Structure of the Membrane-tethering GRASP Domain Reveals a Unique PDZ Ligand Interaction That Mediates Golgi Biogenesis

Abstract

Biogenesis of the ribbon-like membrane network of the mammalian Golgi requires membrane tethering by the conserved GRASP domain in GRASP65 and GRASP55, yet the tethering mechanism is not fully understood. Here, we report the crystal structure of the GRASP55 GRASP domain, which revealed an unusual arrangement of two tandem PDZ folds that more closely resemble prokaryotic PDZ domains. Biochemical and functional data indicated that the interaction between the ligand-binding pocket of PDZ1 and an internal ligand on PDZ2 mediates the GRASP self-interaction, and structural analyses suggest that this occurs via a unique mode of internal PDZ ligand recognition. Our data uncover the structural basis for ligand specificity and provide insight into the mechanism of GRASP-dependent membrane tethering of analogous Golgi cisternae.Golgi biogenesis involves membrane tethering by two multifunctional proteins, GRASP65 and GRASP55, which are differentially localized to cis and medial Golgi cisternae, respectively. GRASP65 is associated with cis Golgi cisternae via both binding to the cis-localized golgin GM130 and insertion of its myristoylated N terminus (1, 2). GRASP55 is primarily on medial Golgi cisternae and binds medial-localized golgin-45 and other proteins and is myristoylated and palmitoylated (3, 4). Each protein contains a conserved N-terminal GRASP domain that mediates self-association, which results in the formation of homotypic tethering complexes that link analogous cisternae in adjacent ministacks (5-8).The GRASP region is predicted to contain two PDZ-like domains (4, 9). PDZ domains are ubiquitous globular proteinprotein interaction modules featuring a hydrophobic binding groove, which interacts with the C terminus of its target ligand, although recognition of internal sequences has also been observed (10, 11). Although recent work supports the presence of PDZ domains within the GRASP module (8), secondary structure predictions indicate significant mismatches to the typical organization of ␤-strands and ␣-helices found in eukaryotic PDZs.Toward elucidating the structural mechanism of GRASPmediated tethering, we solved the crystal structure of the GRASP domain of GRASP55. Although the GRASP domain was indeed composed of two PDZ domains, the domains were circularly permuted, resulting in overall folds that were structurally more similar to prokaryotic PDZs. This unusual arrangement of a metazoan PDZ revealed that the key ␤2 strands of the binding grooves lay outside of the previously predicted PDZ-like regions. Significantly, an internal ligand sequence mapped in GRASP65 that binds to its first PDZ domain formed a surface projection that appeared to fit inside a deep depression within the PDZ1-binding pocket. Taken together, these data suggest a unique internal PDZ ligand interaction on opposite sides of the molecule and imply a multimeric tethering mechanism that mediates Golgi biogenesis. MATERIALS AND METHODSConstructs-GFP-ActA (8) was cloned into GRASP55 pCS-2 (12) using XbaI. To generate His-tagged GRASP55, GR...

Structural Insight into Golgi Membrane Stacking by GRASP65 and GRASP55 Proteins

“…This construct of GRASP55 is very similar to GRASP65(1-210) and seven residues longer than the one used in previous studies, in which the structure of the tandem PDZ domains of GRASP55 was reported (17). The conformation of the PDZ domains in our structure of GRASP55 is nearly identical to that in the previous structure (Fig.…”

“…Our crystal structures show that the CT of the GRASP proteins interacts with the PDZ1 domain in just such a manner; even the unconventional PDZ2-PDZ2 association generates an antiparallel ␤-sheet using the two ␤5s and utilizes hydrophobic packing via a Phe from ␣2. In the previous structural studies of GRASP55, the PDZ1 domain was speculated to bind to an internal ligand, residues 196 -199 of ␤5 in the PDZ2 domain, whereas the PDZ2 domain engaged the C terminus of GM130 (17). Consistent with this proposal, hydrophobic residues from ␣2 and ␤5 of the PDZ1 domain (Leu-59 and Ile-100 in GRASP55) and part of ␤5 of the PDZ2 domain (residues 196 -199) were shown to be critical for the tethering ability of GRASP55 (17).…”

“…In the previous structural studies of GRASP55, the PDZ1 domain was speculated to bind to an internal ligand, residues 196 -199 of ␤5 in the PDZ2 domain, whereas the PDZ2 domain engaged the C terminus of GM130 (17). Consistent with this proposal, hydrophobic residues from ␣2 and ␤5 of the PDZ1 domain (Leu-59 and Ile-100 in GRASP55) and part of ␤5 of the PDZ2 domain (residues 196 -199) were shown to be critical for the tethering ability of GRASP55 (17). In addition, the residues involved in the GRASP65-GM130 association were mapped to ␤5 in the PDZ2 domain (residues 189 -201) (6).…”

“…Mutagenesis studies have suggested that the PDZ domains of GRASP proteins may bind to an internal ligand (16,17). A crystal structure of the GRASP domain of GRASP55 was reported recently (17). The structure reveals canonical folding of the PDZ domains, but no relevant intermolecular interactions were identified.…”

“…The PDZ domain is known to be a peptide-binding module with a hydrophobic cleft formed between an ␣-helix and a ␤-strand as a binding pocket (15). Mutagenesis studies have suggested that the PDZ domains of GRASP proteins may bind to an internal ligand (16,17). A crystal structure of the GRASP domain of GRASP55 was reported recently (17).…”