Structural basis for specific lipid recognition by CERT responsible for nonvesicular trafficking of ceramide

Abstract

In mammalian cells, ceramide is synthesized in the endoplasmic reticulum and transferred to the Golgi apparatus for conversion to sphingomyelin. Ceramide transport occurs in a nonvesicular manner and is mediated by CERT, a cytosolic 68-kDa protein with a C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain. The CERT START domain efficiently transfers natural D-erythro-C16-ceramide, but not lipids with longer (C20) amide-acyl chains. The molecular mechanisms of ceramide specificity, both stereo-specific recognition and length limit, are not well understood. Here we report the crystal structures of the CERT START domain in its apo-form and in complex with ceramides having different acyl chain lengths. In these complex structures, one ceramide molecule is buried in a long amphiphilic cavity. At the far end of the cavity, the amide and hydroxyl groups of ceramide form a hydrogen bond network with specific amino acid residues that play key roles in stereo-specific ceramide recognition. At the head of the ceramide molecule, there is no extra space to accommodate additional bulky groups. The two aliphatic chains of ceramide are surrounded by the hydrophobic wall of the cavity, whose size and shape dictate the length limit for cognate ceramides. Furthermore, local high-crystallographic B-factors suggest that the alpha-3 and the Omega1 loop might work as a gate to incorporate the ceramide into the cavity. Thus, the structures demonstrate the structural basis for the mechanism by which CERT can distinguish ceramide from other lipid types yet still recognize multiple species of ceramides.

Fig. 1.

Overall structure of the CERT START domain. (A) The CERT START domain (apo-form) in a ribbon representation. α-Helices (α1-α4), β-strands (β1-β9), and Ω loops (Ω1, Ω2) are numbered from the N to the C terminus. (B–D) Ribbon representation of the CERT START domain in complex with C₆-, C₁₆-, C₁₈-ceramide, respectively. The ceramide molecules are represented as space-filling spheres in which yellow, blue, and red spheres represent C, N, and O atoms, respectively.

Fig. 2.

Schematic representation of C₁₆-ceramide recognition by the CERT START domain. Residues lining the amphiphilic cavity are shown. Red dashed lines, hydrogen bonds; red circles, water molecules; black, blue, and red dots, C, N, and O atoms, respectively, of the residues involved in the hydrogen network. Green boxes indicate residues contributing to the hydrophobicity of the cavity in general, whereas green boxes with thick borders indicate those with direct hydrophobic interactions, which are represented as green dashed lines. Among these, eight amino acid residues, which are common to all of the C₆-, C₁₆-, C₁₈-ceramide complex structures, are indicated by thick-bordered green boxes filled with light green.

Fig. 3.

Molecular surface of the CERT START domain in complex with C₆- (A), C₁₆- (B), and C₁₈- (C) ceramide cut at the level of the cavity, respectively. Ceramide molecules are drawn as sticks, in which yellow, blue, and red represent C, N, and O atoms, respectively. Hydrophobic and polar/charged amino acid residues inside the cavity are shown in green and blue, respectively. The outer surface and the cross-section of the CERT START domain are drawn in gray and in dark brown, respectively.

Fig. 4.

Hydrogen bond network between the CERT START domain and C₁₆-ceramide (A). Large white letters, amino acid residues interacting with the ceramide; small white letters, α-helix and β-sheet strands, numbered; green meshes, ceramide omit map contoured at 2.5 σ; orange dashed lines, hydrogen bonds; red circles, water molecules. In the wire model, N and O atoms are highlighted by blue and red, respectively. The ceramide molecule (yellow) and the side chains of the CERT START domain are shown by wire models. (B) Water-mediated stabilization of O1 oxygen of ceramide viewed in the direction opposite to A.

Fig. 5.

Activities of the CERT START domain. (A) Ceramide extraction activities of the CERT START domain. (B) Ceramide transfer activities of the CERT START domain. The activities of the indicated constructs for C₁₆-ceramide are shown as mean values with standard deviations calculated from triplicate experiments. WW/AA indicates W473A/W562A double mutant.

Fig. 6.

Temperature factors and a putative entrance to the amphiphilic cavity. (A–D) Ribbon diagrams of the CERT START domain colored according to the crystallographic B-factors for the apo-CERT START domain and in complex with C₆-, C₁₆-, and C₁₈-ceramide, respectively. The color bar below A shows the crystallographic B-factor scale. (A–E) The side chains of Trp-473 and Trp-562 are drawn as stick models. The ceramide molecules are drawn as filled spheres. (E) Ribbon representation of the CERT START domain in complex with C₁₈-ceramide. The structure is rotated by 45° around the y axis with respect to those shown in D. α3 and Ω1 loop are colored cyan and magenta, respectively. C₁₈-ceramide is drawn as space-filling spheres, in which yellow, blue, and red spheres represent C, N, and O atoms, respectively. (F) Molecular surface of the CERT START domain in complex with C₁₈-ceramide, drawn in the same orientation as in E. The hydrophobic surface is painted green, and the residues in α3 and Ω1 loop are highlighted as dotted spheres in cyan and magenta, respectively. C₁₈-ceramide is drawn as in E.