Analysis of the ligand binding properties of recombinant bovine liver-type fatty acid binding protein

Rolf, Burkhard; Oudenampsen-Krüger, Elke; Börchers, Torsten; Færgeman, Nils J.; Knudsen, Jens; Lezius, Axel G.; Spener, Friedrich

doi:10.1016/0005-2760(95)00170-0

Cited by 102 publications

(83 citation statements)

References 51 publications

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“…The binding data produced an apparent K d value of 0.68 M, a figure that is similar to that obtained using DAUDA displacement studies (11) and in the same affinity range as that reported for fatty acids binding at the secondary site (27)(28)(29). A second tryptophan mutant, M74W, was also used to confirm these observations.…”

Section: Fatty Acyl Coas Can Compete With Fatty Acids For Binding Tosupporting

confidence: 78%

“…In fact, the crystal structure of liver FABP with two bound oleates had already indicated that a fully functional (high affinity) site 2 appeared to depend on the presence of oleic acid at site 1, the primary site (12). The K d values range from 0.009 -0.2 M for the primary site and from 0.06 -4.0 M for the secondary site (27)(28)(29). It has been proposed that the very low K d values that have been quoted result from using fatty acid salts rather than free acids as ligands (30).…”

Section: Discussionmentioning

confidence: 99%

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Tryptophan Insertion Mutagenesis of Liver Fatty Acid-binding Protein

Hagan

Worner-Gibbs

Wilton

2005

Journal of Biological Chemistry

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Liver fatty acid-binding protein (FABP) 1 is a member of a family of structurally related small (14 -15 kDa) cytosolic lipidbinding proteins that also include intestinal, heart (muscle), adipocyte, ileal, keratinocyte, and brain FABP (for recent reviews, see Refs. 1-7). The exact physiological functions of these proteins are unclear, although it is generally thought that they may have a potential role in the uptake and targeting of fatty acids to various intracellular organelles and metabolic pathways. All further sites of metabolism of long chain fatty acids in the cell involve membrane proteins. For a targeting role to operate, the FABP must interact with an intracellular structure such as a membrane interface or receptor/docking protein. Targeting could be modulated by the presence of bound fatty acid and be affected by the nature of the fatty acid such as chain length and the degree of unsaturation. In addition, FABP knock-out studies in mice have highlighted the physiological importance of acyl CoAs as ligands for liver FABP (8), and hence competition between these ligands and fatty acids could influence targeting of liver FABP.Evidence for membrane binding has been provided in the case of intestinal, muscle, and adipose FABP. Model fluorescence studies have led to the proposal for interaction-mediated transfer of long-chain fatty acids between the protein and a phospholipid (reviewed in Ref. 3). However, such a process was not observed to operate for liver FABP under the same assay conditions, and an aqueous phase diffusion mechanism was proposed, not requiring interaction of the protein with membrane surfaces (3). In contrast, using different assay conditions involving low ionic strength buffers we have reported the apparent binding of liver FABP to anionic vesicles, monitored as the release of the fluorescent fatty acid ligand (DAUDA) from the protein (9, 10).We have previously demonstrated that the binding of liver FABP to anionic vesicles involves nonspecific electrostatic interactions (9). As a result of studies using charge reversal mutagenesis, the cationic residues on the protein surface that make a significant contribution to such binding have been identified as Lys-31, Lys-36, and Lys-57 (10). These residues are strategically placed around the portal region of the proteins in positions of enhanced protein mobility. Our studies also highlighted the role of cationic residues in ligand binding at site 2 of liver FABP particularly ligands with more bulky anionic head groups such as acyl CoAs, lysophospholipids, and bile acids (11).To investigate further the effect of ligand and membrane binding on the conformation of liver FABP linked to a targeting role for this protein we have used a strategy of tryptophan insertion mutagenesis. Positions 28, 54, and 74 (see Fig. 1) were initially selected for mutagenesis to tryptophan because these positions are strategically placed surrounding the portal region. Fig. 1 is derived from the crystal structure with two bound oleates (12). The oleate at site 1 is buri...

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Section: Fatty Acyl Coas Can Compete With Fatty Acids For Binding Tosupporting

confidence: 78%

Section: Discussionmentioning

confidence: 99%

Tryptophan Insertion Mutagenesis of Liver Fatty Acid-binding Protein

Hagan

Worner-Gibbs

Wilton

2005

Journal of Biological Chemistry

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“…Using radiolabeled cholesterol, a K d of 1.53 M and a stoichiometry of 0.83 mol cholesterol/mol of protein has been reported (15). However, no cholesterol binding was detected by other workers using a similar technique (11).…”

mentioning

confidence: 81%

“…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 (3,4,(11)(12)(13)(14). Whether LFABP binds cholesterol is uncertain.…”

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confidence: 99%

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The Crystal Structure of the Liver Fatty Acid-binding Protein

Thompson

Winter

Terwey

et al. 1997

Journal of Biological Chemistry

253

274

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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-turnhelix that caps one end of the ␤-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.

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Determination of the secondary structural elements of chicken liver fatty acid binding protein by two-dimensional homonuclear NMR

1999

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A conformational study in solution of the fatty acid binding protein from chicken liver is presented. The nearly complete sequence‐specific 1H resonance assignment was achieved from homonuclear two‐dimensional nmr experiments using a sample of native protein. The principal elements of secondary structure were identified: 10 antiparallel β‐strands and one helical segment followed by a turn comprising 5 residues. These elements correspond closely with those of the crystal structure of the related protein, and two new secondary structural features obtained from the nmr data are the β‐sheet conformation between the first and the last β‐strand in the protein sequence, as well as a helical loop at the N‐terminus of the polypeptide chain. © 1999 John Wiley & Sons, Inc. Biopoly 50: 1–11, 1999

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Analysis of the ligand binding properties of recombinant bovine liver-type fatty acid binding protein

Cited by 102 publications

References 51 publications

Tryptophan Insertion Mutagenesis of Liver Fatty Acid-binding Protein

Tryptophan Insertion Mutagenesis of Liver Fatty Acid-binding Protein

The Crystal Structure of the Liver Fatty Acid-binding Protein

Determination of the secondary structural elements of chicken liver fatty acid binding protein by two-dimensional homonuclear NMR

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