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June 9, 2004 Crystal Structure of Human α-Tocopherol Transfer Protein Bound to its Ligand: Implications for Ataxia with Vitamin E DeficiencyK.C. Min1,2,3, R.A. Kovall3, and W.A. Hendrickson1,3 Human α-tocopherol transfer protein (ATTP) plays a central role in vitamin E homeostasis, preventing the degradation of α-tocopherol (α-T), a lipophilic molecule, by routing it for secretion by hepatocytes (liver cells). Mutations in the gene that encodes ATTP have been shown to cause a severe deficiency in α-T, which results in a progressive neurodegenerative spinocerebellar ataxia, a disorder of neurons primarily in the brainstem and spinal cord, known as ataxia with vitamin E deficiency (AVED). We have determined the high-resolution crystal structure of human ATTP with (2R, 4′R, 8′R)-α-T in the binding pocket. One mutation associated with human disease is located directly in the binding pocket, likely disrupting binding to α-T.
ATTP exists in the fluid portion of a cell’s cytoplasm and is expressed mainly in the liver. The heterologous expression of ATTP in cell culture confers its ability to secrete α-T by a non-Golgi pathway, preventing the degradation of α-T. Patients with AVED absorb α-T from their diets normally, but have nearly immeasurable steady-state plasma levels due to the defective incorporation of α-T for transport with very low density lipoprotein. A transgenic mouse in which the ATTP gene was deleted replicated the phenotype of the human disease. There are eight forms of vitamin E (including α-δ tocopherol and α-δ tocotrienol), which differ in two structural features: the degree of methylation (decreasing from α-T to δ-T) of the chroman ring and saturation of the phytyl tail. All forms are potent lipophilic antioxidants, but α-T is the most biologically potent due to the action of ATTP. Of the eight possible stereoisomers, i.e. variations, of α-T, only (2R, 4′R, 8′R)-α-T occurs naturally. Synthetic vitamin E supplements are racemic mixtures, but ATTP is sensitive to stereochemistry and particularly so at the C2 stereocenter.
The high-resolution 1.5Ĺ structure of ATTP in a complex with (2R, 4′R, 8′R)-α-T was determined from multiwavelength anomalous diffraction (MAD) data measured at beamline X4A using a selenomethionyl recombinant protein crystal. The structure of ATTP is composed of two domains: an N-terminal all-helical domain and a C-terminal domain, which at its core is composed of a βαβαβαβ fold and contains the binding pocket for α-T (Figure 1). The most striking feature of the structure is the buried, solvent-inaccessible binding pocket, which would require significant conformational changes to release α-T. Most of the interactions between ATTP and α-T are through van der Waals contacts, although there are three well-ordered water molecules that appear to form a hydrogen bonding network with the hydroxyl group of α-T (Figure 2A). In ATTP, a number of missense mutations – which occur when one DNA base pair is substituted for another – have been associated with AVED. One mutation, L183P – a change from one amino acid, leucine, to another, proline – was found to map directly to the binding pocket (Figure 2A). The sidechain of L183 has extensive van der Waals contacts with α-T, and a change to proline at this position would not only be expected to disrupt these interactions, but would also affect the residues in the nearby α-helix that also form contacts with α-T. When expressed in E. coli, the L183P mutant was insoluble and could not be further characterized.
Three other missense mutations, R59W (arginine-to-tryptophan), R192H (arginine-to-histidine), and R221W, involve a change in positively charged residues, which help to form a prominent positively charged surface on ATTP (Figure 2B). Although the function of this region is not understood, it seems unlikely to affect binding to α-T directly. We have proposed that it may represent a site of protein-protein or protein-lipid interaction that regulates the release of α-T from the binding pocket of ATTP. Interestingly, a similar mutation (R233W) in a related protein, cellular retinaldehyde binding protein, causes a hereditary retinopathy, suggesting that this region of the protein may have a conserved function across multiple members of this family of lipophilic transfer proteins. BEAMLINE FUNDING PUBLICATION FOR MORE INFORMATION |
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facility funded by the U.S. Department of Energy's Office of Basic Energy Science. The NSLS operates two electron
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Ataxia with Vitamin E Deficiency (AVED) is a recessively
inherited condition in which afflicted individuals develop problems
with balance, typically starting in the late teen years. As the
disease progresses, many individuals are limited to a wheelchair,
lose sensation in their hands and feet, and have difficulties with
slurred speech. Some develop retinitis pigmentosa, a blinding
condition caused by the degeneration of the retina. Mutations in α-tocopherol
transfer protein (ATTP) have been shown to cause AVED. 
