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Specificity determinants for lipids bound to beta-barrel proteins
Amy J Reese1, Leonard J Banaszak
1Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, USA.
Journal of Lipid Research
|November 5, 2003
Summary
Altering a key protein loop in adipocyte lipid-binding protein (ALBP) changed how it binds fatty acids. This structural modification impacts ligand positioning and protein stability, offering insights into lipid-binding protein function.
Area of Science:
- Structural biology
- Protein biochemistry
- Lipid metabolism
Background:
- Intracellular lipid transport proteins share a conserved 10-stranded beta-barrel structure with an internal binding cavity.
- Conformational differences in loop regions, specifically the E-F loop, distinguish fatty acid-binding proteins (e.g., adipocyte lipid-binding protein, ALBP) from retinoid-binding proteins (e.g., cellular retinoic acid-binding protein I, CRABP I).
Purpose of the Study:
- To investigate the functional impact of altering the E-F loop in ALBP to mimic CRABP I.
- To analyze the structural consequences of this mutation on ligand binding and protein conformation using X-ray crystallography.
Main Methods:
- Site-directed mutagenesis was employed to introduce a three-residue mutation into wild-type ALBP (WT-ALBP) to create EF-ALBP.
- X-ray crystallography was used to determine the crystal structures of ligand-free EF-ALBP and EF-ALBP bound to oleic acid at 1.5 Å and 1.7 Å resolution, respectively.
- Comparative analysis of the mutant structures with previously determined WT-ALBP structures.
Main Results:
- The three-residue mutation in the E-F loop of ALBP altered the positioning of the bound C18 fatty acid (oleic acid) within the binding cavity, as evidenced by electron density.
- Crystallographic data allowed for direct comparison of protein conformation and ligand positioning between EF-ALBP and WT-ALBP.
- Preliminary chemical characterization revealed alterations in the binding properties and overall stability of the mutant EF-ALBP protein.
Conclusions:
- Modifications in a single loop region of ALBP can significantly influence fatty acid binding and positioning.
- Structural insights from EF-ALBP suggest that ligand atoms tend to occupy planar regions within the binding cavities of both fatty acid and retinoid binding proteins.
- The study provides a structural basis for understanding how subtle conformational changes affect the function and stability of lipid-binding proteins.