Evidence from NMR interaction studies challenges the hypothesis of direct lipid transfer from L‐FABP to malaria sporozoite protein UIS3

UIS3 is a malaria parasite protein essential for liver stage development of Plasmodium species, presumably localized to the membrane of the parasitophorous vacuole formed in infected cells. It has been recently proposed that the soluble domain of UIS3 interacts with the host liver fatty acid binding protein (L‐FABP), providing the parasite with a pathway for importing exogenous lipids required for its rapid growth. This finding may suggest novel strategies for arresting parasite development. In this study, we have investigated the interaction between human L‐FABP and the soluble domain of Plasmodium falciparum UIS3 by NMR spectroscopy. The amino acid residue‐specific analysis of ¹H,¹⁵N‐2D NMR spectra excluded the occurrence of a direct interaction between L‐FABP (in its unbound and oleate‐loaded forms) and Pf‐UIS3. Furthermore, the spectrum of Pf‐UIS3 was unchanged when oleate or phospholipids were added. The present investigation entails a reformulation of the current model of host‐pathogen lipid transfer, possibly redirecting research for early intervention against malaria.     

Two fatty acid‐binding proteins expressed in the intestine interact differently with endocannabinoids

Two different members of the fatty acid‐binding protein (FABP) family are found in enterocyte cells of the gastrointestinal system, namely liver‐type and intestinal fatty acid‐binding proteins (LFABP and IFABP, also called FABP1 and FABP2, respectively). Striking phenotypic differences have been observed in knockout mice for either protein, for example, high fat‐fed IFABP‐null mice remained lean, whereas LFABP‐null mice were obese, correlating with differences in food intake. This finding prompted us to investigate the role each protein plays in directing the specificity of binding to ligands involved in appetite regulation, such as fatty acid ethanolamides and related endocannabinoids. We determined the binding affinities for nine structurally related ligands using a fluorescence competition assay, revealing tighter binding to IFABP than LFABP for all ligands tested. We found that the head group of the ligand had more impact on binding affinity than the alkyl chain, with the strongest binding observed for the carboxyl group, followed by the amide, and then the glycerol ester. These trends were confirmed using two‐dimensional ¹H–¹⁵N nuclear magnetic resonance (NMR) to monitor chemical shift perturbation of the protein backbone resonances upon titration with ligand. Interestingly, the NMR data revealed that different residues of IFABP were involved in the coordination of endocannabinoids than those implicated for fatty acids, whereas the same residues of LFABP were involved for both classes of ligand. In addition, we identified residues that are uniquely affected by binding of all types of ligand to IFABP, suggesting a rationale for its tighter binding affinity compared with LFABP.     

X-ray crystallographic analysis of adipocyte fatty acid binding protein (aP2) modified with 4-hydroxy-2-nonenal

Fatty acid binding proteins (FABP) have been characterized as facilitating the intracellular solubilization and transport of long‐chain fatty acyl carboxylates via noncovalent interactions. More recent work has shown that the adipocyte FABP is also covalently modified in vivo on Cys117 with 4‐hydroxy‐2‐nonenal (4‐HNE), a bioactive aldehyde linked to oxidative stress and inflammation. To evaluate 4‐HNE binding and modification, the crystal structures of adipocyte FABP covalently and noncovalently bound to 4‐HNE have been solved to 1.9 Å and 2.3 Å resolution, respectively. While the 4‐HNE in the noncovalently modified protein is coordinated similarly to a carboxylate of a fatty acid, the covalent form show a novel coordination through a water molecule at the polar end of the lipid. Other defining features between the two structures with 4‐HNE and previously solved structures of the protein include a peptide flip between residues Ala36 and Lys37 and the rotation of the side chain of Phe57 into its closed conformation. Representing the first structure of an endogenous target protein covalently modified by 4‐HNE, these results define a new class of in vivo ligands for FABPs and extend their physiological substrates to include bioactive aldehydes.     

Insight into the interaction sites between fatty acid binding proteins and their ligands

Fatty acid binding proteins (FABPs), are evolutionarily conserved small cytoplasmic proteins that occur in many tissue-specific types. One of their primary functions is to facilitate the clearance of the cytoplasmic matrix from free fatty acids and of other detergent-like compounds. Crystallographic studies of FABP proteins have revealed a well defined binding site located deep inside their β-clam structure that is hardly exposed to the bulk solution. However, NMR measurements revealed that, when the protein is equilibrated with its ligands, residues that are clearly located on the outer surface of the protein do interact with the ligand. To clarify this apparent contradiction we applied molecular dynamics simulations to follow the initial steps associated with the FABP–fatty acid interaction using, as a model, the interaction of toad liver basic FABP, or chicken liver bile acid binding protein, with a physiological concentration of palmitate ions. The simulations (~200 ns of accumulated time) show that fatty acid molecules interact, unevenly, with various loci on the protein surface, with the favored regions being the portal and the anti-portal domains. Random encounters with palmitate at these regions led to lasting adsorption to the surface, while encounters at the outer surface of the β-clam were transient. Therefore, we suggest that the protein surface is capable of sequestering free fatty acids from solution, where brief encounters evolve into adsorbed states, which later mature by migration of the ligand into a more specific binding site.     

Conformational plasticity of the lipid transfer protein SCP2

The nonspecific lipid transfer protein sterol carrier protein 2 (SCP2) is involved in organellar fatty acid metabolism. A hydrophobic cavity in the structure of SCP2 accommodates a wide variety of apolar ligands such as cholesterol derivatives or fatty acyl-coenzyme A (CoA) conjugates. The properties of this nonspecific lipid binding pocket are explored using NMR chemical shift perturbations, paramagnetic relaxation enhancement, amide hydrogen exchange, and 15N relaxation measurements. A common binding cavity shared by different physiological ligands is identified. NMR relaxation measurements reveal that residues in the three C-terminal alpha-helices within the lipid binding region exhibit mobility at fast (picosecond to nanosecond) and slow (microsecond to millisecond) time scales. Ligand binding is associated with a considerable loss of peptide backbone mobility. The observed conformational dynamics in SCP2 may play a role for the access of hydrophobic ligands to an occluded binding pocket. The C-terminal peroxisomal targeting signal of SCP2 is specifically recognized by the Pex5p receptor protein, which conducts cargo proteins toward the peroxisomal organelle. Neither the C-terminal targeting signal nor the N-terminal precursor sequence interferes with lipid binding by SCP2. The alpha-helices involved in lipid binding also mediate a secondary interaction interface with the Pex5p receptor. Silencing of conformational dynamics of the peptide backbone in these helices upon either lipid or Pex5p binding might communicate the loading state of the cargo protein to the targeting receptor.     

The Interaction of Equine Lysozyme:Oleic Acid Complexes with Lipid Membranes Suggests a Cargo Off-Loading Mechanism

The normal function of equine lysozyme (EL) is the hydrolysis of peptidoglycan residues of bacterial cell walls. EL is closely related to α-lactalbumins with respect to sequence and structure and further possesses the calcium binding site of α-lactalbumins. Recently, EL multimeric complexes with oleic acids (ELOAs) were shown to possess tinctorial and morphological properties, similar to amyloidal aggregates, and to be cytotoxic. ELOA's interactions with phospholipid membranes appear to be central to its biological action, similar to human α-lactalbumin made lethal to tumor cells. Here, we describe the interaction of ELOA with phospholipid membranes. Confocal scanning laser microscopy shows that ELOA, but not native EL, accumulates on the surface of giant unilamellar vesicles, without inducing significant membrane permeability. Quartz crystal microbalance with dissipation data indicated an essentially non-disruptive binding of ELOA to supported lipid bilayers, leading to formation of highly dissipative and “soft” lipid membrane; at higher concentrations of ELOA, the lipid membrane desorbs from the surface probably as bilayer sheets of vesicles. This membrane rearrangement occurred to a similar extent when free oleic acid (OA) was added, but not when free OA was removed from ELOA by prior incubation with bovine serum albumin, emphasizing the role of OA in this process. NMR data indicated an equilibrium between free and bound OA, which shifts towards free OA as ELOA is progressively diluted, indicating that OA is relatively loosely bound. Activity measurements together with fluorescence spectroscopy and circular dichroism suggested a conversion of ELOA towards a more native-like state on interaction with lipid membranes, although complete refolding was not observed. Altogether, these results suggest that ELOA may act as an OA carrier and facilitate OA transfer to the membrane. ELOA's properties illustrate that protein folding variants may possess specific functional properties distinct from the native protein.     

Exploring the role of the phospholipid ligand in endothelial protein C receptor: A molecular dynamics study

Endothelial protein C receptor (EPCR) is a CD1‐like transmembrane glycoprotein with important regulatory roles in protein C (PC) pathway, enhancing PC's anticoagulant, anti‐inflammatory, and antiapoptotic activities. Similarly to homologous CD1d, EPCR binds a phospholipid [phosphatidylethanolamine (PTY)] in a groove corresponding to the antigen‐presenting site, although it is not clear if lipid exchange can occur in EPCR as in CD1d. The presence of PTY seems essential for PC γ‐carboxyglutamic acid (Gla) domain binding. However, the lipid‐free form of the EPCR has not been characterized. We have investigated the structural role of PTY on EPCR, by multiple molecular dynamics (MD) simulations of ligand bound and unbound forms of the protein. Structural changes, subsequent to ligand removal, led to identification of two stable and folded ligand‐free conformations. Compared with the bound form, unbound structures showed a narrowing of the A′ pocket and a high flexibility of the helices around it, in agreement with CD1d simulation. Thus, a lipid exchange with a mechanism similar to CD1d is proposed. In addition, unbound conformations presented a reduced interaction surface for Gla domain, confirming the role of PTY in establishing the proper EPCR conformation for the interaction with its partner protein. Single MD simulations were also obtained for 29 mutant models with predicted structural stability and impaired binding ability. Ligand affinity calculations, based on linear interaction energy method, showed that substitution‐induced conformational changes affecting helices around the A′ pocket were associated to a reduced binding affinity. Mutants responsible for this effect may represent useful reagents for experimental tests. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Primary structure of locust flight muscle fatty acid binding protein.

The amino acid sequence of the fatty acid binding protein (FABP) from flight muscle of the locust, Schistocerca gregaria, has been determined. The sequence of the N-terminal 39 amino acid residues, determined by automated Edman degradation, was used to prepare a degenerate oligonucleotide that corresponded to amino acid residues 16-23. cDNA coding for FABP was constructed from flight muscle mRNA and amplified by the polymerase chain reaction using the degenerate oligonucleotide and an oligo dT-NotI primer adapter as primers. The amplification product was cloned and sequenced. Additionally, a cDNA library of flight muscle mRNA was prepared and screened with a 414-bp probe prepared from the clone. The primary structure of locust FABP was compared with the proteins in the Swiss protein databank and found to have significant homology with mammalian FABPs over the entire 133-residue sequence. The best match was versus human heart FABP (41% identity), attesting to the highly conserved nature of this protein. The results suggest that locust muscle FABP is a member of the lipid binding protein superfamily and may provide valuable insight into the evolution of this abundant protein class.     

31P-CP-MAS NMR studies on TPP+ bound to the ion-coupled multidrug transport protein EmrE

The binding of tetraphenylphosphonium (TPP⁺) to EmrE, a membrane-bound, 110 residue Escherichia coli multidrug transport protein, has been observed by ³¹P cross-polarisation–magic-angle spinning nuclear magnetic resonance spectroscopy (CP–MAS NMR). EmrE has been reconstituted into dimyristoyl phosphatidylcholine bilayers. CP–MAS could selectively distinguish binding of TPP⁺ to EmrE in the fluid membrane. A population of bound ligand appears shifted 4 ppm to lower frequency compared to free ligand in solution, which suggests a rather direct and specific type of interaction of the ligand with the protein. This is also supported by the observed restricted motion of the bound ligand. The observation of another weakly bound substrate population arises from ligand binding to negatively charged residues in the protein loop regions.     

Structural and functional studies on different human FABP types

Interaction of various ligands with recombinant proteins of 5 human FABP types was studied by radiochemical and fluorescence procedures. Liver, heart, intestinal and myelin FABP showed a higher affinity for oleic acid than adipocyte FABP. Intestinal and adipocyte FABP had a relatively high Kd value for arachidonic acid. Liver and intestinal FABP showed high affinity for DAUDA in contrast to the other FABP types. ANS was only well bound by liver and adipocyte FABP. Retinol was not bound by any FABP type, retinoic acid only by adipocyte FABP. Data indicate the importance of both electrostatic and hydrophobic interaction for the ligand-FABP binding. The immunological crossreactivity between six human FABP types including epidermal FABP and their respective antibodies raised in rabbit, chicken and mouse appeared to be low and may suggest heterogeneity of protein surface.     

Identification and characterization of a fatty acid binding protein in bovine mammary gland

A fatty acid binding protein (FABP) was isolated from bovine mammary cytosol by gel filtration and ion exchange chromatography. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated a mol. wt. of 12,000. Isoelectric focusing showed two bands at pH 5.6 and 5.8. FABP bound long chain fatty acids and their CoA thioesters, but not medium or short chain fatty acids. Affinity constant (Ka) for 18:1 was about 2 micromolar. Endogenously bound fatty acids included 16:0, 18:0 and 18:1, in both covalent and noncovalent association with FABP. Activities of microsomal phosphatidic acid phosphatase, fatty acid:CoA ligase or diacylglycerol acyltransferase were not affected by purified FABP in vitro.     

The ligand-mediated affinity of brain-type fatty acid-binding protein for membranes determines the directionality of lipophilic cargo transport

The intracellular transport of lipophilic cargoes is a highly dynamic process. In eukaryotic cells, the uptake and release of long-chain fatty acids (LCFAs) are executed by fatty-acid binding proteins. However, how these carriers control the directionality of cargo trafficking remains unclear. Here, we revealed that the unliganded archetypal Drosophila brain-type fatty acid-binding protein (dFABP) possesses a stronger binding affinity than its liganded counterpart for empty nanodiscs (ND). Titrating unliganded dFABP and nanodiscs with LCFAs rescued the broadening of FABP cross-peak intensities in HSQC spectra from a weakened protein–membrane interaction. Two out of the 3 strongest LCFA contacting residues in dFABP identified by NMR HSQC chemical shift perturbation (CSP) are also part of the 30 ND-contacting residues (out of the total 130 residues in dFABP), revealed by attenuated TROSY signal in the presence of lipid ND to apo-like dFABP. Our crystallographic temperature factor data suggest enhanced αII helix dynamics upon LCFA binding, compensating for the entropic loss in the βC-D/βE-F loops. The aliphatic tail of bound LCFA impedes the charge-charge interaction between dFABP and the head groups of the membrane, and dFABP is prone to dissociate from the membrane upon ligand binding. We therefore conclude that lipophilic ligands participate directly in the control of the functionally required membrane association and dissociation of FABPs.     

13C NMR studies of fatty acid-protein interactions: comparison of homologous fatty acid-binding proteins produced in the intestinal epithelium

Summary A high-resolution, solution-state NMR method for characterizing and comparing the interactions between carboxyl ¹³C-enriched fatty acids (FA) and individual binding sites on proteins has been developed. The utility of this method results from the high degree of resolution of carboxyl from other carbon resonances and the high sensitivity of FA carboxyl chemical shifts to intermolecular environmental factors such as degree of hydrogen-bonding or hydration, degree of ionization (pH), and proximity to positively-charged or aromatic side-chain moieties in proteins. Information can be obtained regarding binding heterogeneity (structural as well as thermodynamic), binding stoichiometries, relative binding affinities, the ionization behavior of bound FA and protein side-chain moieties, the physical and ionization states of unbound FA, and the exchange rates of FA between protein binding sites and between protein and non-protein acceptors of FA, such as model membranes.Cytosolic fatty acid binding proteins represent an excellent model system for studying and comparing fatty acid-protein interactions. Prokaryotic expression vectors have been used to direct efficient synthesis of several mammalian intestinal FABPs in E. coli. This has enabled us to isolate gram-quantities of purified FABPs, to introduce NMR-observable isotopes, and to generate FABP mutants.The intestine is the only tissue known to contain abundant quantities of more than one FABP homologue in a single cell type. It is likely that these homologous FABPs serve distinct functional roles in intestinal lipid transport. This paper presents comparative ¹³C NMR results for FA interactions with FABP homologues from intestine, and the functional implications of these analyses are discussed.Abbreviations FA Fatty Acid(s) - FABP Fatty Acid Binding Protein(s) - I-FABP_c Cytosolic rat intestinal Fatty Acid Binding Protein - L-FABP_c Cytosolic rat liver Fatty Acid Binding Protein - CD Circular Dichroic spectroscopy Established Investigator of the American Heart Association     

Conformational exchange of fatty acid binding protein induced by protein-nanodisc interactions

Fatty acid binding proteins (FABPs) can facilitate the transfer of long-chain fatty acids between intracellular membranes across considerable distances. The transfer process involves fatty acids, their donor membrane and acceptor membrane, and FABPs, implying that potential protein-membrane interactions exist. Despite intensive studies on FABP-membrane interactions, the interaction mode remains elusive, and the protein-membrane association and dissociation rates are inconsistent. In this study, we used nanodiscs (NDs) as mimetic membranes to investigate FABP-membrane interactions. Our NMR experiments showed that human intestinal FABP interacts weakly with both negatively charged and neutral membranes, but it prefers the negatively charged one. Through simultaneous analysis of NMR relaxation in the rotating-frame (R_1ρ), relaxation dispersion, chemical exchange saturation transfer, and dark-state exchange saturation transfer data, we estimated the affinity of the protein to negatively charged NDs, the dissociation rate, and apparent association rate. We further showed that the protein in the ND-bound state adopts a conformation different from the native structure and the second helix is very likely involved in interactions with NDs. We also found a membrane-induced FABP conformational state that exists only in the presence of NDs. This state is native-like, different from other conformational states in structure, unbound to NDs, and in dynamic equilibrium with the ND-bound state.     

The purification and characterization of a fatty acid binding protein specific to pig (Sus domesticus) adipose tissue.

Western-blot analysis using antiserum to 3T3-L1-cell fatty acid binding protein (FABP) revealed that pig adipose tissue contains a 15 kDa protein immunologically similar to the murine protein. This 15 kDa protein was purified from pig adipose tissue by sequential application of Sephadex G-50 gel filtration, cation exchange and covalent chromatography on Thiol-Sepharose-4B. The purity of the pig protein was established by two-dimensional polyacrylamide-gel electrophoresis. Isoelectric focusing indicated that the pig adipose FABP (a-FABP) exists with two charge isoforms (pI 5.1 and 5.2), both of which persist after delipidation. The N-terminus of the purified pig a-FABP was blocked; however, cleavage with CNBr allowed recovery of a 12-amino-acid peptide which was identical with the murine a-FABP sequence (residues 36-48) at 10 of 12 positions. The pig a-FABP bound 12-(9-anthroyloxy)oleic acid saturably and stoichiometrically, with an apparent dissociation constant of 1.0 microM. Northern-blot analysis using the cDNA for the murine 3T3-L1 FABP revealed that the pig a-FABP was expressed exclusively in adipose tissue.     

Transport and Utilization of Lipids in Insect Flight Muscles*

In migrating lepidopteran and orthopteran insects, lipid is the preferred fuel for sustained flight activity. Diacylglycerol is delivered by lipophorin to the flight muscle and hydrolyzed to free fatty acid and glycerol. After penetrating the plasma membrane by an unknown mechanism, fatty acids are bound by the intracellular fatty acid binding protein (FABP) and transported through the cytosol. After their conversion to acyl-CoA esters, the fatty acids enter the mitochondrial matrix via the carnitine shuttle for subsequent β-oxidation. This article reviews the current knowledge of lipid metabolism in insect flight muscle, with particular emphasis on the structure and function of FABP and its expression during locust development and flight.     

Isolation, characterization, and expression of fatty acid binding protein in the liver of Gallus domesticus

1. Fatty acid binding protein (FABP) was isolated from chicken liver cytosol. 2. Apparent molecular weight, pI, functional activity, and hybridization of a rat hFABP cDNA probe with chicken liver mRNA suggest that chicken liver FABP is structurally related to hepatic FABP (hFABP) previously isolated and characterized in the rat. 3. Fatty acids bound to liver FABP affect the electrophoretic nature of FABP. 4. Levels of liver FABP mRNA isolated from chickens at various stages of development parallel developmental alterations in lipid metabolism, being highest in day old chicks and laying hens versus juvenile birds.     

Titration and exchange studies of liver fatty acid-binding protein with 13C-labeled long-chain fatty acids

Uniformly (13)C-labeled long-chain fatty acids were used to probe ligand binding to rat liver fatty acid-binding protein (LFABP), an atypical member of the fatty acid-binding protein (FABP) family that binds more than one molecule of long-chain fatty acid, accommodates a variety of diverse ligands, and exhibits diffusion-mediated lipid transport to membranes. Two sets of (1)H-(13)C resonances were found in a titration series of NMR spectra for oleate-LFABP complexes, indicating that two molecules of the fatty acid are situated in the protein cavity. However, no distinct resonances were observed for the excess fatty acid in solution, suggesting that at least one ligand undergoes rapid exchange with oleate in the bulk solution. An exchange rate of 54 +/- 6 s(-1) between the two sets of resonances was measured directly using (13)C z,z-exchange spectroscopy. In light of these NMR measurements, possible molecular mechanisms for the ligand-exchange process are evaluated and implications for the anomalous fatty acid transport mechanism of LFABP are discussed.