The structure of the TRAPP subunit TPC6 suggests a model for a TRAPP subcomplex

Abstract

The TRAPP (transport protein particle) complexes are tethering complexes that have an important role at the different steps of vesicle transport. Recently, the crystal structures of the TRAPP subunits SEDL and BET3 have been determined, and we present here the 1.7 Angstroms crystal structure of human TPC6, a third TRAPP subunit. The protein adopts an alpha/beta-plait topology and forms a dimer. In spite of low sequence similarity, the structure of TPC6 strikingly resembles that of BET3. The similarity is especially prominent at the dimerization interfaces of the proteins. This suggests heterodimerization of TPC6 and BET3, which is shown by in vitro and in vivo association studies. Together with TPC5, another TRAPP subunit, TPC6 and BET3 are supposed to constitute a family of paralogous proteins with closely similar three-dimensional structures but little sequence similarity among its members.

Figure 1

Overall structure of TPC6. (A) Schematic representation of the TPC6 dimer with labelled helices (red and orange) and strands (green) on a semitransparent molecular surface. Unless stated otherwise, pictures were prepared using PyMOL (DeLano, 2003). (B) Multiple sequence alignment of human TPC6, TPC5 and BET3 (GenBank accession numbers AL833179, BC042161 and AF041432, respectively). The secondary structure elements of TPC6 and BET3 are represented above and below the aligned sequences. Identical, strongly similar and weakly similar residues are highlighted in red, green and blue, respectively. Residues of TPC6 and BET3 marked by an asterisk denote amino acids most strongly involved in dimer formation. Sequences corresponding to the BET3 family motif are enclosed in boxes. The multiple sequence alignment was prepared using CLUSTAL (Higgins et al, 1992).

Figure 2

Stereo image of the TPC6 interaction interface. For both monomers (coloured red and orange), the residues most strongly involved in dimerization are shown. Amino acids from different chains are labelled in bold print and italic font.

Figure 3

Structural comparison of TPC6 (red) and BET3 (cyan). (A) Superposition of the TPC6 and BET3 monomers. (B) Placement of palmitate (yellow) from the hydrophobic cavity of BET3 into the corresponding area of TPC6 after least-squares superposition of the protein chains. Six bulky hydrophobic residues of TPC6 occupy the tunnel accommodating the palmitate in BET3. Residues at the corresponding positions of BET3 are labelled in italics. (C) Flat surfaces of the TPC6 and BET3 dimers. An electrostatic potential map calculated with DELPHI (Rocchia et al, 2001) is projected onto the molecular surface of the proteins using GRASP (Nicholls et al, 1991). Positive and negative potential are coloured blue and red, respectively, at the 10 kT level.

Figure 4

Circular dichroism spectroscopy of BET3 family proteins. Spectra of recombinant TPC6, TPC5 and BET3 were recorded, as described.

Figure 5

Interactions between TPC6, BET3 and TPC5. (A) Histidine (His)-pulldown (PD) of recombinant 7 × His-tagged TPC6 with BET3 and TPC5 expressed in HEK 293 cells. Western blots were probed with anti-Myc and anti-HA antibodies and anti-His-HRP conjugate. (B) TPC6, BET3 and TPC5 were expressed in HEK 293 cells. The immunoprecipitation (IP) of Myc–BET3 led to the co-precipitation of Flag–TPC6 and Flag–TPC5. Western blots were probed with anti-Myc and anti-Flag antibodies. Unspecific Flag antibody bands are marked by asterisks. (C) Crosslinking of TPC6, BET3 and TPC5. Proteins alone and as mixtures were incubated with glutaraldehyde, as described. A BET3 degradation product is marked by an asterisk.

Figure 6

Model of a TPC6–BET3 heterodimer. The structure of this TRAPP subcomplex is derived from a superposition of the TPC6 (red) and BET3 (cyan) dimers.