Effect of Charge Reversal Mutations on the Ligand- and Membrane-binding Properties of Liver Fatty Acid-binding Protein

Abstract

. Using a strategy of charge reversal mutagenesis, the potential role of specific cationic residues in promoting interfacial binding of FABP to anionic phospholipid vesicles has been investigated. Cationic residues chosen included those within the ␣-helical region (Lys-20, Lys-31, and Lys-33) and those that make a significant contribution to the positive surface potential of the protein (Lys-31, Lys-36, Lys-47, Lys-57, and Arg-126). Only three cationic residues make a significant contribution to interfacial binding, and these residues (Lys-31, Lys-36, and Lys-57) are all located within the ligand portal region, where the protein may be predicted to exhibit maximum disorder. The binding of tryptophan mutants, F3W, F18W, and C69W, to dioleoylphosphatidylglycerol vesicles, containing 5 mol% of the fluorescent phospholipid dansyldihexadecanoylphosphatidylethanolamine, was monitored by fluorescence resonance energy transfer (FRET). All three mutants showed enhanced dansyl fluorescence due to FRET on addition of phospholipid to protein; however, this fluorescence was considerably greater with the F3W mutant, consistent with the Nterminal region of the protein coming in close proximity to the phospholipid interface. These results were confirmed by succinimide quenching studies. Overall, the results indicate that the portal region of liver FABP and specifically Lys-31, Lys-36, and Lys-57 are involved in the interaction with the interface of anionic vesicles and that the N-terminal region of the protein undergoes a conformational change, resulting in DAUDA release.Liver fatty acid-binding protein (FABP) 1 is a member of a family of structurally related small (14 -15 kDa) cytosolic lipid binding proteins that also include intestinal, heart (muscle), adipocyte, ileal, keratinocyte, and brain FABP (for recent reviews, see Refs. 1-6). 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.A process involving membrane binding has been advocated for intestinal, muscle, and adipose FABP where model fluorescence studies have led to the proposal of a collisional mechanism for explaining the FABP-mediated transfer of fatty acids between phospholipid membranes and vesicles (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 is proposed, not requiring interaction of the protein with membrane surfaces (3). In contrast, using different assay conditions 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 protei...

Tryptophan Insertion Mutagenesis of Liver Fatty Acid-binding Protein

“…The discrepancy with DAUDA displacement studies probably reflects the fact that L28W is reporting on site 2 with an apparent K d value for fatty acids (published range 0.06 -4.0 M) that is an order of magnitude higher than the site 1 K d (published range 0.009 to 0.2 M) (27)(28)(29), whereas DAUDA displacement reports on site 1 (10). Presumably, the replacement of a fatty acid at site 2 by oleoyl CoA compulsorily also results in loss of the fatty acid including DAUDA from a perturbed site 1 to allow the bulky acyl chain of the oleoyl CoA to be accommodated within the binding cavity.…”

“…The titration profiles are not compatible with the apparent K i values that were determined in previous studies. Thus, using DAUDA displacement, an apparent K i value of 1 M was obtained for oleoyl CoA (11), whereas in contrast the apparent K i value for oleic acid was 0.04 M (10). A greater than 20-fold difference in K i between oleic acid and oleoyl CoA should dramatically affect the shape of the fluorescence changes with the L28W mutant with oleoyl CoA being a poor displacer of oleic acid.…”

“…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).…”

“…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.…”

Probing the Interaction of Brain Fatty Acid Binding Protein (B-FABP) with Model Membranes

“…In the diffusional-dependent mechanism, the FA molecule is released from its binding site when the protein is near the acceptor membrane and the FA diffuses in the aqueous phase. There are studies showing that the nature of the vesicle lipid composition influences the diffusional-dependent mechanism [34] and suggest that the membrane-water interface is responsible for inducing protein conformational changes with subsequent release of the FA molecule [35], [36].…”