Structural insights into oligomerization and mitochondrial remodelling of dynamin 1-like protein - PubMed (original) (raw)

Structural insights into oligomerization and mitochondrial remodelling of dynamin 1-like protein

Chris Fröhlich et al. EMBO J. 2013.

Abstract

Dynamin 1-like protein (DNM1L) mediates fission of mitochondria and peroxisomes, and dysfunction of DNM1L has been implicated in several neurological disorders. To study the molecular basis of mitochondrial remodelling, we determined the crystal structure of DNM1L that is comprised of a G domain, a bundle signalling element and a stalk. DNM1L assembled via a central stalk interface, and mutations in this interface disrupted dimerization and interfered with membrane binding and mitochondrial targeting. Two sequence stretches at the tip of the stalk were shown to be required for ordered assembly of DNM1L on membranes and its function in mitochondrial fission. In the crystals, DNM1L dimers further assembled via a second, previously undescribed, stalk interface to form a linear filament. Mutations in this interface interfered with liposome tubulation and mitochondrial remodelling. Based on these results and electron microscopy reconstructions, we propose an oligomerization mode for DNM1L which differs from that of dynamin and might be adapted to the remodelling of mitochondria.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1

Figure 1

Structure of DNM1L. (A) Structure-based domain architecture of human DNM1L. The first and last residue of each domain is labelled as well as the modified residues for crystallization. The classical predicted domain assignment is shown below. (B) Analytical ultracentrifugation sedimentation velocity experiments for DNM1L, DNM1L ΔB-insert, DNM1L 4A and the combined mutant (4A+ΔB). The relative protein concentration c(s) as function of the normalized sedimentation coefficient _s_20,w is plotted. Dimer peaks for the DNM1L 4A and the combined mutant are indicated. Peaks in the wild-type sedimentation profile could not be assigned to oligomeric species as the protein is in a fast equilibrium between different oligomeric states. (C) Ribbon-type presentation of human DNM1L. Regions not resolved in the crystal structure, such as the B-insert, are indicated by dotted lines. Important structural elements are labelled. (D) The stalks of DNM1L molecules A and C were superimposed. A 17.5° rotation of the G domains around hinge 1 is evident. (E) Side and top view on the DNM1L dimer that assembles via the central stalk interface-2. (F) Close-up view of the dimer interface-2. Hydrogen bonds are indicated as dotted lines.

Figure 2

Figure 2

Dimerization via the stalk mediates oligomerization and mitochondrial remodelling. (A) Analytical ultracentrifugation sedimentation velocity experiments for DNM1L and the indicated interface-2 mutants as described in Figure 1B. Monomer and dimer peaks for E490R and K642E are indicated. As DNM1L, the E490A mutant undergoes rapid exchange reactions between different oligomeric species. (B) Left panel: Sedimentation experiments and liposome binding assays for DNM1L and DNM1L K642E. Sedimentation experiments were performed in the absence and presence of 2 mM GTP-γ-S in the absence of liposomes. Liposome co-sedimentation assays were carried out in the presence of 2 mM GDP, and in the presence or absence of PS liposomes. Lanes from SDS–PAGE are representative for three independent experiments. Boxed lanes are from the same gel. SN, supernatant. P, pellet fraction. Right panel: Quantification of sedimentation and liposome binding assays (error bars represent the s.e.m.). Bars show the percentage of protein found in the pellet with respect of total protein applied on gel. The statistical significance was calculated with respect to the corresponding DNM1L experiments. ***P<0.001; **P<0.01; *P<0.05 (also for all subsequent statistical analyses). (C) Basal and PS liposome-stimulated GTPase activities of DNM1L and the K642E mutant were determined at 37°C (_n_=2 for each experiment, error bars represent the s.e.m.). The statistical significance was calculated with respect to the corresponding DNM1L experiments. (D) Negative-stain electron microscopic analysis of DNM1L in the presence of PS liposomes and different nucleotides. The K642E mutant did not show any tubulation in the absence and presence of nucleotides. (E) Cellular localization and mitochondrial morphology studies in mito-dsRed expressing COS-7 cells. Cells depleted of DNM1L by siRNA were co-transfected with GFP, siRNA-resistant GFP-DNM1L or GFP-DNM1L K642E, respectively. Scrambled siRNA and co-transfected GFP were used as a control. Magnified boxed regions and a line scan plot with the relative fluorescence of the indicated GFP constructs and mito-dsRed are shown to the lower right of each subpanel. Scale bars: 50 μm. (F) Western blot showing efficient siRNA-mediated knock down of endogenous DNM1L. Scrambled siRNA was used as a control. Actin was stained as a loading control. Antibody efficiency was monitored using a COS cell lysate and recombinant DNM1L in a separate western blot. (G) FRAP assay for mitochondrial network connectivity. Mito-dsRed in an ROI (_d_=6 μm) containing multiple mitochondria was photobleached and its fluorescence recovery monitored for 90 s. Curves show mean values from 20 independent experiments under the indicated conditions. Prebleach intensities were normalized. For clarity, only three representative error bars are shown for each experiment. Source data for this figure is available on the online supplementary information page

Figure 3

Figure 3

Functional characterization of the GPRP motif and the B-insert. (A) Ribbon-type representation of the DNM1L monomer illustrating the positions of the B-insert and the GPRP motif, the known post-translational modifications (green, black and magenta) and disease and functionally inactivating mutations (orange). The first and last visible residues of the B-insert and L2S are indicated. (B) Upper panel: Oligomerization experiments and liposome binding assays as in Figure 2B for DNM1L, DNM1L ΔB-insert, DNM1L 4A and the combined DNM1L variant (4A+ΔB). Examples are representative for three independent experiments. SN, supernatant; P, pellet fraction. Lower panel: Quantification of sedimentation and liposome binding assays. (C) Basal and PS liposome-stimulated GTPase activities of DNM1L and DNM1L mutants were determined at 37°C, as in Figure 2C. (D) Cellular localization and mitochondrial morphology studies, as in Figure 2E. COS-7 cells depleted of DNM1L by siRNA were co-transfected with mito-dsRed and siRNA-resistant DNM1L ΔB-insert and the 4A mutant. Magnified boxed regions and a line scan plot with the relative fluorescence of the indicated eGFP fusion proteins and mito-dsRed are shown at the lower right of each subpanel. Scale bars: 50 μm. (E) Mitochondrial network connectivity quantified by an FRAP assay, as in Figure 2G.

Figure 4

Figure 4

DNM1L oligomerizes via an alternative interface. (A) Left and middle panels: Top and side views on a surface representation of the DNM1L oligomer in the crystal. Interface-4 is indicated (white box). The direction of the oligomer is indicated by arrows. Right panel: close-up view of interface-4. Hydrogen bonds are indicated with dotted lines. (B) Oligomerization experiments and liposome binding assays for DNM1L and interface-4 mutants (E426A, R430D), as in Figure 2B. Lanes are representative of three independent experiments. SN, supernatant; P, pellet fraction. (C) Basal and PS liposome-stimulated GTPase activities of DNM1L and interface-4 mutants were determined, as in Figure 2C. (D) Representative negative-stain electron micrographs of DNM1L interface-4 mutants, as in Figure 2D. Scale bars=2 μm. (E) Cellular localization and mitochondrial morphology studies in COS-7 cells depleted of endogenous DNM1L by siRNA and co-transfected with mito-dsRed and siRNA-resistant interface-4 mutants, as in Figure 2E. Scale bars: 50 μm. (F) Mitochondrial network connectivity quantified by an FRAP assay, as in Figure 2G.

Figure 5

Figure 5

Structural model of the DNM1L oligomer. (A) DNM1L tetramers were manually fitted into the EM reconstruction of yeast Dnm1 (Mears et al, 2007). In this model, the stalks of DNM1L are oriented tangentially to the lipid tubule, with the B-insert pointing towards the tubule. Oligomerization of DNM1L into a filament proceeds via stalk interface-1, -2 and -3, as in dynamin and MxA. Additionally, stalk interface-4 mediates assembly of a double stalk filament. G domains could dimerize across neighbouring double filaments. (B) Schematic illustration of the DNM1L molecules in the oligomeric model. The repeating unit of the reconstruction (boxed) contains eight DNM1L monomers.

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