In collaboration with scientists from the Gladstone Institute of Cardiovascular Disease (GDIC) in San Francisco, we have recently determined the structural basis for a novel mechanism of defective receptor binding of apolipoprotein E2. The results have been published in Nature Structural Biology 3(8), 718 (1996)
Human apolipoprotein (apo)E plays an important role in cholesterol metabolism through its interaction with the low density lipoprotein (LDL) receptor. The receptor binding region of apoE is located in the vicinity of residues 136-150. The basic residues within this region are thought to interact directly with the LDL receptor. The wild type apoE3 (Arg-158) binds with high affinity to LDL receptors, whereas apoE2 (Cys-158) binds with only 1% of apoE3 activity. The defective apoE2 is an underlying cause of the familial plasma lipid disorder type III hyperlipoproteinemia. Because the Arg/Cys substitution occurs outside the receptor binding region, this substitution was suggested to disrupt receptor binding indirectly.
Cross-eyed stereo picture showing a ribbon model of the ApoE-3 four helix bundle-like structure. The location of the receptor binding region and the key residues Arg 158 and Arg 150 are marked.
In apoE3, Arg-158 forms a salt bridge with Asp-154, whereas in ApoE2 loss of Arg-158 results in the formation of a new salt bridge between Asp-154 and Arg-150. As a result, Arg-150, known to be critical for receptor binding, is shifted out of the receptor binding region, suggesting that the salt bridge rearrangement may be key for disrupting normal binding. To test this hypothesis, Asp-154 was substituted, by site-directed mutagenesis, with Ala to eliminate the 154- 150 salt bridge and the recombinant variant was tested for receptor binding activity. Substitution of Asp-154 resulted in a marked increase in receptor binding activity to near normal levels. The position of the Arg-150 in the mutant protein was determined by x-ray crystallography, demonstrating that Arg- 150 was returned to a position within the receptor binding region identical to that in ApoE3. The crystal structures prove that the defective binding of ApoE2 to the LDL receptor results from the replacement of one salt bridge with another by a novel mechanism.
Stereo view of apoE2-Ala154 mutant structure modeled into experimental electron density. The alanine residue 154 in the center has no salt bridge to Arg 150 and thus allows this important residue to move back into the receptor binding region located at the bottom in this picture.
Contributing Scientists: Karl H. Weisgraber, Li-Ming Dong, Sergei D. Trakhanov, Trey Simmons, Kay S. Arnold, Yvonne M. Newhouse and Thomas L. Innerarity, Gladstone Institute of Cardiovascular Disease (GDIC), San Francisco : Molecular biology and protein engineering
Sean Parkin and Bernhard Rupp, LLNL-BBRP : Crystallography
Click the file names to access the PDB entry of ApoE3 (1nfn.pdb) or of the mutant Asp154Ala (1nfo.pdb).
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Copyright © Bernhard Rupp
Last modified: Dezember 27, 2009 01:40