The Crystal Structure of the Liver Fatty Acid-binding Protein

The crystal structure of the recombinant form of rat liver fatty acid-binding protein was completed to 2.3 Å and refined to an R factor of 19.0%. The structural solution was obtained by molecular replacement using superimposed polyalanine coordinates of six intracellular lipid-binding proteins as a search probe. The entire amino acid sequence of rat liver fatty acid-binding protein along with an amino-terminal formyl-methionine was modeled in the crystal structure. In addition, the crystal was obtained in the presence of oleic acid, and the initial electron density clearly showed two fatty acid molecules bound within a central cavity. The carboxylate of one fatty acid molecule interacts with arginine 122 and is shielded from free solvent. It has an overall bent conformation. The more solvent-exposed carboxylate of the other oleate is located near the helix-turn-helix that caps one end of the β-barrel, while the acyl chain lies in the interior. The cavity contains both polar and nonpolar residues but also shows extensive hydrophobic character around the nonpolar atoms of the ligands. The primary and secondary oleate binding sites appear to be totally interdependent, mainly because favorable hydrophobic interactions form between both aliphatic chains. The crystal structure of the recombinant form of rat liver fatty acid-binding protein was completed to 2.3 Å and refined to an R factor of 19.0%. The structural solution was obtained by molecular replacement using superimposed polyalanine coordinates of six intracellular lipid-binding proteins as a search probe. The entire amino acid sequence of rat liver fatty acid-binding protein along with an amino-terminal formyl-methionine was modeled in the crystal structure. In addition, the crystal was obtained in the presence of oleic acid, and the initial electron density clearly showed two fatty acid molecules bound within a central cavity. The carboxylate of one fatty acid molecule interacts with arginine 122 and is shielded from free solvent. It has an overall bent conformation. The more solvent-exposed carboxylate of the other oleate is located near the helix-turn-helix that caps one end of the β-barrel, while the acyl chain lies in the interior. The cavity contains both polar and nonpolar residues but also shows extensive hydrophobic character around the nonpolar atoms of the ligands. The primary and secondary oleate binding sites appear to be totally interdependent, mainly because favorable hydrophobic interactions form between both aliphatic chains. INTRODUCTIONLiver fatty acid-binding protein (LFABP), 1The abbreviations used are:LFABPliver fatty acid-binding proteiniLBPintracellular lipid-binding proteinALBPadipocyte lipid-binding proteinIFABPintestinal fatty acid-binding proteinHFABPheart muscle fatty acid-binding proteinL-MFABPlocust muscle fatty acid-binding proteinP2myelin P2 lipid-binding proteinMFB2Manduca sexta fatty acid-binding proteinCRBPIcellular retinol-binding protein ICRBPIIcellular retinol-binding protein IICRABPIcellular retinoic acid-binding protein ICRABPIIcellular retinoic acid-binding protein IIDAUDA11-(5-dimethylaminonaphthalene-1-sulfonyl-amino) undecanoic acidr.m.s.root mean square. is a member of the intracellular lipid-binding protein (iLBP) family (1Banaszak L. Winter N. Xu Z. Bernlohr D.A. Cowan S. Jones T.A. Adv. Protein Chem. 1994; 45: 89-151Crossref PubMed Google Scholar,2Veerkamp J. Maatman R. Prog. Lipid Res. 1995; 34: 17-52Crossref PubMed Scopus (304) Google Scholar). The functions of LFABP, like other family members, are thought to include lipid uptake, lipid transport, regulation of lipid metabolism, and cellular protection by maintaining the concentrations of free cytosolic fatty acids below toxic levels (3Kaikaus R.M. Bass N.M. Ockner R.K. Experientia. 1990; 46: 617-630Crossref PubMed Scopus (156) Google Scholar). The iLBP family is characterized by their small size of approximately 130 amino acids, their affinity for hydrophobic molecules, and their tertiary structure. Both crystal and solution structures have been determined for different iLBP family members that primarily bind fatty acids in adipocytes (ALBP), intestine (IFABP), heart muscle (HFABP), locust muscle (L-MFABP), myelin (P2), and hornworm midgut (MFB2); retinol in liver (CRBPI) and intestine (CRBPII); or retinoic acid in testis (CRABPI) and skin (CRABPII). References to these structural data for the iLBP family are cited in Table III. Although the homology of the amino acid sequences vary, all iLBPs are composed of a 10-stranded, antiparallel β-barrel with two short antiparallel α-helices positioned over one end of the barrel. Within the confines of the β-strands is a cavity that forms the lipid binding site.Table IIILFABP and iLBP structural comparisonProteinCodeCαr.m.s. deviationReferenceÅP21pmp1201.3455Cowan S.W. Newcomer M.E. Jones A.T. J. Mol. Biol. 1993; 230: 1225-1246Crossref PubMed Scopus (160) Google ScholarALBP1adl1221.4556LaLonde J.M. Levenson M.A. Roe J.J. Bernlohr D.A. Banaszak L.J. J. Biol. Chem. 1994; 269: 25339-25347Abstract Full Text PDF PubMed Google ScholarHFABP1hmr1211.4657Young A.C.M. Scapin G. Kromminga A. Patel S.B. Veerkamp J.H. Sacchettini J.C. Structure. 1994; 2: 523-534Abstract Full Text Full Text PDF PubMed Scopus (111) Google ScholarCRBPII1opb1211.4658Winter N.S. Bratt J.M. Banaszak L.J. J. Mol. Biol. 1993; 230: 1247-1259Crossref PubMed Scopus (94) Google ScholarL-MFABP1ftp1241.4959Haunerland N.H. Jacobson B.L. Wesenberg G. Rayment I. Holden H.M. Biochemistry. 1994; 33: 12378-12385Crossref PubMed Scopus (38) Google ScholarCRABPII1cbq1251.6241Kleywegt G.J. Bergfors T. Senn H. Le Motte P. Gsell B. Shudo K. Jones A.T. Structure. 1994; 2: 1241-1258Abstract Full Text Full Text PDF PubMed Scopus (196) Google ScholarCRABPI1cbr1241.6741Kleywegt G.J. Bergfors T. Senn H. Le Motte P. Gsell B. Shudo K. Jones A.T. Structure. 1994; 2: 1241-1258Abstract Full Text Full Text PDF PubMed Scopus (196) Google ScholarCRBPI1crb1261.7055Cowan S.W. Newcomer M.E. Jones A.T. J. Mol. Biol. 1993; 230: 1225-1246Crossref PubMed Scopus (160) Google ScholarIFABP1ifc1251.8161Scapin G. Gordon J.I. Sacchettini J.C. J. Biol. Chem. 1992; 267: 4253-4269Abstract Full Text PDF PubMed Google ScholarMFB21mdc1141.8962Benning M.M. Smith A.F. Wells M.A. Holden H.M. J. Mol. Biol. 1992; 228: 208-219Crossref PubMed Scopus (45) Google Scholar Open table in a new tab The various genes for iLBPs have expression levels that are related to the cell or tissue type. Several of the different genotypes have been cloned and are available in expression vectors. In some instances, measurements have been made of their expression levels in different cell types. For example, LFABP has been detected in abundance in tissue from liver, adipose deposits, myocardium, kidney, and large and small intestinal epithelia of rats (4Ockner R.K. Manning J.A. Poppenhausen R.B. Ho W.K. Science. 1972; 177: 56-58Crossref PubMed Scopus (516) Google Scholar, 5Bass N.M. Manning J.A. Biochem. Biophys. Res. Commun. 1986; 137: 929-935Crossref PubMed Scopus (100) Google Scholar). In the small intestine, it is expressed at highest concentrations in tips of the villi in the jejunum, where it can make up to 5% of the soluble protein (5Bass N.M. Manning J.A. Biochem. Biophys. Res. Commun. 1986; 137: 929-935Crossref PubMed Scopus (100) Google Scholar, 6Bass N.M. Manning J.A. Ockner R.K. Gordon J.I. Seetharam S. Alpers D.H. J. Biol. Chem. 1985; 260: 1432-1436Abstract Full Text PDF PubMed Google Scholar). In the liver, it also comprises up to 5% of the cytoplasmic protein (7Ockner R.K. Manning J.A. Kane J.P. J. Biol. Chem. 1982; 257: 7872-7878Abstract Full Text PDF PubMed Google Scholar).The gene for rat LFABP contains three introns, and the initial study was followed by the insertion of the corresponding cDNA into an expression system (8Lowe J.B. Sacchettini J.C. Laposata M. McQuillan J.J. Gordon J.I. J. Biol. Chem. 1984; 262: 5931-5937Abstract Full Text PDF Google Scholar, 9Sweetser D.A. Lowe J.B. Gordon J.I. J. Biol. Chem. 1986; 261: 5553-5561Abstract Full Text PDF PubMed Google Scholar). The expression system has been used to provide protein for the structural studies described in this report. Previously, introductory structural data was described for LFABP from chicken (10Scapin G. Spadon P. Mammi M. Zanotti G. Monaco H. Mol. Cell. Biochem. 1990; 98: 95-99Crossref PubMed Scopus (54) Google Scholar). Although the crystal structure of chicken liver LFABP suggested that few conformational differences existed between it and other family members, no information was given for any bound fatty acid or on the full structure with side chains (10Scapin G. Spadon P. Mammi M. Zanotti G. Monaco H. Mol. Cell. Biochem. 1990; 98: 95-99Crossref PubMed Scopus (54) Google Scholar).Beyond its ubiquity across a variety of cell types, LFABP is unique within the iLBP family in its broad ligand binding specificity. In addition to fatty acids, their CoA esters, and lysophosphatidic acid, LFABP also binds heme, squalene, certain eicosanoids, bilirubin, and a host of other hydrophobic compounds (3Kaikaus R.M. Bass N.M. Ockner R.K. Experientia. 1990; 46: 617-630Crossref PubMed Scopus (156) Google Scholar, 4Ockner R.K. Manning J.A. Poppenhausen R.B. Ho W.K. Science. 1972; 177: 56-58Crossref PubMed Scopus (516) Google Scholar, 11Rolf B. Oudenampsen-Krüger E. Börchers T. Faergeman N.J. Knudsen J. Lezius A. Spener F. Biochim. Biophys. Acta. 1995; 1259: 245-253Crossref PubMed Scopus (87) Google Scholar, 12Thumser A.E.A. Voysey J.E. Wilton D.C. Biochem. J. 1994; 301: 801-806Crossref PubMed Scopus (115) Google Scholar, 13Kahn S.H. Sorof S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9401-9405Crossref PubMed Scopus (67) Google Scholar, 14Levi A.J. Gatmaitan Z. Area I.M. J. Clin. Invest. 1969; 48: 2156-2167Crossref PubMed Scopus (452) Google Scholar). Whether LFABP binds cholesterol is uncertain. Using radiolabeled cholesterol, a Kd of 1.53 μM and a stoichiometry of 0.83 mol cholesterol/mol of protein has been reported (15Nemecz G. Schroeder F. J. Biol. Chem. 1991; 266: 17180-17186Abstract Full Text PDF PubMed Google Scholar). However, no cholesterol binding was detected by other workers using a similar technique (11Rolf B. Oudenampsen-Krüger E. Börchers T. Faergeman N.J. Knudsen J. Lezius A. Spener F. Biochim. Biophys. Acta. 1995; 1259: 245-253Crossref PubMed Scopus (87) Google Scholar).Another distinguishing feature of LFABP is its ability to bind two fatty acids per protein molecule; other iLBPs bind only one. For the dissociation of oleate from rat and bovine LFABP, one site has a Kd ranging from 0.009 to 0.2 μM, and the other has a Kd of 0.06-4.0 μM (11Rolf B. Oudenampsen-Krüger E. Börchers T. Faergeman N.J. Knudsen J. Lezius A. Spener F. Biochim. Biophys. Acta. 1995; 1259: 245-253Crossref PubMed Scopus (87) Google Scholar, 16Miller K.R. Cistola D.P. Mol. Cell. Biochem. 1993; 123: 29-37Crossref PubMed Scopus (65) Google Scholar, 60Richieri G.V. Ogata R.T. Kleinfeld A.M. J. Biol. Chem. 1994; 269: 23918-23930Abstract Full Text PDF PubMed Google Scholar). The weaker binding was determined by titration calorimetry (11Rolf B. Oudenampsen-Krüger E. Börchers T. Faergeman N.J. Knudsen J. Lezius A. Spener F. Biochim. Biophys. Acta. 1995; 1259: 245-253Crossref PubMed Scopus (87) Google Scholar, 16Miller K.R. Cistola D.P. Mol. Cell. Biochem. 1993; 123: 29-37Crossref PubMed Scopus (65) Google Scholar), while the small Kd values were measured with a fluorescent reagent, itself a fatty acid-binding protein (60Richieri G.V. Ogata R.T. Kleinfeld A.M. J. Biol. Chem. 1994; 269: 23918-23930Abstract Full Text PDF PubMed Google Scholar). In some studies, a third molecule has even been reported to bind to LFABP (17Cistola D.P. Sacchettini J.C. Banaszak L.J. Walsh M.T. Gordon J.I. J. Biol. Chem. 1989; 264: 2700-2710Abstract Full Text PDF PubMed Google Scholar). Considering the unusual stoichiometry of fatty acid binding, the variety of lipids that can bind to LFABP, and the greater solvent accessibility of the backbone amides (18Muga A. Cistola D. Mantsch H. Biochim. Biophys. Acta. 1993; 1162: 291-296Crossref PubMed Scopus (24) Google Scholar), it has been hypothesized that the cavity size of LFABP is larger than that of other members of this family.The multiple binding sites found in LFABP have distinct chemical shifts from the single site in IFABP according to NMR studies using acids (17Cistola D.P. Sacchettini J.C. Banaszak L.J. Walsh M.T. Gordon J.I. J. Biol. Chem. 1989; 264: 2700-2710Abstract Full Text PDF PubMed Google Scholar). the differences it is that the fatty acids are bound to LFABP with their in a more than in the NMR data as a of it has also been suggested that the fatty acid have values bound to In IFABP and other the of bound fatty acids have values because of with arginine residues within the cavity. In the of LFABP, of the are more to the that are at two binding sites (17Cistola D.P. Sacchettini J.C. Banaszak L.J. Walsh M.T. Gordon J.I. J. Biol. Chem. 1989; 264: 2700-2710Abstract Full Text PDF PubMed Google are a of other that LFABP from other iLBP family For example, the by LFABP binds fatty acids also from and J. Mol. Cell. Biochem. 1993; 123: PubMed Scopus (45) Google Scholar). to fluorescent studies, the of fatty acids from LFABP to is on the is the for and J. Mol. Cell. Biochem. 1993; 123: PubMed Scopus (45) Google Scholar). The studies that while and form a with LFABP soluble fatty acids the binding J. Mol. Cell. Biochem. 1993; 123: PubMed Scopus (45) Google calorimetry that a larger of affinity for fatty acids from with binding by IFABP K.R. Cistola D.P. Mol. Cell. Biochem. 1993; 123: 29-37Crossref PubMed Scopus (65) Google Scholar). that the hydrophobic a more in ligand binding to LFABP than it in IFABP and other iLBPs that bind fatty In all iLBP members that bind two are in the binding cavity. The between the and the carboxylate of the ligand is a factor in the to the binding of LFABP is with one of these in other fatty acid-binding It is also from chemical by oleic acid is bound T. Spener F. Mol. Cell. Biochem. 1993; 123: PubMed Scopus Google Scholar). for in ligand binding from site where a to a affinity for fatty acids A.E.A. Voysey J.E. Wilton D.C. Biochem. J. 1994; 301: 801-806Crossref PubMed Scopus (115) Google Scholar, A.E.A. A.F. Wilton D.C. Biochem. J. 1994; PubMed Scopus (38) Google Scholar, A.E.A. Voysey J. Wilton D.C. Biochem. J. PubMed Scopus (24) Google LFABP is of an iLBP family It binds more than mol of The chemical of the ligand can more than members of the fatty acid-binding fatty acids or their are LFABP to have different from other in the iLBP data some of the and studies were in but over a of few were The initial to the structure using molecular replacement were a was used as described INTRODUCTIONLiver fatty acid-binding protein (LFABP), 1The abbreviations used are:LFABPliver fatty acid-binding proteiniLBPintracellular lipid-binding proteinALBPadipocyte lipid-binding proteinIFABPintestinal fatty acid-binding proteinHFABPheart muscle fatty acid-binding proteinL-MFABPlocust muscle fatty acid-binding proteinP2myelin P2 lipid-binding proteinMFB2Manduca sexta fatty acid-binding proteinCRBPIcellular retinol-binding protein ICRBPIIcellular retinol-binding protein IICRABPIcellular retinoic acid-binding protein ICRABPIIcellular retinoic acid-binding protein IIDAUDA11-(5-dimethylaminonaphthalene-1-sulfonyl-amino) undecanoic acidr.m.s.root mean square. is a member of the intracellular lipid-binding protein (iLBP) family (1Banaszak L. Winter N. Xu Z. Bernlohr D.A. Cowan S. Jones T.A. Adv. Protein Chem. 1994; 45: 89-151Crossref PubMed Google Scholar,2Veerkamp J. Maatman R. Prog. Lipid Res. 1995; 34: 17-52Crossref PubMed Scopus (304) Google Scholar). The functions of LFABP, like other family members, are thought to include lipid uptake, lipid transport, regulation of lipid metabolism, and cellular protection by maintaining the concentrations of free cytosolic fatty acids below toxic levels (3Kaikaus R.M. Bass N.M. Ockner R.K. Experientia. 1990; 46: 617-630Crossref PubMed Scopus (156) Google Scholar). The iLBP family is characterized by their small size of approximately 130 amino acids, their affinity for hydrophobic molecules, and their tertiary structure. Both crystal and solution structures have been determined for different iLBP family members that primarily bind fatty acids in adipocytes (ALBP), intestine (IFABP), heart muscle (HFABP), locust muscle (L-MFABP), myelin (P2), and hornworm midgut (MFB2); retinol in liver (CRBPI) and intestine (CRBPII); or retinoic acid in testis (CRABPI) and skin (CRABPII). References to these structural data for the iLBP family are cited in Table III. Although the homology of the amino acid sequences vary, all iLBPs are composed of a 10-stranded, antiparallel β-barrel with two short antiparallel α-helices positioned over one end of the barrel. Within the confines of the β-strands is a cavity that forms the lipid binding site.Table IIILFABP and iLBP structural comparisonProteinCodeCαr.m.s. deviationReferenceÅP21pmp1201.3455Cowan S.W. Newcomer M.E. Jones A.T. J. Mol. Biol. 1993; 230: 1225-1246Crossref PubMed Scopus (160) Google ScholarALBP1adl1221.4556LaLonde J.M. Levenson M.A. Roe J.J. Bernlohr D.A. Banaszak L.J. J. Biol. Chem. 1994; 269: 25339-25347Abstract Full Text PDF PubMed Google ScholarHFABP1hmr1211.4657Young A.C.M. Scapin G. Kromminga A. Patel S.B. Veerkamp J.H. Sacchettini J.C. Structure. 1994; 2: 523-534Abstract Full Text Full Text PDF PubMed Scopus (111) Google ScholarCRBPII1opb1211.4658Winter N.S. Bratt J.M. Banaszak L.J. J. Mol. Biol. 1993; 230: 1247-1259Crossref PubMed Scopus (94) Google ScholarL-MFABP1ftp1241.4959Haunerland N.H. Jacobson B.L. Wesenberg G. Rayment I. Holden H.M. Biochemistry. 1994; 33: 12378-12385Crossref PubMed Scopus (38) Google ScholarCRABPII1cbq1251.6241Kleywegt G.J. Bergfors T. Senn H. Le Motte P. Gsell B. Shudo K. Jones A.T. Structure. 1994; 2: 1241-1258Abstract Full Text Full Text PDF PubMed Scopus (196) Google ScholarCRABPI1cbr1241.6741Kleywegt G.J. Bergfors T. Senn H. Le Motte P. Gsell B. Shudo K. Jones A.T. Structure. 1994; 2: 1241-1258Abstract Full Text Full Text PDF PubMed Scopus (196) Google ScholarCRBPI1crb1261.7055Cowan S.W. Newcomer M.E. Jones A.T. J. Mol. Biol. 1993; 230: 1225-1246Crossref PubMed Scopus (160) Google ScholarIFABP1ifc1251.8161Scapin G. Gordon J.I. Sacchettini J.C. J. Biol. Chem. 1992; 267: 4253-4269Abstract Full Text PDF PubMed Google ScholarMFB21mdc1141.8962Benning M.M. Smith A.F. Wells M.A. Holden H.M. J. Mol. Biol. 1992; 228: 208-219Crossref PubMed Scopus (45) Google Scholar Open table in a new tab The various genes for iLBPs have expression levels that are related to the cell or tissue type. Several of the different genotypes have been cloned and are available in expression vectors. In some instances, measurements have been made of their expression levels in different cell types. For example, LFABP has been detected in abundance in tissue from liver, adipose deposits, myocardium, kidney, and large and small intestinal epithelia of rats (4Ockner R.K. Manning J.A. Poppenhausen R.B. Ho W.K. Science. 1972; 177: 56-58Crossref PubMed Scopus (516) Google Scholar, 5Bass N.M. Manning J.A. Biochem. Biophys. Res. Commun. 1986; 137: 929-935Crossref PubMed Scopus (100) Google Scholar). In the small intestine, it is expressed at highest concentrations in tips of the villi in the jejunum, where it can make up to 5% of the soluble protein (5Bass N.M. Manning J.A. Biochem. Biophys. Res. Commun. 1986; 137: 929-935Crossref PubMed Scopus (100) Google Scholar, 6Bass N.M. Manning J.A. Ockner R.K. Gordon J.I. Seetharam S. Alpers D.H. J. Biol. Chem. 1985; 260: 1432-1436Abstract Full Text PDF PubMed Google Scholar). In the liver, it also comprises up to 5% of the cytoplasmic protein (7Ockner R.K. Manning J.A. Kane J.P. J. Biol. Chem. 1982; 257: 7872-7878Abstract Full Text PDF PubMed Google Scholar).The gene for rat LFABP contains three introns, and the initial study was followed by the insertion of the corresponding cDNA into an expression system (8Lowe J.B. Sacchettini J.C. Laposata M. McQuillan J.J. Gordon J.I. J. Biol. Chem. 1984; 262: 5931-5937Abstract Full Text PDF Google Scholar, 9Sweetser D.A. Lowe J.B. Gordon J.I. J. Biol. Chem. 1986; 261: 5553-5561Abstract Full Text PDF PubMed Google Scholar). The expression system has been used to provide protein for the structural studies described in this report. Previously, introductory structural data was described for LFABP from chicken (10Scapin G. Spadon P. Mammi M. Zanotti G. Monaco H. Mol. Cell. Biochem. 1990; 98: 95-99Crossref PubMed Scopus (54) Google Scholar). Although the crystal structure of chicken liver LFABP suggested that few conformational differences existed between it and other family members, no information was given for any bound fatty acid or on the full structure with side chains (10Scapin G. Spadon P. Mammi M. Zanotti G. Monaco H. Mol. Cell. Biochem. 1990; 98: 95-99Crossref PubMed Scopus (54) Google Scholar).Beyond its ubiquity across a variety of cell types, LFABP is unique within the iLBP family in its broad ligand binding specificity. In addition to fatty acids, their CoA esters, and lysophosphatidic acid, LFABP also binds heme, squalene, certain eicosanoids, bilirubin, and a host of other hydrophobic compounds (3Kaikaus R.M. Bass N.M. Ockner R.K. Experientia. 1990; 46: 617-630Crossref PubMed Scopus (156) Google Scholar, 4Ockner R.K. Manning J.A. Poppenhausen R.B. Ho W.K. Science. 1972; 177: 56-58Crossref PubMed Scopus (516) Google Scholar, 11Rolf B. Oudenampsen-Krüger E. Börchers T. Faergeman N.J. Knudsen J. Lezius A. Spener F. Biochim. Biophys. Acta. 1995; 1259: 245-253Crossref PubMed Scopus (87) Google Scholar, 12Thumser A.E.A. Voysey J.E. Wilton D.C. Biochem. J. 1994; 301: 801-806Crossref PubMed Scopus (115) Google Scholar, 13Kahn S.H. Sorof S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9401-9405Crossref PubMed Scopus (67) Google Scholar, 14Levi A.J. Gatmaitan Z. Area I.M. J. Clin. Invest. 1969; 48: 2156-2167Crossref PubMed Scopus (452) Google Scholar). Whether LFABP binds cholesterol is uncertain. Using radiolabeled cholesterol, a Kd of 1.53 μM and a stoichiometry of 0.83 mol cholesterol/mol of protein has been reported (15Nemecz G. Schroeder F. J. Biol. Chem. 1991; 266: 17180-17186Abstract Full Text PDF PubMed Google Scholar). However, no cholesterol binding was detected by other workers using a similar technique (11Rolf B. Oudenampsen-Krüger E. Börchers T. Faergeman N.J. Knudsen J. Lezius A. Spener F. Biochim. Biophys. Acta. 1995; 1259: 245-253Crossref PubMed Scopus (87) Google Scholar).Another distinguishing feature of LFABP is its ability to bind two fatty acids per protein molecule; other iLBPs bind only one. For the dissociation of oleate from rat and bovine LFABP, one site has a Kd ranging from 0.009 to 0.2 μM, and the other has a Kd of 0.06-4.0 μM (11Rolf B. Oudenampsen-Krüger E. Börchers T. Faergeman N.J. Knudsen J. Lezius A. Spener F. Biochim. Biophys. Acta. 1995; 1259: 245-253Crossref PubMed Scopus (87) Google Scholar, 16Miller K.R. Cistola D.P. Mol. Cell. Biochem. 1993; 123: 29-37Crossref PubMed Scopus (65) Google Scholar, 60Richieri G.V. Ogata R.T. Kleinfeld A.M. J. Biol. Chem. 1994; 269: 23918-23930Abstract Full Text PDF PubMed Google Scholar). The weaker binding was determined by titration calorimetry (11Rolf B. Oudenampsen-Krüger E. Börchers T. Faergeman N.J. Knudsen J. Lezius A. Spener F. Biochim. Biophys. Acta. 1995; 1259: 245-253Crossref PubMed Scopus (87) Google Scholar, 16Miller K.R. Cistola D.P. Mol. Cell. Biochem. 1993; 123: 29-37Crossref PubMed Scopus (65) Google Scholar), while the small Kd values were measured with a fluorescent reagent, itself a fatty acid-binding protein (60Richieri G.V. Ogata R.T. Kleinfeld A.M. J. Biol. Chem. 1994; 269: 23918-23930Abstract Full Text PDF PubMed Google Scholar). In some studies, a third molecule has even been reported to bind to LFABP (17Cistola D.P. Sacchettini J.C. Banaszak L.J. Walsh M.T. Gordon J.I. J. Biol. Chem. 1989; 264: 2700-2710Abstract Full Text PDF PubMed Google Scholar). Considering the unusual stoichiometry of fatty acid binding, the variety of lipids that can bind to LFABP, and the greater solvent accessibility of the backbone amides (18Muga A. Cistola D. Mantsch H. Biochim. Biophys. Acta. 1993; 1162: 291-296Crossref PubMed Scopus (24) Google Scholar), it has been hypothesized that the cavity size of LFABP is larger than that of other members of this family.The multiple binding sites found in LFABP have distinct chemical shifts from the single site in IFABP according to NMR studies using acids (17Cistola D.P. Sacchettini J.C. Banaszak L.J. Walsh M.T. Gordon J.I. J. Biol. Chem. 1989; 264: 2700-2710Abstract Full Text PDF PubMed Google Scholar). the differences it is that the fatty acids are bound to LFABP with their in a more than in the NMR data as a of it has also been suggested that the fatty acid have values bound to In IFABP and other the of bound fatty acids have values because of with arginine residues within the cavity. In the of LFABP, of the are more to the that are at two binding sites (17Cistola D.P. Sacchettini J.C. Banaszak L.J. Walsh M.T. Gordon J.I. J. Biol. Chem. 1989; 264: 2700-2710Abstract Full Text PDF PubMed Google are a of other that LFABP from other iLBP family For example, the by LFABP binds fatty acids also from and J. Mol. Cell. Biochem. 1993; 123: PubMed Scopus (45) Google Scholar). to fluorescent studies, the of fatty acids from LFABP to is on the is the for and J. Mol. Cell. Biochem. 1993; 123: PubMed Scopus (45) Google Scholar). The studies that while and form a with LFABP soluble fatty acids the binding J. Mol. Cell. Biochem. 1993; 123: PubMed Scopus (45) Google calorimetry that a larger of affinity for fatty acids from with binding by IFABP K.R. Cistola D.P. Mol. Cell. Biochem. 1993; 123: 29-37Crossref PubMed Scopus (65) Google Scholar). that the hydrophobic a more in ligand binding to LFABP than it in IFABP and other iLBPs that bind fatty In all iLBP members that bind two are in the binding cavity. The between the and the carboxylate of the ligand is a factor in the to the binding of LFABP is with one of these in other fatty acid-binding It is also from chemical by oleic acid is bound T. Spener F. Mol. Cell. Biochem. 1993; 123: PubMed Scopus Google Scholar). for in ligand binding from site where a to a affinity for fatty acids A.E.A. Voysey J.E. Wilton D.C. Biochem. J. 1994; 301: 801-806Crossref PubMed Scopus (115) Google Scholar, A.E.A. A.F. Wilton D.C. Biochem. J. 1994; PubMed Scopus (38) Google Scholar, A.E.A. Voysey J. Wilton D.C. Biochem. J. PubMed Scopus (24) Google LFABP is of an iLBP family It binds more than mol of The chemical of the ligand can more than members of the fatty acid-binding fatty acids or their are LFABP to have different from other in the iLBP data some of the and studies were in but over a of few were The initial to the structure using molecular replacement were a was used as described