A membrane-inserted structural model of the yeast mitofusin Fzo1

Abstract

Mitofusins are large transmembrane GTPases of the dynamin-related protein family, and are required for the tethering and fusion of mitochondrial outer membranes. Their full-length structures remain unknown, which is a limiting factor in the study of outer membrane fusion. We investigated the structure and dynamics of the yeast mitofusin Fzo1 through a hybrid computational and experimental approach, combining molecular modelling and all-atom molecular dynamics simulations in a lipid bilayer with site-directed mutagenesis and in vivo functional assays. The predicted architecture of Fzo1 improves upon the current domain annotation, with a precise description of the helical spans linked by flexible hinges, which are likely of functional significance. In vivo site-directed mutagenesis validates salient aspects of this model, notably, the long-distance contacts and residues participating in hinges. GDP is predicted to interact with Fzo1 through the G1 and G4 motifs of the GTPase domain. The model reveals structural determinants critical for protein function, including regions that may be involved in GTPase domain-dependent rearrangements.

Figure 1

Architecture and domain organization of the Fzo1 model. (a) Scheme showing the domains of Fzo1 from S. cerevisiae. Residue numbers for each domain and the deletion mutants are indicated. The red arrow highlights the deletion mutant that causes a defect in respiration. HRN, N-terminal truncated heptad repeat (violet, residues 101–190); GTPase, GTPase domain (red, residues 194–373); HR1, heptad repeat 1 (green, residues 484–547); transmembrane segments (yellow, residues 706–757), HR2, heptad repeat 2 (orange, residues 769–831). The fragments between the hinges are indicated below the alignment and are designated A to E. (b) Dextrose and glycerol growth spot assay for the 30-, 60- and 91-amino acid N-terminal FZO1 deletion mutant strains, namely fzo1Δ ^1–30, fzo1Δ ^1–60 and fzo1Δ ^1–91, respectively. The fzo1Δ and FZO1-Myc strains were used as negative and positive controls, respectively. (c) Anti-Myc and Anti-Pgk1 immunoblots of whole-cell extracts prepared from the strains used in (b). The FZO1-Myc mdm30Δ strain was used as a control for lack of Fzo1 ubiquitylation. Molecular weight markers are indicated on the right of long or short exposures of the immunoblots. (d) Fzo1 model after the equilibration phase (as described in the Methods section). The blue surface represents POPE and POPC lipids from a portion of the bilayer. The GDP nucleotide and the Mg²⁺ ion are depicted in the space-filled representation.

Figure 2

Stable secondary structure motifs in simulations. Comparison of the Fzo1 domain organization shown in Fig. 1a with the stable secondary structures (persistence greater than 90%) in the three replicate simulations. The colour code is blue, α-helix; red, β-sheet; yellow, turn; green, bend; black, β-bridge; and violet, π-helix.

Figure 3

Insights into the Fzo1 transmembrane domain. (a) PREDDIMER ab initio prediction of the helical dimer (F _SCOR 3.113, crossing angle χ 119.7°). (b,c) Snapshots from the Fzo1.I trajectory showing the most representative structure (i.e., the centroid). Glycine residues within the GxxxG motif are presented as a space-filled representation, whereas residues involved in interactions are depicted in stick form. Phosphate atoms from POPC and POPE (orange) and water molecules (cyan) are indicated, whereas the rest of the protein is omitted for clarity.

Figure 4

Cartoon representation of the Fzo1 model and its functional domains. (top) Residue numbers delimiting the domains, (bottom) secondary structure elements are annotated with the Fzo1 mutations performed in this study. Mutants considered for the charge swap strategy are connected by a bar. The colour code is cyan, loss of function (LOF) and maroon, wild-type phenotypes. Putative hinge regions are indicated by blue arrows. Previously reported mutations across Fzo1 functional domains are shown in Supplementary Fig. 9. Green and pink horizontal bars above the secondary structure elements depict the N- and C-terminal halves, respectively. The topology diagram was generated using the HERA tool.

Figure 5

Swap mutations across the predicted salt bridge D335-K464. (a) Relative positions of D335 (red) and K464 (blue) in the Fzo1 model. The green and pink regions correspond to the N- and C-terminal halves, respectively (Supplementary Fig. 9). (b) Dextrose and glycerol growth spot assay. (c) Anti-Fzo1 and anti-Pgk1 immunoblots of whole-cell extracts prepared from the strains analysed in (b). Molecular weight markers are indicated on the right. (d) Anti-Myc and Anti-Pgk1 immunoblots of whole-cell extracts prepared from the indicated strains. Molecular weight markers are indicated on the right of long or short exposures of the immunoblots.

Figure 6

Swap mutations across the predicted salt bridge D523-H780. (a) Location of D523 (red) and H780 (blue) in the Fzo1 model. The green and pink regions correspond to the N- and C-terminal halves, respectively (Supplementary Fig. 9). The positions of HR1 and HR2 are indicated. (b) Dextrose and glycerol growth spot assay. (c) Immunoblots of whole-cell extracts prepared from the strains analysed in (b). The Pgk1 immunoblot was used as a loading control. Molecular weight markers are indicated on the right.

Figure 7

Critical residues in the Fzo1 model hinge region. (a) Detailed structure of the putative hinge region. The colour code is: cyan: Leu819, yellow: Tyr490, purple: Leu802, red: Glu818, grey: Leu501, and light grey: Leu504. The domains are: violet: HRN, green: HR1, and orange: HR2. The location of this hinge region within the model is presented in the right panel. (b), (c) and (e) Dextrose and glycerol growth spot assay with indicated strains. (d) and (f) Anti-Fzo1 and anti-Pgk1 immunoblots of whole-cell extracts prepared from the strains used in (b), (c) and (e). Molecular weight markers are indicated on the right of immunoblots. The graph in (d) represents the quantified ratio of Mdm30-dependent degradation for wild-type Fzo1 or the Fzo1 E818P mutant.

Figure 8

Analysis of the key protein-GDP interactions in the Fzo1 nucleotide-binding site. The colour code is grey during the equilibration phase, cyan for simulation Fzo1.I, green for Fzo1.II and purple for Fzo1.III. Distance restraints and persistent H-bonds over 50% of the simulation time are indicated with orange and green dotted lines, respectively. The G-boxes from G1 to G4 are highlighted. The evolution of the donor-acceptor distance network for the residues contacting the ligand is presented on the right. The last three blocks show the new interactions observed in Fzo1 with respect to BDLP, and the red line delimits the end of the equilibration phase. Donor-acceptor distances are depicted using a colour gradient from the darkest (below or equal to 3.5 Å) to lightest colours (up to 4.0 Å).

Figure 9

Swap mutations across the predicted salt bridge K200-D313. (a) Location of D313 (red) and K200 (blue) in the Fzo1 model. The most representative structure after the cluster analysis on the Fzo1.I trajectory is shown (i.e., the centroid). The green and pink regions correspond to the N- and C-terminal halves, respectively (Supplementary Fig. 9). The GDP atoms and the bound magnesium are indicated. (b) Dextrose and glycerol growth spot assay. Single mutations across the predicted interaction cause a severe respiration defect, and the double mutant (charge swap) does not rescue these defects. (c) Dextrose and glycerol growth spot assay. The K200A and K200D point mutations affect respiration, whereas the K200R mutation does not impact growth on glycerol media. (d) Anti-Fzo1 immunoblot of whole-cell extracts prepared from the strains used in (c). The Pgk1 immunoblot was used as a loading control. Molecular weight markers are indicated on the right.