Full-length structural model of RET3 and SEC21 in COPI: identification of binding sites on the appendage for accessory protein recruitment motifs

Abstract

COPI, a 600 kD heptameric complex (consisting of subunits α, β, γ, δ, ε, ζ, and β') "coatomer," assembles non-clathrin-coated vesicles and is responsible for intra-Golgi and Golgi-to-ER protein trafficking. Here, we report the three-dimensional structures of the entire sequences of yeast Sec21 (γ-COPI mammalian ortholog), yeast Ret3 (ζ-COPI mammalian ortholog), and the results of successive molecular dynamics investigations of the subunits and assembly based on a protein-protein docking experiment. The three-dimensional structures of the subunits in their complexes indicate the residues of the two subunits that impact on assembly, the conformations of Ret3 and Sec21, and their binding orientations in the complexed state. The structure of the appendage domain of Sec21, with its two subdomains--the platform and the β-sandwich, was investigated to explore its capacity to bind to accessory protein recruitment motifs. Our study shows that a binding site on the platform is capable of binding the Eps15 DPF and epsin DPW2 peptides, whereas the second site on the platform and the site on the β-sandwich subdomain were found to selectively bind to the amphiphysin FXDXF and epsin DPW1 peptides, respectively. Identifying the regions of both the platform and sandwich subdomains involved in binding each peptide motif clarifies the mechanism through which the appendage domain of Sec21 engages with the accessory proteins during the trafficking process of non-clathrin-coated vesicles.

Fig. 1

Ret3 structural features and frames as extracted from MD trajectories. a Residues of Ret3 present in the template structure (green block), and the missing residues of the template (blue block). b Full-length structural model of Ret3. c, d Structural conformation of Ret3 at 0 ps (gray) superimposed onto the conformations at c 1000 ps (pink) and d 4200 ps (red)

Fig. 2

Tertiary structure of Sec21. a Residues of Sec21 present in the template structure (green block), and the missing residues of the template (blue block). b Cartoon representation of the modeled structure of full-length Sec21, with trunk domain (residues 1–600), linker (residues 601–645), and appendage (646–935). c Model of the Sec21 appendage, with the sandwich subdomain and the fragmented platform subdomain. Fragment 1 is similar to the γ-appendage in γ-COP (gray), while the additional fragment of the appendage, residues 875–935, is fragment 2 (blue)

Fig. 3

The complex of Ret3 and Sec21 as part of the tetrameric F subcomplex obtained from docking. a The complex of Ret3 and Sec21 obtained from protein–protein docking; Ret3 is shown in pink and Sec21 in gray. b A close-up view of the interacting amino acids of the subunits (spheres); amino acids of Sec21 are shown in green, blue, violet, and olive, while those of Ret3 are shown in white)

Fig. 4

Molecular dynamics simulations for the Ret3–Sec21 complex. a Variations in the distances between the centers of mass of the paired groups of amino acids from each of the four interaction nodes (subsites) at the interface of the two subunits during 20 ns of MD simulation. The dashed lines represent the distances between centers of mass for each node in the docking structure (the reference), and the fluctuating lines show the distance variations during MD. The dashed lines relating to node 2 (blue) and node 3 (violet) overlap with the fluctuating MD plot of node 2 (blue) b The amino acids of the two subunits at the binding site in the structure of the complex obtained from docking. The conformation at the interface of the subunits and the interacting amino acids from each node are shown at time steps of c 10 ns and d 16 ns. In both the plot and the figures, node 1 is dark green, node 2 is blue, node 3 is violet, and node 4 is light green

Fig. 5

Binding site on the platform subdomain of the Sec21 appendage. a The deep pocket at the interface of the platform subdomain (gray surface) and the sandwich (green ribbon) on Sec21. b Phe836 and Phe712 are on two opposite sides of the pocket entrance. c Superposition of the conformation of the appendage obtained at 3 ns (orange ribbon) onto the one at 6 ns (green ribbon). d A close up view of the frames at 3 ns and 6 ns. Phe712 and Phe836 block the entrance to the binding pocket at 3 ns by displacing the β8–β9 loop of the sandwich subdomain and the β12–β13 loop of the platform

Fig. 6

Peptide binding sites on the platform subdomain of the Sec21 appendage. a The ∼20 Å deep binding pocket of the platform subdomain and the residues for accommodating a ligand. b Residues of Sec21 that interact with DPW2 (magenta sticks). c Residues of the binding site that interact with DPF (pink sticks). d DPW1 (green sticks) binds to a second binding site on the platform. e Residues of the ∼14 Å deep binding pocket that interact with DPW1

Fig. 7

FXDXF binds to the sandwich subdomain of the Sec21 appendage. a The binding site of FXDXF (red sticks) on the surface of the sandwich subdomain. b Interaction profile of FXDXF with binding site residues (yellow sticks)