MFN1 structures reveal nucleotide-triggered dimerization critical for mitochondrial fusion

Abstract

Mitochondria are double-membraned organelles with variable shapes influenced by metabolic conditions, developmental stage, and environmental stimuli1,2,3,4. Their dynamic morphology is a result of regulated and balanced fusion and fission processes5,6. Fusion is crucial for the health and physiological functions of mitochondria, including complementation of damaged mitochondrial DNAs and the maintenance of membrane potential6,7,8. Mitofusins are dynamin-related GTPases that are essential for mitochondrial fusion9,10. They are embedded in the mitochondrial outer membrane and thought to fuse adjacent mitochondria via combined oligomerization and GTP hydrolysis11,12,13. However, the molecular mechanisms of this process remain unknown. Here we present crystal structures of engineered human MFN1 containing the GTPase domain and a helical domain during different stages of GTP hydrolysis. The helical domain is composed of elements from widely dispersed sequence regions of MFN1 and resembles the ‘neck’ of the bacterial dynamin-like protein. The structures reveal unique features of its catalytic machinery and explain how GTP binding induces conformational changes to promote GTPase domain dimerization in the transition state. Disruption of GTPase domain dimerization abolishes the fusogenic activity of MFN1. Moreover, a conserved aspartate residue trigger was found to affect mitochondrial elongation in MFN1, probably through a GTP-loading-dependent domain rearrangement. Thus, we propose a mechanistic model for MFN1-mediated mitochondrial tethering, and our results shed light on the molecular basis of mitochondrial fusion and mitofusin-related human neuromuscular disorders14.

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Figure 1: Overall structure of MFN1IM.
Figure 2: A tryptophan switch mediates nucleotide binding.
Figure 3: Dimerization of MFN1IM via the G domain.
Figure 4: Catalytic machinery of MFN1.

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Acknowledgements

We thank the staff at beamline BL17U1 of SSRF for the help with the collection of diffraction data, W.-L. Huang and H.-Y. Wang for technical assistance, J. Hu and X. Guo for advice on liposome tethering assay, and O. Daumke for comments on the manuscript. This work was supported by grants of National Basic Research Program of China (2013CB910500), National Natural Science Foundation of China (31200553), Natural Science Foundation of Guangdong Province (2014TQ01R584 and 2014A030312015), New Century Excellent Talents in University (NCET-12-0567) and the Recruitment Program of Global Youth Experts to S.G., and the National Institutes of Health (GM110039 and GM119388) to D.C.C.

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Authors

Contributions

S.G. and D.C.C. conceived the project. Y.-L.C. made the constructs, purified proteins, and performed crystallographic and biochemical experiments. S.M. carried out mitochondrial elongation assays. Y.C. performed ITC measurements and helped with collection of X-ray diffraction data. J.-X.F., B.Y. and Y.-J.L. performed cloning and purification for some of the MFN1IM mutants. D.-D.G. performed some of the SEC-RALS experiments, D.-D.G., J.-Y.Y. and S.L. helped with crystallization experiments. Y.-L.C., S.L. and S.G. solved the structures. Y.-L.C., D.C.C. and S.G. wrote the paper.

Corresponding author

Correspondence to Song Gao.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks M. Ford and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 MFN1 constructs and their biochemical properties.

a, Schematic representation showing the strategy of generating human MFN1 constructs for crystallization. Indication of the labels and numbers are the same as in Fig. 1a. HR1T and HR2T denote truncated HR1 and HR2, respectively. We removed the transmembrane (TM) region and flanking residues from human MFN1 and inserted artificial linkers as illustrated. Three different constructs used for crystallization are named MFN1IMA, MFN1IMB and MFN1IMC, respectively (collectively termed MFN1IM). b, Summary of the crystal structures. ‘Initial ligands’ denotes ligands added to the protein solution before crystallization, whereas ‘final state’ denotes the contents from the refined structure. Resolutions for the structure are specified. c, ITC results showing that MFN1IM constructs have no binding affinity to GMPPNP or GMPPCP. Only the result of the MFN1IMC construct is shown here as representative. d, Electron density of the guanine nucleotides in corresponding structures. The electron density maps are all shown at a contour level of 1.2σ. The residues involved in ligand coordination are shown as ball-and-stick models. e, Details of the MFN1IM active site in the GTP-bound state. Key hydrogen bonds for coordinating the GTP were indicated by dotted lines. In the bottom panel, details of the Mg2+ coordination is depicted. The electron density for Mg2+ ion, water and GTP was shown as grey mesh at a contour level of 1.2σ. f, GTP turnover rates of wild-type MFN1IM and MFN1IM(T109A). MFN1IM(T109A) shows greatly impaired GTPase activity that facilitates the co-crystallization with GTP. Results from two separated experiments are presented for each protein. g, ITC results showing that MFN1IM(T109A) binds both GTP and GDP.

Extended Data Figure 2 Overall structure of MFN1IM.

a, The topology diagrams of the G domains of Ras, MFN1 and BDLP. Secondary structural elements were not drawn to scale and positions of G1–G4 motifs are indicated. Elements of MFN1 are named and coloured as in Fig. 1c. For BDLP, elements extra than Ras in light blue. The helices of BDLP are named as in ref. 21. b, Helical wheel diagrams of HD1. Hydrophobic residues are coloured yellow and other residues are coloured with the corresponding helices as in Fig. 1d. The plots are arranged according to the positions of the four helices of HD1 in the crystal structure, showing a massive hydrophobic core of HD1. c, Intramolecular association of MFN1IM. For the G-domain–HD1 interaction, Leu8, Met76, Val333 and Phe337 embrace Phe11, whereas Lys15 forms a salt bridge with Asp173 and a hydrogen bond with the main chain oxygen of Arg74. The MFN1(L705P) mutant was previously found to be non-functional in mediating mitochondrial fusion13. Leu705 is surrounded by several hydrophobic residues including Ile45, Ile48, Ala362 and Ile708, as well as a salt bridge formed by Arg365 and Glu701. The proline mutation of Leu705 may disrupt α4H and the local hydrophobic interactions, thereby impeding the folding of the protein. d, GTP turnover rates of wild-type MFN1IM and MFN1IM(K15A) and MFN1IM(L705P). Results from two separated experiments are presented for each protein. e, Mitochondria elongation assays of wild-type MFN1 and MFN1(K15A). The Myc-tagged MFN1 constructs were assayed for mitochondrial elongation activity by expression in Mfn1/2-null MEFs, which have completely fragmented mitochondria. Overexpression of wild-type MFN1 in Mfn1/2-null MEFs induces the formation of mostly tubular mitochondria, indicating normal elongation activity, whereas MFN1(K15A) induces substantially less mitochondrial tubulation. Green fluorescence is from immunostaining against the Myc epitope; red fluorescence is from mito-DsRed. The data are quantified on the right. For each construct, 100 cells were scored in biological triplicate; representative images are shown. Error bars indicate s.e.m. Scale bars, 10 μm.

Extended Data Figure 3 Sequence alignment of mitofusins and BDLP.

Sequence alignment of mitofusins and BDLP. Amino acid sequences of human (hs) MFN1 (UniProt accession Q8IWA4) and MFN2 (O95140), mouse (mm) MFN1 (Q811U4) and MFN2 (Q80U63), fruitfly (Drosophila melanogaster, dm) Marf (Q7YU24), fruitfly Fzo (O18412) and BDLP from N. punctiforme (B2IZD3) are aligned using Clustal W43. Residues with a conservation of 100% are in red shades, greater than 80% in green shades and 50% in grey shades, respectively. α-helices are shown as cylinders and β-strands as arrows for both nucleotide-free human MFN1 (above the sequences) and nucleotide-free BDLP (2J, under the sequences). In the case of human MFN1, the secondary structure signs are coloured as in Fig. 1b and labelled as in Fig. 1b–d for MFN1IM regions. Secondary structural elements of the missing HD2 and transmembrane domain predicted from the PHYRE2 server44 (exclusively α-helices) are depicted as shaded cylinders with dashed outlines. For BDLP, the secondary structure signs are coloured grey and labelled according to the previous report21. The G1–G4 elements are specified in the sequences. Key residues on human MFN1 are also indicated, including those involved in the hydrophobic core of HD1 (diamond symbol), hinges (downwards triangle), guanine nucleotide binding and hydrolysis (circle), G interface (upwards triangle), and the plausible HD1–HD2 conformational change (square).

Extended Data Figure 4 Structural comparison of MFN1IM with other dynamin family members.

Structural comparison of nucleotide-free MFN1IMB with nucleotide-free BDLP (PDB code 2J69)21, GDP-bound atlastin-1 (3Q5D)19, nucleotide-free GBP1 (1DG3)45, nucleotide-free dynamin-1 (3SNH)17, nucleotide-free DNM1L (4BEJ)46, nucleotide-free MxA (3SZR)16, and AMPPNP-bound EHD2 (2QPT)15. For these molecules, the region N-terminal to the G domain is in red, the G domain itself in orange, the conventional middle domain is in green, the conventional GTPase effector domain (GED) in marine, the paddle region of BDLP and the pleckstrin homology (PH) domain of dynamin-1 is in cyan, and the Eps15 homology (EH) domain of EHD2 is in magenta. The hinges between the G domains and middle domains are depicted by grey spheres. Nucleotides are shown as ball-and-stick models.

Extended Data Figure 5 Structural comparison of MFN1IM with BDLP and dynamin-1.

a, Structural comparison of the G domains between MFN1IM and BDLP (left) or dynamin-1 (right) in the nucleotide-free state. The MFN1 G domain (coloured as in Fig. 1b) is separately superimposed with G domains of BDLP (PDB code 2J69, light blue) and rat dynamin-1 (2AKA, wheat). The root mean standard deviation (r.m.s.d.) values of aligned Cα atoms are shown. α-helices on the two lobes are labelled. The G domain of MFN1IM resembles the BDLP G domain, except that at lobe 1, dynamin-1 is similar to MFN1IM in lobe 1, but at lobe 2 the αC tilts 60° from its counterpart α2’G in MFN1IM. b, Structural comparison of MFN1IM in different nucleotide-loading states. Structures of nucleotide-free MFN1IMB, GTP-bound MFN1IMC(T109A), transition-like state MFN1IMC and GDP-bound MFN1IMC(T109A) are colour-specified and superimposed on their G domains. c, Architectures of MFN1IMA•GTPγS and MFN1IMA•GDP. Shown are the corresponding Cα traces and electron density maps (contoured at 1.2σ), by which molecule outlines are clearly discernible. These two structures are presented to exclude possible influence of the Thr109Ala mutation in the GTP- and GDP-bound structures shown in b. d, GTP turnover rates of wild-type MFN1IM and the hinge 2 mutants. Results from two experiments are shown for each protein. e, Mitochondrial elongation assay for wild-type MFN1 and the hinge mutants. For each construct, 100 cells were scored in biological triplicate; representative images are shown. Error bars indicate s.e.m. Scale bars, 10 μm. f, Full-length MFN1 models showing the plausible hinge 1 between HD1 and HD2. Models were based on nucleotide-free (PDB code 2J69, top) and GMPPNP-bound (2W6D, bottom) BDLP. G domain and HD1 are coloured as in Fig. 1b; HD2 is in light blue. Hinge 1 is shown as dashed lines. Yellow triangles indicate approximate position for the Pro695 insert. g, Extra support of the guanine base in MFN1 (GDP-bound MFN1IMC(T109A), coloured as in Fig. 1c), BDLP (PDB code 2J68, light blue) and dynamin-1 (5D3Q, wheat). The α-helices that support the guanine base are specified. Parts of the G domains are removed for clarity. Note the similarity between MFN1IM and BDLP, as well as the difference between MFN1IM and dynamin-1 in nucleotide coordination.

Extended Data Figure 6 Dimerization of MFN1IM G domains in the transition-like state.

a, Oligomerization states of MFN1IM in different nucleotide-loading conditions by RALS. MFN1IM is monomeric in nucleotide-free, GTPγS-bound and GDP-bound states, and forms dimers in the presence of GDP•AlF4. Data are as in Fig. 3d. b, Liposome tethering assay for wild-type MFN1IM and corresponding mutants. Representative images from five separate experiments are shown. Wild-type MFN1IM tethered liposomes carrying fluorescence in the presence of GTP hydrolysis-dependent manner as large aggregated liposomes were observed (first left). In GTPγS-present condition the liposome aggregation was largely attenuated, suggesting that tethering is dependent on GTP hydrolysis (second left). When proteins were washed off the liposome by imidazole, the liposomes became homogeneously scattered (middle), indicating that the liposomes were tethered but did not merger. MFN1IM(E209A) and MFN1IM(R238A) displayed suppressed tethering activity (right two). Scale bars, 50 μM. c, Dimerization test of the G interface mutants in the presence of GDP•AlF4. d, GTP turnover rates of the G interface mutants compared with wild-type MFN1IM. Results from two separated experiments are presented for each protein. e, Mitochondrial elongation assay for MFN1(E245A) and related MFN2(E266A). For each construct, 100 cells were scored in biological triplicate; representative images are shown. Error bars indicate s.e.m. Scale bars, 10 μm. Both mutants lost fusogenic activity. f, Rearrangement of residues in the G interface upon nucleotide binding. Structures shown from left to right are: nucleotide-free MFN1IMB; GTP-bound MFN1IMC(T109A); transition-like state MFN1IMC; and GDP-bound MFN1IMC(T109A). Key residues involved in the structural rearrangement of the G interface are shown as ball-and-stick models. Yellow surface representation is used for GTP and GDP.

Extended Data Figure 7 Analysis of the switch I conformations.

a, Configuration of switch I of MFN1IM in nucleotide-free and the transition-like states (molecule A of the dimer is used). Switch I is coloured yellow. Residues involved in the hydrophobic networks are shown as ball-and-stick models. Note the rearrangements of this region between the two states. b, Stability of switch I region of MFN1IM at different states. The stability of switch I is reflected by the mean B factor of the main-chain atoms of switch I compared to that of the whole peptide chain. TransA and TransB denote molecules A and B of the MFN1IMC dimer in the transition-like state, respectively. The switch I regions in both nucleotide-free and transition-like (TransA) states have relatively stable conformations with regard to the whole molecule. c, Superposition of the GTPase catalysis centres of two molecules of the MFN1IMC dimer in the transition-like state. The G1–G4 elements are as in Fig. 2b, except that the G2 element of the molecule B in pale green. His107 and is shown as ball-and-stick models. d, The electron density of the switch I regions in the two molecules of the MFN1IMC dimer. The density is shown as blue mesh at a contour level of 1.2σ for both molecules A (left) and B (right). His107 is shown as ball-and-stick models.

Extended Data Figure 8 Characterization of MFN1ΔTM and the Asp189 trigger.

a, Schematic representation showing the strategy of generating the MFN1ΔTM construct. Colour as in Fig. 1a, and HD2 is in purple. b, Comparison of GTPase activity between MFN1IMC and MFN1ΔTM. Results from two separated experiments are presented for each protein. c, RALS analysis of MFN1ΔTM showing that it is a stable dimer in nucleotide-free state. d, Analytical gel filtration results of MFN1ΔTM in the GTPγS, GDP•AlF4 and GDP-bound states. e, Analytical gel filtration results of MFN1ΔTM(E209A) and MFN1ΔTM(R238A) in nucleotide-free and GDP•AlF4-bound states. Note that in the GDP•AlF4-bound state, no peak at the exclusion volume is observed, indicating that both mutants do not oligomerize. f, Structural comparison of MFN1IM in different nucleotide-loading states at α2G. Note the distinct orientation of Asp189 in the GTP-bound state, and the uniformly oriented Asp193. Asp193 is a conserved residue that also faces the predicted HD2. Colour as in Fig. 4g. g, Electron density of Asp189 and Asp193 on α2G in MFN1IM structures contoured at 1.0σ. Note the difference in orientations of α2G in these structures as revealed by the density maps. Although the side chain of Asp189 is not fully traceable in some non-GTP-bound cases, their locations would differ from that in the GTP-bound form. h, Mitochondrial elongation assay for the mutants in the plausible G-domain–HD2 contact. For each construct, 100 cells were scored in biological triplicate; representative images are shown. Error bars indicate s.e.m. Scale bars, 10 μm. Note that the clumping mitochondria for MFN1(D189A) and anticipated normal mitochondria for MFN1(D193A). Arg455, Arg460, Gln473 and Arg594 are conserved residues in the predicted HD2 which were screened for contacting Asp189 based on sequence alignment of mitofusins and BDLP. Corresponding mutants increased mitochondrial fragmentation or aggregation. It seems that either they are not the right residues interacting with D189, or a single point mutation was not sufficient to break the plausible interaction.

Extended Data Figure 9 Proposed model for MFN1-mediated OMM fusion.

a, Model for nucleotide-regulated OMM fusion mediated by MFN1. The G domain, HD1, predicted HD2 and transmembrane domain are indicated in the top left MFN1 molecule, and coloured orange, green, grey and blue, respectively. During GTP hydrolysis, HD2s of tethered MFN1 molecules may fold back via intrinsic mechanistic potential analogous to the BSE-stalk of MxA protein (Y.C. et al., unpublished observations) to bring opposing membrane in close proximity. Repeating tethering reactions by appropriate numbers of MFN1 would promote docking of opposing OMMs, presumably as described in a recent in vitro electron cryo-tomography study where discrete electron densities representing yeast FZO1 displayed a ring-like arrangement surrounding docked OMMs47. If this ‘docking ring’ exists in mammals, MFN1 may contribute to its formation through hydrolysis-dependent in trans oligomerization (shown in c). Subsequent membrane merger may rely on local membrane curvature, as reported in many cellular events such as synaptic vesicle fusion and cell-to-cell fusion48,49. As the space between docked OMMs (approximately 2 nm) is too small to accommodate MFN1 molecules47, these molecules may gather at the rim of the docking site, resulting in a crowding effect that possibly generates bending on local OMMs to facilitate fusion50,51. b, Schematic drawing shows the GTP-loading-induced conformational rearrangement of the MFN1 HD1–HD2 region via the Asp189 trigger. c, Possible organization of the plausible in trans cross oligomer of MFN1 around the docking site. This process is dependent on GTP hydrolysis.

Extended Data Table 1 Crystallographic data collection and refinement statistics

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Cao, YL., Meng, S., Chen, Y. et al. MFN1 structures reveal nucleotide-triggered dimerization critical for mitochondrial fusion. Nature 542, 372–376 (2017). https://doi.org/10.1038/nature21077

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