the calyx, was also carried out for palmitic acid. Two external sites with a lower affinity were identified as secondary sites, one consisting in a hydrophobic cavity allowing two distinct binding modes for the fatty acid, and the other corresponding to a surface crevice close to the protein α‐helix. The overall results provide a comprehensive picture of the dynamical behavior of βLG in complex with fatty acids, and elucidate the structural basis of the binding of these physiological ligands. Proteins 2014; 82:2609–2619. © 2014 Wiley Periodicals, Inc.     

The interaction of lipophilic drugs with intestinal fatty acid-binding protein

Intestinal fatty acid-binding protein (I-FABP) is a small protein that binds long-chain dietary fatty acids in the cytosol of the columnar absorptive epithelial cells (enterocytes) of the intestine. The binding cavity of I-FABP is much larger than is necessary to bind a fatty acid molecule, which suggests that the protein may be able to bind other hydrophobic and amphipathic ligands such as lipophilic drugs. Herein we describe the binding of three structurally diverse lipophilic drugs, bezafibrate, ibuprofen (both R- and S-isomers) and nitrazepam to I-FABP. The rank order of affinity for I-FABP determined for these compounds was found to be R-ibuprofen approximately bezafibrate > S-ibuprofen > nitrazepam. The binding affinities were not directly related to aqueous solubility or partition coefficient of the compounds; however, the freely water-soluble drug diltiazem showed no affinity for I-FABP. Drug-I-FABP interaction interfaces were defined by analysis of chemical shift perturbations in NMR spectra, which revealed that the drugs bound within the central fatty acid binding cavity. Each drug participated in a different set of interactions within the cavity; however, a number of common contacts were observed with residues also involved in fatty acid binding. These data suggest that the binding of non-fatty acid lipophilic drugs to I-FABP may increase the cytosolic solubility of these compounds and thereby facilitate drug transport from the intestinal lumen across the enterocyte to sites of distribution and metabolism.     

Structural properties of the adipocyte lipid binding protein

The adipocyte lipid binding protein, ALBP (also adipocyte fatty acid binding protein, A-FABP, 422 protein, aP2, and p15 protein), is one of the most studied of the intracellular lipid binding protein family. Here we sequentially compare the different sources of ALBP and describe the idea that one-third of the amino acid side chains near the N-terminal end appear to play a major role in conformational dynamics and in ligand transfer. Crystallographic data for mouse ALBP are summarized and the ligand binding cavity analyzed in terms of the overall surface and conformational dynamics. The region of the proposed ligand portal is described. Amino acid side chains critical to cavity formation and fatty acid interactions are analyzed by comparing known crystal structures containing a series of different hydrophobic ligands. Finally, we address ALBP ligand binding affinity and thermodynamic studies.     

Intestinal fatty acid binding protein may favor differential apical fatty acid binding in the intestine

The intestinal mucosa metabolizes fatty acids differently when presented to the lumenal or basolateral membrane. Expression of both liver and intestinal fatty acid binding proteins (L- and I-FABPs) uniquely in the enterocyte offers a possible explanation of this phenomenon. An organ explant system was used to analyze the relative binding of fatty acids to each protein. More fatty acid was bound to L-FABP than to I-FABPs (28% vs. 6% of cytosolic radioactivity), no matter on which side the fatty acid was added. However, a 2-3-fold increase in fatty acid binding to the intestinal paralog was noted after apical addition of palmitic or oleic acid in mucosa from chow fed rats. When oleic acid was added apically, a 1.4-fold increase in binding to I-FABP was observed in mucosa derived from chronically fat fed rats, consistent with the previously observed 50% increase in the content of that protein. Immunocytochemical localization of both FABPs in vivo demonstrated an apical cytoplasmic localization in the fasting state, and redistribution to the entire cytoplasm after fat feeding. These data are consistent with the hypothesis that I-FABP may contribute to the metabolic compartmentalization of apically presented fatty acids in the intestine.     

NMR studies of 4-19F-phenylalanine-labeled intestinal fatty acid binding protein: evidence for conformational heterogeneity in the native state

(19)F-Nuclear magnetic resonance (NMR) studies have been carried out after incorporation of 4-(19)F-phenylalanine into the intestinal fatty acid binding protein (IFABP), a protein composed of two beta-sheets containing a large hydrophobic cavity into which ligands bind. NMR spectra have been obtained with both the ligand-free and ligand-bound (oleate) forms. There are 29 residues involved in van der Waals or hydrophobic interactions or both to form a U-shaped ligand binding pocket (Sacchettni J. C., Scapin G., Gopaul D., and Gordon J. I. (1992) J. Biol. Chem. 267, 23534-23545). The protein contains eight phenylalanines, and all are included in those residues that line the pocket. Peak assignments were made using site-specific incorporation of 4-(19)F-phenylalanine. Fluorine is a highly sensitive probe to monitor the conformation and dynamics of the side chains in native state. We find that chemical exchange in the binding pocket exists in the native apo- and holo-state. Of the eight phenylalanine residues, Phe2, Phe47, Phe62, Phe68, and Phe93 are arranged on one side of the binding pocket, and all exist in two conformations with Phe2, Phe47, and Phe62 showing exchange cross-peaks with minor conformation in (19)F-(19)F nuclear Overhauser effect (NOESY) spectra. The line widths of Phe68 and Phe93 are broader than those of other phenylalanine residues and can be deconvoluted into two peaks. Phe47, Phe62, Phe68, Phe93, and Trp82 have been proposed to be involved in the early stage of collapse (Ropson, I. J., and Frieden, C. (1992) Proc. Natl. Acad. Sci U.S.A. 89, 7222-7226), but a temperature study suggests that Phe47 behaves differently than other residues and may be more involved in a later stage of folding, for example, side chain stabilization. In the holo-form, Phe17 shows an extra exchange cross-peak in addition to those exchange cross-peaks observed in apo-form. Holo-IFABP exhibits broader line width than the apo-form, suggesting more flexibility of the binding cavity upon ligand binding.     

Polyene fatty acid interactions with recombinant intestinal and liver fatty acid-binding proteins. Spectroscopic studies

Binding and proximity relationships of fatty acids with recombinant rat liver fatty acid-binding protein (L-FABP) and intestinal fatty acid-binding protein (I-FABP) were studied with absorption and fluorescence spectroscopy. Protein aromatic amino acids were examined in the absence and presence of bound fatty acid. Second derivative absorbance spectroscopy of the apo- and holoproteins suggested that fatty acid binding altered the conformation of L-FABP, but not of I-FABP. Fatty acid binding also blocked the accessibility of L-FABP tyrosine and I-FABP tryptophan to Stern-Volmer quenching by acrylamide, indicating that these amino acids were present in the fatty acid-binding pocket. Forster energy transfer from I-FABP tryptophan to bound cis-parinaric acid resulted in quenching of tryptophan lifetime and appearance of sensitized lifetime of bound cis-parinaric acid. The calculated donor-acceptor distances were 16.9 +/- 0.6 and 19.2 +/- 0.3 A for I-FABP and L-FABP, respectively. Absorbance spectral shifts and ratios of fluorescence excitation maxima indicated that the parinaric acid microenvironment in the fatty acid-binding site of I-FABP was much less polar than that of L-FABP. Parinaric acids displayed similar rotational correlation time and limiting anisotropy when bound to I-FABP and to L-FABP. These results are consistent with a close proximity of bound fatty acids to the tyrosine and tryptophan residues and with immobilization of the polyene fatty acids in the fatty acid-binding site(s) of L-FABP and I-FABP. The two proteins differ in that only L-FABP has two fatty acid-binding sites and appears to undergo significant conformational change upon fatty acid binding.     

Characterization of the sources of protein-ligand affinity: 1-sulfonato-8-(1'')anilinonaphthalene binding to intestinal fatty acid binding protein.

1-Sulfonato-8-(1')anilinonaphthalene (1,8-ANS) was employed as a fluorescent probe of the fatty acid binding site of recombinant rat intestinal fatty acid binding protein (1-FABP). The enhancement of fluorescence upon binding allowed direct determination of binding affinity by fluorescence titration experiments, and measurement of the effects on that affinity of temperature, pH, and ionic strength. Solvent isotope effects were also determined. These data were compared to results from isothermal titration calorimetry. We obtained values for the enthalpy and entropy of this interaction at a variety of temperatures, and hence determined the change in heat capacity of the system consequent upon binding. The ANS-1-FABP is enthalpically driven; above approximately 14 degrees C it is entropically opposed, but below this temperature the entropy makes a positive contribution to the binding. The changes we observe in both enthalpy and entropy of binding with temperature can be derived from the change in heat capacity upon binding by integration, which demonstrates the internal consistency of our results. Bound ANS is displaced by fatty acids and can itself displace fatty acids bound to I-FABP. The binding site for ANS appears to be inside the solvent-containing cavity observed in the x-ray crystal structure, the same cavity occupied by fatty acid. From the fluorescence spectrum and from an inversion of the Debye-Hueckel formula for the activity coefficients as a function of added salt, we inferred that this cavity is fairly polar in character, which is in keeping with inferences drawn from the x-ray structure. The binding affinity of ANS is considered to be a consequence of both electrostatic and conditional hydrophobic effects. We speculate that the observed change in heat capacity is produced mainly by the displacement of strongly hydrogen-bonded waters from the protein cavity.     

A fluorescently labeled intestinal fatty acid binding protein. Interactions with fatty acids and its use in monitoring free fatty acids.

The fatty acid-binding protein from rat intestine (I-FABP) has been covalently modified with the fluorescent compound Acrylodan. Acrylodan was found to label Lys27, one of the few amino acid residues found by x-ray diffraction studies to change orientation upon fatty acid (FA) binding to I-FABP. Binding of FA to this Acrylodan-modified I-FABP (ADIFAB) induces a large shift in fluorescence emission wavelength from 432 to 505 nm. As a consequence, the ratio of emission intensities provides a direct measure of the concentration of FA bound to the protein. Binding of FA is well described by single site equilibrium for FA concentrations below the critical micelle concentration. ADIFAB dissociation constants (Kd) determined at 37 degrees C and at concentrations below the critical micelle concentration for oleate, palmitate, linoleate, arachidonate, and linolenate were, respectively, 0.28, 0.33, 0.97, 1.6, and 2.5 microM. The variation of these Kd values with FA molecular species is highly correlated with the solubility of the FA in water, suggesting that all these FA bind with a similar conformation in the I-FABP binding site. The ADIFAB response together with the measured equilibrium constants allows a direct determination of the concentration of long chain free fatty acid (FFA) in the concentration range, depending upon the FA molecular species, between 1 nM and > 20 microM. As an example of its use as a probe to measure FFA levels, ADIFAB is used here to monitor the time course for FFA release from IgE receptor- and ionomycin-activated rat basophilic leukemia (RBL) cells.     

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.     

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.     

Insights into binding of fatty acids by fatty acid binding proteins

Members of the phylogenetically related intracellular lipid binding protein (iLBP) are characterized by a highly conserved tertiary structure, but reveal distinct binding preferences with regard to ligand structure and conformation, when binding is assessed by the Lipidex method (removal of unbound ligand by hydrophobic polymer) or by isothermal titration calorimetry, a true equilibrium method. Subfamily proteins bind retinoids, subfamily II proteins bind bulky ligands, examples are intestinal bile acid binding protein (I-BABP) and liver fatty acid binding protein (L-FABP) which binds 2 ligand molecules, preferably monounsaturated and n-3 fatty acids. Subfamily III intestinal fatty acid binding protein (I-FABP) binds fatty acid in a bent conformation. The fatty acid bound by subfamily IV FABPs has a U-shaped conformation; here heart (H-) FABP preferably binds n-6, brain (B-) FABP n-3 fatty acids. The ADIFAB-method is a fluorescent test for fatty acid in equilibrium with iLBP and reveals some correlation of binding affinity to fatty acid solubility in the aqueous phase; these data are often at variance with those obtained by the other methods. Thus, in this review published binding data are critically discussed, taking into account on the one hand binding increments calculated for fatty acid double bonds on the basis of the solubility hypothesis, on the other hand the interpretation of calorimetric data on the basis of crystallographic and solution structures of iLBPs.     

Human liver fatty acid binding protein cDNA and amino acid sequence. Functional and evolutionary implications

Human liver fatty acid binding protein (L-FABP) cDNA clones were identified in a liver cDNA library. The two longest clones were completely sequenced. The nucleotide sequence predicts a protein of 127 amino acid residues. Identity of the clones was confirmed by limited amino acid sequence analysis of purified human L-FABP peptides and Edman degradation of radiolabeled in vitro translated FABP. Statistical analysis of the amino acid and mRNA sequences of human L-FABP, rat L-FABP, rat intestinal (I-) FABP, and mouse 422 protein indicates that the human and rat L-FABPs are highly homologous and that L-FABP and I-FABP diverged a long time ago (approximately 650-690 million years ago), although they are more closely related to each other than either of them is to 422 protein. Secondary structure predictions from the primary sequence of human and rat L-FABP reveal a region (residues 12-30) that might be the putative fatty acid binding domain of the two L-FABPs. Knowledge of the primary amino acid sequence of L-FABP and possible functional domains will be pivotal in further defining and understanding the mechanism of ligand binding and transfer by this protein.     

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.     

Expression of rat intestinal fatty acid binding protein in E. coli and its subsequent structural analysis: a model system for studying the molecular details of fatty acid-protein interaction

A prokaryotic expression vector containing the rec A promoter and a translational enhancer element from the gene 10 leader of bacteriophage T7 was used to direct efficient synthesis of rat intestinal fatty acid binding protein (I-FABP) in E. coli. Expression of I-FABP in E. coli has no apparent, deleterious effects on the organism. High levels of expression of I-FABP mRNA in supE⁺ strains of E. coli, such as JM101, is associated with suppression of termination at its UGA stop codon. This can be eliminated by using a sup-Estrain as MG1655 and by site-directed mutagenesis of the cDNA to create an in frame UAA stop codon. E. coli-derived rat I-FABP lacks its initiator Met residues. It has been crystallized with and without bound palmitate. High resolution x-ray crystallographic studies of the 131 residue apo- and holo-proteins have revealed the following. I-FABP contains 10 anti-parallel -strands organized into two orthogonally situated -sheets. The overall conformation of the protein resembles that of a clam — hence the term -clam. The bound ligand is located in the interior of the protein. Its carboxylate group forms part of a unique five member hydrogen bonding network consisting of two ordered solvent molecules as well as the side chains of Arg¹⁰⁶ and Gln¹¹⁵. The hydrocarbon chain of the bound C16:0 fatty acid has a distinctive bent conformation with a slight left-handed helical twist. This conformation is maintained by interactions with the side chains of a number of hydrophobic and aromatic amino acids. Apo-I-FABP has a similar overall conformation to holo-I-FABP indicating that the -clam structure is stable even without bound ligand. The space occupied by bound ligand in the core of the holo-protein is occupied by additional ordered solvent molecules in the apo-protein. Differences in the side chain orientations pf several residues located over a potential opening to the cores of the apo- and holo-proteins suggest that solvent may play an important role in the binding mechanism. Comparison of the C coordinates of apo- and holo-I-FABP with those of other proteins indicates it is a member of a superfamily that currently includes (i) 10 mammalian intracellular lipid binding proteins, (ii) the photoactive yellow protein from the purple photoautotrophic bacterium Ectothiorhodospira halophila and (iii) a group of extracellular lipid binding proteins from a diverse number of phyla that have a common barrel consisting of 8 anti-parallel -strands stacked in two nearly orthogonal sheets. In summary, E. coli-derived I-FABP not only represents a useful model for assessing the atomic details of fatty acid-protein interactions and the mechanisms which regulate acquisition and release of this type of ligand, but also structure/function relationships in other superfamily members.Abbreviations I-FABP Intestinal Fatty Acid Binding Protein - r.m.s root mean square     

Binding kinetics of engineered mutants provide insight about the pathway for entering and exiting the intestinal fatty acid binding protein

To better understand the mechanism by which fatty acids bind to and dissociate from the binding cavities of fatty acid binding proteins (FABPs), we constructed 31 single amino acid mutants of the intestinal FABP (I-FABP) and determined the rate constants for binding and dissociation, primarily for long-chain fatty acids (FA). FA dissociation from these proteins was measured both by the ADIFAB method and by the change in tryptophan fluorescence of the FABPs. Rate constants for binding (kon) were calculated from the rate constants for dissociation (koff) and the equilibrium binding affinities. Amino acid substitutions were made at locations within the binding cavity, in the region of the gap between the betaD- and betaE-strands, and within the "portal" region of the protein. The koff values for the mutant proteins ranged from about 20-fold slower to 4-fold faster than the wild-type (WT) protein. Values for kon were as much as 20-fold slower than the WT protein, but in no case was kon significantly faster than the WT. Mutants with slower and faster koff values were generally those involving sites within the binding cavity and, relative to the WT protein, revealed higher and lower affinities, respectively. Reduced rates of binding were generally, but not exclusively, associated with sites within the portal region. For example, for F68A which is located closer to the opposite end of the protein from the portal region, the kon is more than 10-fold slower than WT. Even for these distal sites, however, the evidence is consistent with reductions in kon being due to alterations of the portal region. Binding affinities and rate constants measured as a function of ionic strength also suggest that the FA initially binds, through an electrostatic interaction, to Arg-56 on the surface of the protein, before inserting into the binding cavity. Thus, the results of this study are consistent with FA binding to I-FABP involving an initial interaction with Arg-56 followed by insertion of the FA, through the portal region, into the binding cavity and with a reversal of these steps for the dissociation reaction.