Structural basis for the nucleotide-dependent dimerization of the large G protein atlastin-1/SPG3A

Abstract

The large GTPase atlastin belongs to the dynamin superfamily that has been widely implicated in facilitating membrane tubulation, fission, and in select cases, fusion. Mutations spread across atlastin isoform 1 (atlastin-1) have been identified in patients suffering from hereditary spastic paraplegia (HSP), a neurodegenerative disorder affecting motor neuron function in the lower extremities. On a molecular level, atlastin-1 associates with high membrane curvature and fusion events at the endoplasmic reticulum and cis-Golgi. Here we report crystal structures of atlastin-1 comprising the G and middle domains in two different conformations. Although the orientation of the middle domain relative to the G domain is different in the two structures, both reveal dimeric assemblies with a common, GDP-bound G domain dimer. In contrast, dimer formation in solution is observed only in the presence of GTP and transition state analogs, similar to other G proteins that are activated by nucleotide-dependent dimerization. Analyses of solution scattering data suggest that upon nucleotide binding, the protein adopts a somewhat extended, dimeric conformation that is reminiscent of one of the two crystal structures. These structural studies suggest a model for nucleotide-dependent regulation of atlastin with implications for membrane fusion. This mechanism is affected in several mutants associated with HSP, providing insights into disease pathogenesis.

Fig. 1.

Structure of atlastin-1. (A) Domain organization of atlastin-1. The large G domain (orange) is connected to the middle domain (blue) by a short flexible linker (green) and is followed by two transmembrane helices (black) that span the membrane and a C-terminal domain. A topological model is shown (Right). The fragment used in this study consists of residues 1–446 of human atlastin-1. (B) Protomer structure of atlastin-1 in crystal form 1. (Inset) GDP/Mg²⁺-coordinating residues as sticks. (C) Protomer structure of atlastin-1 in crystal form 2. The G domain is shown in a similar orientation as shown in B. GDP and Mg²⁺ are shown as sticks and spheres, respectively.

Fig. 2.

Crystallographic dimers of atlastin-1. (A) GBP1. A model for GTP-bound, full-length GBP1 was constructed according to ref. . (B) Dynamin. A dynamin G domain-GED fusion dimer bound to is shown (PDB ID code 2X2E) (4). (C) Atlastin-1. Dimers observed in crystal lattices with P2₁2₁2₁ symmetry (form 2, Upper) and with P6₅22 symmetry (form 1, Lower) are shown. GDP and Mg²⁺ positions are indicated and are shown as sticks and spheres, respectively. Hypothetical membrane positions based on the location of the C terminus of the cytosolic domain are indicated. Positions of missense mutation associated with HSP are shown as residues displayed as red spheres in both forms. (D) Nucleotide arrangement and G domain dimer interface. The G domain dimer interaces of atlastin-1 (form 1; Left) and GBP1 (Right; PDB ID code 2BC9) (3) are shown. The protomers are colored in light and dark gray, with the nucleotide-binding regions (G1–G4) shown in shades of blue. Nucleotide and Mg²⁺ are shown as sticks and spheres, respectively. The dashed, red line separates the dimer halves, marking the interfacial region.

Fig. 3.

Atlastin-1 oligomerization in solution. SEC-MALS data are shown for wild-type atlastin-1 (residues 1–446) in its apo-state (red) or bound to GppNHp (green), GDP (purple), or GDP•AlF_x (orange). The signal from the 90°-scattering detector and refractive index detector are shown as solid, colored lines and black dashed lines, respectively (Left, Y axis). Average molecular weight as calculated every second across the protein elution peak is shown as black circles (Right, Y axis). Theoretical molecular weights based on primary sequence for the monomer and dimer are indicated as horizontal, dashed lines. Proteins (20 μM) were incubated with nucleotides (2 mM) prior to SEC-MALS analysis.

Fig. 4.

Small-angle X-ray scattering. (A) Solution scattering data for atlastin-1. Intensity plots and P(r) functions for apo (red), GDP-bound (purple), GppNHp-bound (green), and GDP•AlF_x-bound (orange) atlastin-1 (residues 1–446) are shown. (Left, Inset) Corresponding Guinier plots. (B) SAXS-based shape reconstructions for nucleotide-bound solution states of atlastin-1. The dimeric form 2 crystal structure was docked in the models as a reference and is shown as a cartoon presentation.

Fig. 5.

Characterization of HSP-associated mutations in atlastin-1. (A) Mapping of HSP mutant residues that affect dimerization (red and orange) onto the crystal structures of atlastin-1. Protomers are colored in light and dark gray. R²¹⁷ (red) is located in the nucleotide-binding pocket. Dimer interface mutant R⁷⁷ (blue) is not associated with HSP but was chosen based on the crystallographic dimer interface. (B) Nucleotide-dependent dimerization of HSP-associated atlastin-1 mutants. Molecular weight distributions for GppNHp- or AlF_x-bound atlastin-1 variants (residues 1–446) were determined by static multiangle light scattering. Errors correspond to calculated errors in the fitting function parameters. (C) GTPase activity. GTPase activity was determined by measuring the production of inorganic phosphate upon GTP hydrolysis.