The Crystal Structure of the Liver Fatty Acid-binding Protein

Thompson, James R.; Winter, N.; Terwey, Daniel P; Bratt, Judy M.; Banaszak, Leonard J.

doi:10.1074/jbc.272.11.7140

Cited by 251 publications

(300 citation statements)

References 64 publications

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“…As expected, myristic acid showed a much lower affinity consistent with previous published work (20). All the fatty acids tested demonstrated a modest cooperativity in terms of fluorescence response, a result consistent with the prediction from the crystal structure that the second fatty acid binding site may not be completely formed until after the primary site is filled (12).…”

Section: Fluorescent Characteristics Of the L28w Tryptophan Mutant-supporting

confidence: 79%

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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: Fluorescent Characteristics Of the L28w Tryptophan Mutant-supporting

confidence: 79%

“…1 is derived from the crystal structure with two bound oleates (12). The oleate at site 1 is buried within the cavity with its carboxyl group neutralized by a hydrogen bonding network linked to Arg-122 (12). In contrast, the oleate at site 2 has the carboxyl group located in the portal region and exposed to the external environment.…”

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confidence: 99%

Tryptophan Insertion Mutagenesis of Liver Fatty Acid-binding Protein

Hagan

Worner-Gibbs

Wilton

2005

Journal of Biological Chemistry

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“…Intestinal and liver FABP have very similar overall conformations, as shown by the comparison of rms distance differences, the superimposition of crystallographic structural models [3], and the secondary structure assignments from their solution structures [10]. The construction of a pair of chimeric proteins by swapping the whole α helical domain between I-and LFABP demonstrated the importance of this domain in the determination of FA transfer mechanism in both proteins.…”

Section: Discussionmentioning

confidence: 99%

“…LFABP is expressed in both small intestine and liver, whereas IFABP is found exclusively in the small intestine [1]. IFABP has a single binding site for long chain FA [2], while LFABP contains at least two FA binding sites [3]. LFABP binds a number of other endogenous hydrophobic ligands [4][5][6][7], whereas IFABP appears to bind exclusively long chain FA [8].…”

Section: Introductionmentioning

confidence: 99%

The integrity of the α-helical domain of intestinal fatty acid binding protein is essential for the collision-mediated transfer of fatty acids to phospholipid membranes

Franchini

Storch

Córsico

2008

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biolog

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SummaryIntestinal FABP (IFABP) and liver FABP (LFABP), homologous proteins expressed at high levels in intestinal absorptive cells, employ markedly different mechanisms of fatty acid transfer to acceptor model membranes. Transfer from IFABP occurs during protein-membrane-collisional interactions, while for LFABP transfer occurs by diffusion through the aqueous phase. In addition, transfer from IFABP is markedly faster than from LFABP. The overall goal of this study was to further explore the structural differences between IFABP and LFABP which underlie their large functional differences in ligand transport. In particular, we addressed the role of the αI-helix domain in the unique transport properties of intestinal FABP. A chimeric protein was engineered with the 'body' (ligand binding domain) of IFABP and the αI-helix of LFABP (α(I)LβIFABP), and the fatty acid transfer properties of the chimeric FABP were examined using a fluorescence resonance energy transfer assay. The results showed a significant decrease in the absolute rate of FA transfer from α(I)LβIFABP compared to IFABP. The results indicate that the αI-helix is crucial for IFABP collisional FA transfer, and further indicate the participation of the αII-helix in the formation of a protein-membrane "collisional complex". Photo-crosslinking experiments with a photoactivable reagent demonstrated the direct interaction of IFABP with membranes and further supports the importance of the αI helix of IFABP in its physical interaction with membranes.

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“…Fritz Spener's group published studies on the binding behavior of L-FABP, with meticulous biochemical proof for the presence of two independent binding sites [6]. As this went against the current dogma of a single fatty acid binding site in FABP, these findings were largely ignored for many years, until finally the three-dimensional structure of L-FABP confirmed his conclusions [7].…”

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confidence: 74%