Structures of the fungal dynamin-related protein Vps1 reveal a unique, open helical architecture

Abstract

Dynamin-related proteins (DRPs) are large multidomain GTPases required for diverse membrane-remodeling events. DRPs self-assemble into helical structures, but how these structures are tailored to their cellular targets remains unclear. We demonstrate that the fungal DRP Vps1 primarily localizes to and functions at the endosomal compartment. We present crystal structures of a Vps1 GTPase-bundle signaling element (BSE) fusion in different nucleotide states to capture GTP hydrolysis intermediates and concomitant conformational changes. Using cryoEM, we determined the structure of full-length GMPPCP-bound Vps1. The Vps1 helix is more open and flexible than that of dynamin. This is due to further opening of the BSEs away from the GTPase domains. A novel interface between adjacent GTPase domains forms in Vps1 instead of the contacts between the BSE and adjacent stalks and GTPase domains as seen in dynamin. Disruption of this interface abolishes Vps1 function in vivo. Hence, Vps1 exhibits a unique helical architecture, highlighting structural flexibilities of DRP self-assembly.

Figure 1.

Domain architecture of Vps1. Schematic of the domain structures of Vps1 and select other DRPs. Domain coloration shown in this figure is used throughout this study. The Stalk is a composite domain consisting of Middle and GED. sc, S. cerevisiae; ct, C. thermophilum; hs, H. sapiens; rn, Rattus norvegicus; InsA, Insert A; InsB, Insert B. The lengths of the sequences are shown to the right of the schematics.

Figure 2.

Cells lacking Vps1 are defective in vacuolar and endosomal morphology and trafficking. (A) WT (W303A) and Δvps1 cells expressing GFP-FYVE were stained with FM 4–64 and were visualized by confocal microscopy. (B) WT and Δvps4 cells expressing Vps1-EGFP were stained with FM 4–64. (C) Visualization of WT cells expressing Vps1-EGFP and mCherry-FYVE. Vps1 puncta partially colocalized with FYVE puncta (67.8 ± 4.9%). (D) CPY maturation in Δvps1 cells. mCPY is the mature form, and P1 and P2 are intermediates. PGK1 is the loading control. The ratio of mature to total CPY was determined for each sample (n = 3). The means of the ratios of mature to total CPY were significantly heterogeneous (one-way ANOVA, F_4,10 = 10.1; P = 0.002). A Tukey-Kramer post hoc test was used to assess the significance of differences between means: those not significantly different from one another are indicated below the graph (P > 0.05), whereas selected pairs of means that are significantly different are indicated above the graph: *, P < 0.05; **, P < 0.01. Molecular masses are given in kilodaltons. (E) Visualization of cells expressing the indicated Vps1 mutant. Bars, 5 µm.

Figure 3.

In vitro characterization of full-length C. thermophilum Vps1 and a minimal Vps1 GG construct. (A) Growth of WT (W303A) or Δvps1 cells expressing the indicated constructs on YPD plates at 30 or 37°C. The leftmost spot in each case corresponds with 2 µl of a culture of OD₆₀₀ 0.5. Spots to the right of this correspond with 2 µl sequential fivefold dilutions. (B) Lipid nanotubes containing 40% DOPS and 20% PI3P were incubated with C. thermophilum Vps1 before staining and imaging. Top: Naked nanotube. Bottom: Nanotube decorated with Vps1. Bar, 50 nm. (C) Molecular weight determination of C. thermophilum Vps1 GG. Absolute molecular weights (shown in blue across the elution peaks and plotted on the right-hand axes) were determined using SEC-MALS. Elution peaks are plotted on the left-hand axes. Calculated molecular weights of monomers and dimers are shown with dotted red lines. (D) Kinetic analysis of the basal rate of GTP hydrolysis by C. thermophilum Vps1 GG. Initial rates of GTP hydrolysis at 37°C were plotted against the GTP concentration. The fit to the curve yielded kcat and K_M. Each point is shown as mean ± SD (n = 3).

Figure 4.

The crystal structures of C. thermophilum Vps1 GG in complex with GMPPCP, GDP.AlF₄⁻, and GDP. (A–C) The structure of Vps1 GG_GMPPCP. GMPPCP is shown in stick representation, and the Mg²⁺ is shown in green. Ordered waters are shown in red. (D–F) The structure of Vps1 GG_GDP.AlF4-. GDP and AlF₄⁻ are shown as stick representations. Mg²⁺ and Na⁺ are shown as green and purple spheres, respectively. (G–I) The structure of Vps1 GG_GDP. A crystallographic twofold axis is shown as a gray arrow. No ordered density for BSE was observed. Coloring as in A. (B, E, and H) Nucleotide binding pockets for Vps1 GG_GMPPCP, GG_GDP.AlF4-, and GG_GDP shown in A, D, and G with the molecular surface overlaid and shown transparently in gray. (C, F, and I) Details of the nucleotide-binding pockets of the structures shown in A, D, and G. (J) 2F_obs-F_calc maps (contoured at 1 σ) of the nucleotide, nucleotide analogue, or transition state analog of each structure.

Figure 5.

Nucleotide-dependent conformational changes in the Vps1 GG structures. (A) Superposition of Vps1 GG_GMPPCP (pale blue) and GG_GDP (pink). For ease of visualization, residues 270–299 have been omitted from both structures. (B) Superposition of GG_GDP.AlF4- (gold) and GG_GDP (pink). As in A, residues 270–299 have been omitted. (C) An alternative view of the superposition shown in A. (D) Detail of the conformational shifts at the bases of the sheets of the core GTPase fold that bring Y126, W114, Y159, and I86 into alignment in GG_GMPPCP (blue) to create a hydrophobic ridge.

Figure 6.

CryoEM reconstruction of the C. thermophilum Vps1 helical assembly bound to GMPPCP and comparison with dynamin 1 ΔPRD bound to GMPPCP and PS. (A) Effects of nucleotide binding on assembly of purified Vps1. EM projection images of negatively stained Vps. Bars, 100 nm. (B) CryoEM density map of Vps1 bound to GMPPCP at 10.97 Å resolution and shown at a contour level of 1.82 σ. (C and D) Comparison of cross sections through the Vps1 (gray) and dynamin 1 ΔPRD (blue; contoured at 1.38 σ) assemblies. Slices parallel to the helical axis at the widest point of the helix were taken. (E and F) Comparison of the helical parameters of Vps1 and dynamin 1 ΔPRD. The angles subtended by the pairs of red and blue lines illustrate the angles of the GTPases and BSEs to a plane perpendicular to the helical axis, respectively. (G and H) End-on views of the Vps1 and dynamin 1 ΔPRD helices.

Figure 7.

A pseudoatomic model of the C. thermophilum Vps1 assembly. (A) The building blocks used to assemble the pseudoatomic model: the Vps1 GG_GMPPCP dimer and the stalk domain dimer from rat dynamin 1 ΔPRD. (B) Assembled pseudoatomic structure generated by sequential docking and application of the refined helical parameters for the Vps1 GG_GMPPCP dimer and the stalk dimer. The fit of each of the positioned dimers was subsequently locally refined. (C) As for B but shown without the density from the reconstruction. (D) End-on view of the pseudoatomic model overlaid with the density. The protrusion of unfilled density facing the lumen of the helix, likely occupied by the start and end of Insert B, is indicated. (E) Assembly Interfaces 1 and 3 form between adjacent dimers at the stalk region (inner density layer). Four stalk dimers are shown. (F and G) Detail of the Vps1 assembly interfaces. (F) The fit of the pseudoatomic model showing two adjacent dimers within the Vps1 helical assembly, showing the canonical GG GTPase interface and the novel αB interface between neighboring GTPase domains. The C_GED helix, which directly connects to the linker at its C terminus, is labeled. (G) The αB interface, circled in red, is formed by the αB and the β2A–αB loops from related GTPases.

Figure 8.

Disruption of the αB loop interface abrogates Vps1 function in vivo. (A) Plasmids containing C. thermophilum Vps1 harboring mutations in either the αB loop (Loop Mut 1 and 2) or in the αB helix (Control Mut 1 and 2) were expressed in Δvps1 cells. Growth was assessed on YPD plates at 30 or 37°C after 2 d. (B) Vacuolar morphology was assessed in Δvps1 cells expressing the loop and control mutants as in A. Vacuoles were visualized using FM 4–64. (C and D) As in A and B but using corresponding mutants in S. cerevisiae Vps1. Bars, 5 μm.