Secondary structure of ATTP. (A) Ribbon diagram of ATTP viewed down the β-sheet. (B) View after rotation of A by 90° on horizontal axis. α-T is colored in yellow. The N-terminal domain helices are indicated in green, and the C-terminal domain helices are indicated in blue and strands are indicated in red. The 2 F_o – F_c electron density that covers α-T is drawn as a blue mesh at 1 σ. Images were generated with povscript (36) and rendered with povray.

Sequence alignment of four CRAL-TRIO proteins. The sequences of human ATTP and yeast Sec14p were aligned by using structural information, after which the sequences of human CRALBP and supernatant protein factor (SPF) over the relevant region were then also aligned. Secondary structure assignments are indicated above for ATTP and below for Sec14p. White lettering boxed with a red background indicates residues that are identical in all four proteins, and red lettering indicates similar residues. Figure was generated by espript (37).

Comparison of ATTP and Sec14p. (A) Stereo diagram of the C^α backbone of ATTP. α-T is shown in ball-and-stick representation in yellow. Helix 9 (residues 173–184) is in contact with helix 10 (residues 201–213), separated by ≈10 Å. (B) Stereo diagram of the C^α backbone of Sec14p oriented by alignment of the C-terminal domain to ATTP (see Materials and Methods). Two β-OG molecules are shown in ball-and-stick representation in yellow. Helix 10 (residues 184–204) does not have contact with helix 11 (residues 219–231), being separated by ≈25 Å, leading to exposure of the ligand-binding pocket. (C) Stereodiagram of C^α backbone of ATTP in red (residues 100–239) superimposed with sec14p in black (residues 108–257). The amino-terminal domain is excluded for clarity. Images were generated by bobscript (38).

Comparison of molecular surfaces of ATTP and Sec14p. (A) Electrostatic potential surface map of ATTP. The position of helix 10 is indicated. A positively charged cleft is indicated where a number of disease-causing mutations are located. (B) Electrostatic potential surface map of Sec14p. Here, the exposure of the ligand-binding pocket is apparent from the difference in position of helix 11, which is equivalent to helix 10 of ATTP. Surface potentials were calculated in grasp and are indicated from red (–) to dark blue (+)(>+15 kT). Images were rendered with raster3d (39).

Ligand-binding pocket of ATTP. (A) Cut-surface view of the ligand-binding pocket of ATTP. α-T is shown in ball-and-stick representation. The ligand-binding pocket is buried deep from the exposed surface of the protein. Surface was generated in grasp and rendered in raster3d. (B) Close-up view of the ligand binding pocket. Side chains (in green) that form van der Waals contacts with α-T (in yellow) or participate in hydrogen-bonding networks in the ligand-binding pocket are shown. Three well-ordered water molecules located in the binding pocket are shown as red spheres. The protein backbone is shown as a worm model, excluding residues 216–220 and 249–275 for clarity. Electron densities that cover the residues of interest, α-T, and three water molecules are drawn as mesh at 1 σ in the 2 F_o – F_c map. Dashed red lines indicate hydrogen bonds. Image was generated in povscript (36) and rendered in povray. (C) Schematic representation of the ligand-binding pocket. α-T is shown with the C2, C4′, and C8′ stereocenters labeled. The C5 methyl is lacking in γ-T and δ-T, and the C7 methyl is lacking in β-T and δ-T. Tocotrienols contain double bonds at C3′,C7′, and C11′. The carbonyl oxygen atoms of Val-182 and Leu-189 are drawn to illustrate the hydrogen-bond interactions with one of the water molecules. Three well-ordered water molecules located in the binding pocket are depicted as blue circles. Dashed red lines indicate hydrogen bonds, and the lengths of the bonds are indicated. Dashed green lines indicate van der Waals interactions.

Missense mutations associated with AVED. The side chain of residues that are mutated in AVED are shown as stick representations on a worm model of the protein backbone. α-T is shown in yellow. Image was generated in povscript and rendered with povray.