Abstract
Fusion of the outer mitochondrial membrane is mediated by the dynamin-like GTPase mitofusin (MFN). Here, we determined the structure of the minimal GTPase domain (MGD) of human MFN1 in complex with GDP-BeF3–. The MGD folds into a canonical GTPase fold with an associating four-helix bundle, HB1, and forms a dimer. A potassium ion in the catalytic core engages GDP and BeF3– (GDP-BeF3–). Enzymatic analysis has confirmed that efficient GTP hydrolysis by MFN1 requires potassium. Compared to previously reported MGD structures, the HB1 structure undergoes a major conformational change relative to the GTPase domains, as they move from pointing in opposite directions to point in the same direction, suggesting that a swing of the four-helix bundle can pull tethered membranes closer to achieve fusion. The proposed model is supported by results from in vitro biochemical assays and mitochondria morphology rescue assays in MFN1-deleted cells. These findings offer an explanation for how Charcot–Marie–Tooth neuropathy type 2 A (CMT2A)-causing mutations compromise MFN-mediated fusion.
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Acknowledgements
We thank J. Shaw (University of Utah) and D. Chan (Caltech) for providing the yeast strains and MEF cell lines, Dr. A. M. Prater for proofreading, L. Wu from BL17B for help with structure determination, and the Tsinghua University Branch of China National Center for Protein Sciences (Beijing) for technical support. J.H. is supported by the National Key Research and Development Program (Grant No. 2016YFA0500201), the National Natural Science Foundation of China (Grant No. 31225006), and an International Early Career Scientist grant from Howard Hughes Medical Institute. L.Y. is supported by the National Natural Science Foundation of China (Grant No. 31700659). Z.L. is supported by the National Natural Science Foundation of China (Grant No. 81322023) and the National Basic Research Program of China (973 Program, Grants 2013CB911103, 2014CBA02003 and 2014CB542802).
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Z.L. and J.H. contributed to the overall study design. L.Y., Y.Q., X.H., and C.Y. performed most experiments, with help from L.L. and X.G. Data were analyzed by L.Y., Y.Q., X.H., C.Y., L.L., X.G., Z.R., Z.L., and J.H. The manuscript was written primarily by J.H., with contributions from the other authors.
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Supplementary Figure 1 Comparison of different MFN1 structures.
(A) Crystal packing of the MFN1 MGD in complex with GDP and BeF3-. Different views of molecule packing in the crystal are shown. Three MFN1 molecules (in lime, orange and blue) in the unit cell (green lines) and three from symmetry operation (purple, green and cyan) are shown in cartoon representation. (B) Superposition of the non-crystallographic dimer with the crystallographic dimer. (C) Superposition of the new MGD dimer (in pink) with previous structures (5GOM in cyan and 5GNT in green). (D) Comparison of the dimer interface of the two MGD dimers. Residues at the interface are highlighted and labeled. Buried surface areas and dimensions of the dimers are indicated.
Supplementary Figure 2 Comparison of MFN1 and dynamin superfamily proteins.
(A) Dimers of dynamin superfamily GTPases are shown in surface representation, with two protomers colored in pink and green, respectively. Nucleotide states and conformational states are labeled. BSE, bundle signaling element. (B) Monomers of dynamin superfamily GTPases are shown in cartoon representation and colored as in Fig. 1B. PDB codes and key structural elements are labeled.
Supplementary Figure 3 Catalytic cores of MFN1 and other dynamin superfamily proteins.
(A) Cutaway views of the catalytic core of MFN1, dynamin and ATL1. The nucleotides are shown in sticks and ions in spheres. (B) Catalytic core of MFN1, dynamin and MnmE. Monovalent ions and their ligands are shown. (C) GTPase activity of MFN1 measured with varied [K+]. 5 μM protein was used for each sample. The activities were measured by phosphate release at saturating GTP concentrations (0.5 mM) using the MGD. GTPase activity of MFN1 measured with varied [GTP] is shown on the right. Each point is the mean and SD of eight measurements. (D) As in C, but with 150 mM K+ and varied [Mg2+] and or [Ca2+]. (E) As in C, but with 150 mM K+, 4 mM Mg2+, and varied [Na+]. (F) As in C, but with 150 mM K+, 4 mM Mg2+, and varied pH. (G) As in C, but with various amounts of MFN1-MGD, 150 mM K+ and 4 mM Mg2+. (H) As in C, but with 2 μM proteins, 150 mM K+ or Na+, and 4 mM Mg2+.
Supplementary Figure 4 Nucleotide binding and dimerization of MFN1.
(A) Stopped-flow analysis of mGDP-BeF3- binding to MGD in the presence of different salts. Preincubated mGDP-MGD (10μM mGDP and 60μM MGD) was mixed in a 1:1 ratio with BeF3- in a stopped-flow apparatus. The mant-group was excited at 360nm and fluorescence change was monitored through a 395nm cutoff filter with time. (B) The binding of indicated nucleotides to S85A MFN1 (0.03 mM) was determined by ITC by stepwise injection of a 0.45 mM nucleotide solution. The estimated dissociation constants KD are given. See Supplementary Table 1 for more details. (C) The sizes of wild-type MFN1-MGD (theoretical molecular mass 49.5 kDa) were determined by MALS coupled with gel filtration in the presence of indicated nucleotides. The estimated molecular masses are shown by the right axis. The data are representative of at least three repetitions. (D) The sizes of wild-type MFN1-MGD (theoretical molecular mass 49.5 kDa) in complex with indicated nucleotides were determined by AUC. The estimated molecular masses are shown on top of the peaks. (E) As in B, but with mutants of the dimer interface in the presence of GDP and BeF3-. (F) As in B, but with mutants of the catalytic core in the presence of GDP and BeF3-. (G) HA-tagged MFN1 H147A or Q255A was transfected into MFN1-deleted MEF cells. Its localization was determined by anti-HA antibodies (green) and compared to MitoDsRed using confocal microscopy. The right images show enlargements of the boxed regions. The mitochondria morphology of indicated samples was categorized and shown on the right with data for “wt” and “vector” from Fig. 2F for comparison. A total of 100-120 cells were counted for each sample. All graphs are representative of at least three repetitions. Scale bars, 10 μm.
Supplementary Figure 5 Sequence conservation of MFN, Fzo1p and BDLP.
(A) Sequence alignment of MFNs, Fzo1p and BDLP. Predicted and observed secondary structural elements and GTPase motifs are highlighted (predicted α helices, yellow; predicted β strands, light green; observed α helices, orange; observed β strands, green). The α helices are shown in bars and β strands with arrows. Heptad repeat 1 (HR1) of MFN is underlined in brown, HR2 in purple, and TMs in red. (B) GTPase activity of disease-causing mutants measured with 150 mM K+ and 4 mM Mg2+. 5 μM protein was used for each sample. The activities were measured by phosphate release at saturating GTP concentrations (0.5 mM) using the MGD. Each point is the mean and SD of eight measurements. (C) Expression levels of HA-Fzo1p used in Fig. S5D are shown with PGK as a loading control. (D) Serial dilution assay to analyze the growth of fzo1Δ yeast expressing the indicated alleles of FZO1. Growth on rich dextrose (YPD) and glycerol (YPG) plates is shown. These strains were generated through a plasmid-shuffle strategy in which pRS416-FZO1 is replaced by a pRS315-2XHA-FZO1 plasmid containing the indicated mutations.
Supplementary Figure 6 Controls for FRET-based assay and tethering assay of MFN1.
(A) Structural elements in “HB-open” and “HB-closed” states are superimposed. Key components involved in conformational changes are shown. (B) Purified MGD, with or without YBBR tag, was labelled with CoA-Cy3, and separated by SDS-PAGE, and analysed by fluorescent imaging (top) or Coomassie staining (bottom). (C) GTPase activity of MGD, with or without YBBR tag, was measured by phosphate release at saturating GTP concentrations (0.5 mM). (D) Mitochondrial morphology rescue in MFN1-deleted MEF cells was tested using wt or MFN1 with YBBR tag insertion. E, MFN1-MGD, containing a YBBR tag at either GTPase site or HB1 site, was purified and labelled with CoA-Cy3 or CoA-Cy5 fluorescent dyes using Sfp phosphopantetheinyl transferase. Cy3- and Cy5-labeled proteins were mixed 1:1 (0.5 μM each), and the indicated nucleotide (nt), 50 μM unlabeled MGD, or buffer was added at the indicated time point. FRET was determined by exciting the donor fluorophore (Cy3) at 537 nm and measuring the emission of the donor and acceptor dyes (Cy5) at 570 nm and 667 nm, respectively. FRET is expressed as I A /(I A + I D ), where I A and I D are fluorescence intensities of the acceptor and donor emission, respectively. (F) Purified TM-containing MGDs were analyzed by SDS-PAGE and Coomassie staining.
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Supplementary Text and Figures
Supplementary Figures 1–6 and Supplementary Table 1
Supplementary Video 1
Conformational changes of MFN1–MGD from open to closed state. MFN1–MGD structures are shown in cartoon representation. Key residues that stabilize each state are labelled. Buried surface areas of the dimers are indicated.
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Yan, L., Qi, Y., Huang, X. et al. Structural basis for GTP hydrolysis and conformational change of MFN1 in mediating membrane fusion. Nat Struct Mol Biol 25, 233–243 (2018). https://doi.org/10.1038/s41594-018-0034-8
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DOI: https://doi.org/10.1038/s41594-018-0034-8
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