Crystal structure of human GGA1 GAT domain complexed with the GAT-binding domain of Rabaptin5

Abstract

GGA proteins coordinate the intracellular trafficking of clathrin-coated vesicles through their interaction with several other proteins. The GAT domain of GGA proteins interacts with ARF, ubiquitin, and Rabaptin5. The GGA-Rabaptin5 interaction is believed to function in the fusion of trans-Golgi-derived vesicles to endosomes. We determined the crystal structure of a human GGA1 GAT domain fragment in complex with the Rabaptin5 GAT-binding domain. In this structure, the Rabaptin5 domain is a 90-residue-long helix. At the N-terminal end, it forms a parallel coiled-coil homodimer, which binds one GAT domain of GGA1. In the C-terminal region, it further assembles into a four-helix bundle tetramer. The Rabaptin5-binding motif of the GGA1 GAT domain consists of a three-helix bundle. Thus, the binding between Rabaptin5 and GGA1 GAT domain is based on a helix bundle-helix bundle interaction. The current structural observation is consistent with previously reported mutagenesis data, and its biological relevance is further confirmed by new mutagenesis studies and affinity analysis. The four-helix bundle structure of Rabaptin5 suggests a functional role in tethering organelles.

Figure 1

GAT–Rabaptin5 complex. (A) Schematic diagram of the constructs of GAT and Rabatin5 fragments. Known domains are shown as boxes with the residue numbers and names (if existing) marked on top. Relevant binding partners are listed below. Regions included in the crystal are shaded. (B) Stereo view of a ribbon diagram of the complex of GGA1 GAT three-helix bundle domain (white) and Rabaptin5^551–661 (blue and green). The crystallographically visible peptide termini are labeled either as N/C or with residue numbers. The major local two-fold axis of the Rabaptin5^551–661 homodimer is depicted as a yellow dash line, and the two-fold axis of the GAT-binding region is shown as a red dash line. (C) Molecular surface model of the complex. The color intensity corresponds to the electrostatic potential calculated with the program GRASP and its default parameters, from −8 kT e⁻¹ (intense red) to +8 kT e⁻¹ (intense blue). Figures 1B, 2A, 4A, and 6 were drawn with the programs MolScript and Raster3D. Figures 1C and 2B were drawn with the program GRASP.

Figure 2

GAT–Rabaptin5 interaction. (A) Stereo view of the interface. Helix backbones are shown in a ribbon representation and colored as white, cyan, and green for GAT and the two Rabaptin5 monomers, respectively. Side chains of residues directly involved in the interface are shown in stick models and colored in blue (for nitrogen atoms), red (oxygen), and magenta (sulfur), or according to their backbone colors (carbon). Helices α₃ and α₄ of GAT are labeled as A3 and A4 (Zhu et al, 2003). The kink of helix α₃ is depicted by the axes of the two portions of the helix (black dash lines). (B) Molecular surface models of the GAT three-helix bundle (left) and N-terminal part of Rabaptin5^551–661 dimer (right). The color scheme is the same as Figure 1C. The interface regions, based on a calculation of buried SAS, are enclosed with yellow lines. Selected residues and (N- or C-) termini are labeled.

Figure 3

Binding affinity between Rabaptin5 fragment and GGA1 GAT domain. (A) GST-mediated pull-down assay. Purified recombinant GST-GGA1^141–326 is immobilized to GSH resin. Rabaptin5^551–661 variants from cell lysate (i.e. samples labeled as ‘before pull-down') were pulled down by the GAT fusion protein and visualized with CBB stain (labeled as ‘after pull-down'). (B) Competition of Rabaptin5^551–661 with full-length Rabaptin5 for GGA1 GAT. GST-GGA1^141–326 (10 μg) were preincubated with GSH-Sepharose beads and cell lysate containing full-length Rabaptin5 in the presence of different concentrations of Rabaptin5^551–661 (0–100 μg) to a final volume of 400 μl. The material bound to the beads was subjected to SDS–PAGE and CBB stain (for GST-GGA1^141–326 and Rabaptin5^551–661) or anti-Rabaptin5 Western blot (for full-length Rabaptin5). The experiment was repeated multiple times and was reproducible.

Figure 4

Rabaptin5 tetramerization. (A) Stereo view of the central section of the four-helix bundle of Rabaptin5^551–661. Backbones of the four helices are shown in ribbon representation and colored in yellow, green, cyan, and white, respectively. Side chains are shown in stick models and their carbon atoms are colored corresponding to their respective backbones. Other atoms are colored as in Figure 2A. Selected residues from the hydrophobic core are labeled. The yellow and white molecules belong to one parallel dimer, as do the cyan and green molecules, whereas the two dimers are antiparallel to each other. (B) Chemical crosslink. Tubes containing equal amounts of His₆-Rabaptin5^551–661 WT (left) or L610^RW/L613^RW mutant (right) were incubated with varied concentrations of BS³ (0–0.6 mM) for 30 min at 22°C. The final crosslinked products were then subjected to 12% homogenous SDS–PAGE and analyzed with anti-His Western blot.

Figure 5

Sequence alignment of Rabaptin5 homolog proteins in the ‘GAT-binding' region. The order from top to bottom is human Rabaptin5 (HR5, residues 551–663), chicken Rabaptin5 (CR5, residues 549–661, GenBank ID BAA21785), and human Rabaptin5β (HR5b, residues 287–383, GenBank ID RNU34932). The region of residues visible in the GAT–Rabaptin5 complex crystal is represented with capital letters, and mobile residues are in lowercase letters. HR5 residue numbers are labeled above the sequence. Residues identical to that of HR5 are boxed. Positions involved in the GAT binding from the two monomers of the Rabaptin5 dimer are marked with open and filled triangles, respectively. Positions involved in the N-terminal dimerization and C-terminal tetramerization (4 Å cutoff) are marked with open and filled circles, respectively. Residues of HR5 subjected to mutagenesis studies are highlighted.

Figure 6

Ribbon diagram of a hypothetical ARF–GAT–(Rabaptin5)₄–GAT–ARF complex. The ARF molecule and ARF–GAT interaction are modeled according to the crystal structure of ARF–GAT peptide complex (PDB file 1J2J). The GAT domain is copied from the crystal structure of GGA1 GAT domain (1OXZ). The GAT–Rabaptin5 interaction and Rabaptin5 tetramer are based on the current crystal structure. Since the two ARF-binding sites are independent of each other, in solution an alterative arrangement is also possible, that is, the right side GAT–ARF complex could flip 180° about the dyad axis of the yellow Rabaptin5 dimer.