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. 2015 Oct 30;290(44):26373-82.
doi: 10.1074/jbc.M115.657940. Epub 2015 Sep 11.

Structural basis for the interaction between the Golgi reassembly-stacking protein GRASP65 and the Golgi matrix protein GM130

Fen Hu  1 , Xiaoli Shi  1 , Bowen Li  1 , Xiaochen Huang  1 , Xavier Morelli  2 , Ning Shi  3
Affiliations

Structural basis for the interaction between the Golgi reassembly-stacking protein GRASP65 and the Golgi matrix protein GM130

Fen Hu et al. J Biol Chem. .

Abstract

GM130 and GRASP65 are Golgi peripheral membrane proteins that play a key role in Golgi stacking and vesicle tethering. However, the molecular details of their interaction and their structural role as a functional unit remain unclear. Here, we present the crystal structure of the PDZ domains of GRASP65 in complex with the GM130 C-terminal peptide at 1.96-Å resolution. In contrast to previous findings proposing that GM130 interacts with GRASP65 at the PDZ2 domain only, our crystal structure of the complex indicates that GM130 binds to GRASP65 at two distinct sites concurrently and that both the PDZ1 and PDZ2 domains of GRASP65 participate in this molecular interaction. Mutagenesis experiments support these structural observations and demonstrate that they are required for GRASP65-GM130 association.

Keywords: GM130, GRASP65, PDZ domain, Golgi stacking, vesicle tethering; Golgi; protein assembly; protein complex; protein-protein interaction; vesicles.

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Figures

FIGURE 1.
FIGURE 1.
Sequence alignment and crystal structures of GRASP65. A, sequence alignment of GRASP domains from human (hG65, NP_114105; hG55, Q9H8Y8.3), rat (rG65, AAI61850.1), and mouse (mG55, NP_081628.3). The strands are numbered according to their GRASP55 PDZ domain structure (27). The figure was generated with ESPript (47). B, electron density map of the bound GM130 C-terminal peptide (left) and a sequence alignment of GM130 from human (AAF65550.1), rat (NP_072118.2), mouse (NP_598613), zebra fish fish (XP_005168139.1), honey bee(XP_623918.3), and Drosophila (CAB77666.1) as well as human GOLGIN45 (NP_003657.1) C-terminal peptides (right). C, schematic representation of the overall structure of GRASP65 (colored blue) bound with the GM130 C-terminal peptide (colored salmon). D, schematic representation of the unbound structure of GRASP65 (Protein Data Bank code 4KFV, colored wheat) illustrating the neighboring molecular C terminus (colored salmon) interacting with the PDZ1 classic peptide binding groove.
FIGURE 2.
FIGURE 2.
Conformational changes of GRASP65 upon GM130 C-terminal peptide binding. The software program Chimera was used for superimposing the structures, measuring the angles of rotation of the domains and generating the figures. The gray rod denotes the rotation axis parallel to the paper plane, and the gray spots denote the rotation axis perpendicular to the paper plane. A, the superimposed structures of GRASP65 with and without the GM130 C-terminal peptide using the PDZ1 domain to pinpoint a rotation of 32.6 degrees at the PDZ2 along its axis. The two representations are rotated by 90 degrees. The unbound GRASP65 structure is colored wheat; the bound GRASP65 structure is colored blue. B, measurement of the angle between the PDZ1 and PDZ2 domains of GRASP65 upon GM130 binding. C, measurement of the angle between the PDZ1 and PDZ2 domains of GRASP65 without GM130 binding.
FIGURE 3.
FIGURE 3.
Detailed interactions between GRASP65 and the GM130 C-terminal peptide. A, detailed interactions between GRASP65 and the GM130 C-terminal residues at the conventional peptide-binding groove of the PDZ1 domain. The residues involved in the PDZ1-GM130 peptide interaction are shown with a stick model, in which the GRASP65 and GM130 peptide backbones are colored white and green, respectively, and the nitrogen and oxygen atoms are colored blue and red, respectively. The surface of the GRASP65 PDZ1 domain is also illustrated. B, detailed hydrophobic interactions between GRASP65 and the GM130 C-terminal peptide at the cleft between the PDZ1 and PDZ2 domains. The residues involved in the PDZ1-GM130 peptide hydrophobic interaction are shown with a stick model. The GRASP65 and GM130 peptide backbones are colored white and green, respectively, and the nitrogen and oxygen atoms are colored blue and red, respectively. The GRASP65 surface is also illustrated. C, detailed hydrogen bond interactions between GRASP65 and the GM130 C-terminal peptide at the cleft between the PDZ1 and PDZ2 domains. The schematic represents the backbone structure of GRASP65. The residues involved in the hydrogen bond interaction are shown as a stick model. The GRASP65 and GM130 peptide backbones are colored white and green, respectively, and the nitrogen and oxygen atoms are colored blue and red, respectively. D, stereo view of the GM130 C-terminal peptide inserting into the cleft between the PDZ1 and PDZ2 domains. The residues of the observable portion of the GM130 C-terminal peptide as well as the electron density of the omit map are illustrated (1.5 σ).
FIGURE 4.
FIGURE 4.
ITC experiments and GM130-induced mitochondrial clustering analysis of the GRASP65-GM130 C-terminal peptide interaction. A, the top graph illustrates the raw data of GRASP65 with GM130 C-terminal peptides and their mutants (GM130(F975A), GM130(I990R), and GRASP65(G97D)), and the y axis indicates the heat released per second during GM130 and GRASP65 binding. The bottom graph illustrates the integrated heat for each injection of GRASP65 together with a fit, whereas the y axis represents the heat released per mole for each injection. The dissociation constants were obtained from curves obtained by the titration of GM130 with GRASP65 (WT). 24 h after transfection, HeLa cells expressing the TOM20-Cherry-GM130 F975A mutant (B), the TOM20-Cherry-GM130 I990R mutant (C), and TOM20-Cherry-GM130 WT (D) were analyzed by confocal microscopy. The bar scale is 10 μm. The pixel density and surface area of GM130 signal were quantified using NIH ImageJ version 1.48 software. The results are plotted for the wild type GM130-expressed cells (n = 13), the GM130 F975A mutant expressed cells (n = 14), and the GM130 I990R mutant expressed cells (n = 15).
FIGURE 5.
FIGURE 5.
Oligomerization of the GM130 coiled-coil domain. The coiled-coil region (amino acids 447 to 897) of GM130 was analyzed by 6% Native and SDS-PAGE. A, the GM130 coiled-coil domain analyzed by 6% native PAGE evidences the formation of hexamers. A trace amount of the monomeric form is also evidenced (lane 1). B, the GM130 coiled-coil domain analyzed by 6% SDS-PAGE evidences a monomeric and dimeric form using native loading buffer (lane 2) and a solely monomeric form using SDS loading buffer (lane 3).
FIGURE 6.
FIGURE 6.
The GRASP65 superhelix in the crystal of the complex and the GRASP65 hexameric ring model. The GM130 C-terminal peptide is colored red; GRASP65 is colored green. A, GRASP65 generates a superhelix arranged from top to tail in a circular manner along the crystallographic 61 screw axis. The schematic illustrates one full turn plus one additional molecule of the superhelix, which contains 7 GRASP65 complex molecules in total. B, crystal packing of the bound form of GRASP65 is illustrated above the 61 screw axis. The superhelical arrangement extends along the entire length of the crystal, resulting in long, solvent-filled central channels. C, side view of one superhelix in the crystal of the bound GRASP65. The crystallographic 61 screw axis is illustrated. D, top view of one superhelix. The aligned GM130 C-terminal peptides point to the ring center. For clarity, GRASP65 is represented in various colors. E, ribbon representation of the GRASP65 ring model containing 6 GRASP65 bound molecules. Both the front and side views of the GRASP65 ring model are illustrated. The N termini of GRASP65 are represented as magenta spheres located at the same side of the ring.
FIGURE 7.
FIGURE 7.
Molecular models of GRASP65/GM130/P115-mediated cis-cisternae membrane stacking and vesicle tethering. A, “double wheel” model of GRASP65/GM130/P115-mediated cis-cisternae membrane stacking. The P115 dimer and GM130 hexamer bridge the two rings formed by the six GRASP65 molecules at opposing cisternae membranes. B, “single wheel” model of GRASP65/GM130/P115/Giantin-mediated vesicle tethering. The structure at one side of the cisternae membrane is represented by a double wheel model. At the other side, P115 binds to the Giantin N terminus on the vesicle instead of GM130 and bridges the vesicles and the cisterna membrane.
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