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Structural basis of mitochondrial receptor binding and constriction by DRP1

Abstract

Mitochondrial inheritance, genome maintenance and metabolic adaptation depend on organelle fission by dynamin-related protein 1 (DRP1) and its mitochondrial receptors. DRP1 receptors include the paralogues mitochondrial dynamics proteins of 49 and 51 kDa (MID49 and MID51) and mitochondrial fission factor (MFF); however, the mechanisms by which these proteins recruit and regulate DRP1 are unknown. Here we present a cryo-electron microscopy structure of full-length human DRP1 co-assembled with MID49 and an analysis of structure- and disease-based mutations. We report that GTP induces a marked elongation and rotation of the GTPase domain, bundle-signalling element and connecting hinge loops of DRP1. In this conformation, a network of multivalent interactions promotes the polymerization of a linear DRP1 filament with MID49 or MID51. After co-assembly, GTP hydrolysis and exchange lead to MID receptor dissociation, filament shortening and curling of DRP1 oligomers into constricted and closed rings. Together, these views of full-length, receptor- and nucleotide-bound conformations reveal how DRP1 performs mechanical work through nucleotide-driven allostery.

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Fig. 1: Architecture of the DRP1–MID49 linear filament.
Fig. 2: Key DRP1–MID49 receptor interfaces and regulatory phosphorylation site.
Fig. 3: Nucleotide-driven allosteric elongation of DRP1 exposes MID49 and MID51 receptor binding sites.
Fig. 4: Dynamic instability of the DRP1–MID49 linear assembly and curling into closed DRP1 rings.
Fig. 5: Drp1(G362D) cannot bind MID49 and forms rings exclusively with GMPPCP or GTP.

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Acknowledgements

We thank M. Braunfeld, C. Kennedy, D. Bulkley, and A. Myasnikov and the University of California San Francisco (UCSF) Center for Advanced Cryo-EM, which is supported in part from National Institutes of Health (NIH) grants S10OD020054 and 1S10OD021741 and the Howard Hughes Medical Institute (HHMI). We thank the QB3 shared cluster and NIH grant 1S10OD021596-01, J.-P. Armache, N. Talledge for microscopy advice, C. Greenberg for consulting on structural modelling, D. Winge for discussions and M. Gu for facilitating mass spectrometry of proteins. This work was further supported by a Faculty Scholar grant from the HHMI (A.F.), the Searle Scholars Program (A.F.), NIH grant 1DP2GM110772-01 (A.F.), NIH grants GM53466 and GM84970 (J.M.S.), the Sandler Family Foundation through the UCSF Program for Breakthrough Biomedical Research and the American Asthma Foundation, and the HHMI (R.Y.-R.W., J.M.S. and D.A.A.). R.Y.-R.W. is an HHMI Fellow of the Life Sciences Research Foundation. A.F. is a Chan Zuckerberg Biohub investigator.

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Nature thanks M. Ryan, H. Zhou and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Authors and Affiliations

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Contributions

R.K., J.M.S. and A.F. conceived the study. R.K., A.Y. and P.V.T. performed all experiments. R.K., R.Y.-R.W. and A.F. performed the computational analyses. D.A.A. advised R.Y.-R.W. and R.K. on model building. All authors evaluated the results and edited the manuscript. R.K. and A.F. wrote the manuscript with input from all authors.

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Correspondence to Adam Frost.

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Extended data figures and tables

Extended Data Fig. 1 DRP1 and MID49 assembly states.

aj, DRP1 assembly states visualized with negative-stain electron microscopy in the presence of different guanine nucleotides and MID49(126–454). Both proteins were incubated at concentrations of 2 μM. Scale bars, 100 nm. km, MID49(126–454) and MID51(132–463) form indistinguishable assemblies with DRP1: DRP1 + MID49(126–454) and GMPPCP (k), DRP1 + MID51(132–463) and GMPPCP (l), DRP1 + both equimolar MID49 and MID51 (m). Scale bars, 100 nm.

Extended Data Fig. 2 Cryo-EM and 3D reconstruction.

a, A cryo-EM micrograph of DRP1–MID49(126–454) filaments formed with GMPPCP. Scale bar, 100 nm. Inset, a representative 2D class average. Scale bar, 10 nm. b, Cross-section of the 3D reconstruction of the filament and the distribution of views determined during helical reconstruction. The length of the cylinders and the colour code correspond to the number of particles for that viewing direction (from few to many, blue to red). The 3D structure has been segmented and coloured with DRP1 in grey and MID49 in golden yellow. c, Particle numbers and workflow for the reconstruction protocol. DRP1 density is shown in grey and MID49 is in golden yellow.

Extended Data Fig. 3 Intra- and inter-filament interactions.

a, The triangular structure seen in cross section. Side 1 of the triangular structure has the atomic model placed in the density. The G-domain-to-G-domain contact between adjacent sides is circled. b, The sum of the three sides with the model fit in density. c, Ribbon diagram of the same atomic model as in b. The rotated view shows eight chains each of DRP1 and MID49. The chains further from the reader are rendered transparent. d, An isolated tetramer of DRP1 from the filament, rendered to highlight the stalk interfaces 1, 2 and 3 observed for DRP1 and other GTPases of the dynamin family. e, Expanded view of the circular region in a illustrating a salt bridge between adjacent G domains. fi, Negative-stain micrographs of: DRP1-only wild-type polymers incubated with GMPPCP (f), DRP1 co-assembly with wild-type MID49(126–454) and GMPPCP (g; inset, high-magnification view), DRP1(E116R) mutant polymers (h), DRP1(E116R) mutant co-assembly with MiD49(126–454) (i). Shorter, single-sided filaments predominate. Disordered ‘triangular assemblies’ were also observed, but were much shorter and infrequent compared with the wild-type proteins. Scale bars, 50 nm.

Extended Data Fig. 4 Resolution estimates.

a, b, Local resolution estimates computed by Resmap74. Histogram of voxel values (a), and results in a depicted as a heat map of a cross-section through the reconstruction (b). c, d, Fourier shell correlation plots for the half-maps with and without symmetry (c) and model-to-map correlations for each sub-region of the structure (d).

Extended Data Fig. 5 Rosetta-based model refinement.

a, Complete assembly used for Rosetta-based model building with the asymmetric unit shown in green. b, Atomic B-factors for one asymmetric unit, DRP1 versus MID49 models (ribbon) and bound GMPPCP (space filling). c, Sequence alignment between human and mouse MID49 sequences. d, Overlay of the homology model of human MID49(126–454) (blue, with DRR in yellow, ribbon) modelled within the cryo-EM density overlaid with the mouse MID49 crystal structure (PDB: 4WOY, grey ribbon)37 which was used as a constraint. No density attributable to ADP within the nucleotidyltransferase domain was observed.

Extended Data Fig. 6 Map-to-model fits and role of the L1NS and L2S loops.

ad, Examples of models fit within B-factor-sharpened cryo-EM density for a helix from the DRP1 stalk (a), the backbone of the L1NS loop from the stalk (b), an elongated helix and turn found in MID49 (c) and GMPPCP and Mg within the G domain (d). e, Roles of L1NS and L2S in linear filament formation. Top, an isolated Drp1 tetramer from the cryo-EM model. The circled region is expanded in the lower panel. Bottom, interactions that the conserved loop L1Ns makes within the assembly. G362 is highlighted with an arrow. f, DRP1 stalk and MID49 at receptor interface-3. g, Dyn3 stalk and pleckstrin homology domain from PDB 5A3F40. h, Overlay of f and g.

Extended Data Fig. 7 Sequence conservation and key interaction sites.

a, Multiple sequence alignment of the regions near and including the DRP1 residues mutated in this study: D190, D221 and G362, G363. The residue numbers apply to human DRP1, isoform 2 (UNIPROT identifier: O00429-3 which is also known as DLP1a). b, Sequence alignment of MID49 and MID51 at the region around residue R235 of MID49. R235 of MID49 corresponds to R243 of MID51. c, Sequence alignment of DRP1 isoform 1 and isoform 2 showing the correspondence of S637 (isoform 1) and S611 (isoform 2).

Extended Data Fig. 8 Biochemical and structural characterization of mutants.

ac, Size-exclusion chromatography traces for DRP1 wild-type and mutants used in the study. Comparison between wild-type (WT) and G362D (a), wild-type and D221A (b), and wild-type and D190A (c). d, Gel filtration standards with annotated molecular weights from the same column and chromatography system. eh, DRP1 assembly and co-assembly reactions with GMPPCP for DRP1(D190A) alone (e), DRP1(D190A) + MID49(126–454) (f), DRP1(D221A) alone (g) and DRP1(D221A) + MID49(126–454) (h). Scale bars, 100 nm. il, DRP1(G362D) assembly and co-assembly reactions with GMPPCP or GTP. DRP1(G362D) forms rings but not linear filaments without MID49 (i, k), with MID49 (j, l), with GMPPCP (i, j) or with GTP (k, l). Scale bars, 100 nm.

Extended Data Fig. 9 DRP1(G362D) rings on model membranes.

a, DOPS liposomes used in the study. b, DOPS liposomes after incubation with DRP1(G362D) showing ring-like assemblies on the membrane and in the background. c Lipid nanotubes incubated with DRP1(G362D). d, Power spectrum of the area shown within the dashed square in b. e, Power spectrum of the area shown within the dashed square in c. In both d and e, layer lines indicative of helical geometry are not detectable. Scale bars, 50 nm.

Extended Data Fig. 10 DRP1(G362D) forms 12-dimer closed rings.

a, 2D class average of the rings. The red dashed circle indicates density that may be attributable to the variable domain. b, 2D class average of infrequent, orthogonal or side views used as a constraint during model building. cf, Top (c) and side (d) projections of the model; top (e) and side (f) views of the final model rendered as ribbons. Scale bars, 100 Å. Green, G domain; red, BSE; purple, stalk.

Supplementary information

Supplementary Table 1

Cryo-EM data collection, refinement and validation statistics.

Reporting Summary

Video 1: Nucleotide-induced opening of hinge 2 between the G-domain and Bundle Signaling.

Element (BSE): Green: G-domain, Red: BSE. G2/Switch1 is indicated by an arrow and label. GTP/GMPPCP indicated as labeled. When the nucleotide binds, the G2/Switch1 loop of the G-domain closes like a lid over the nucleotide. This motion propagates through the associated beta sheet to push open hinge 2 of the BSE.

Video 2: Nucleotide-induced opening of hinges 1 and 2, receptor binding versus curling into rings.

Green: G-domain, Red: BSE, Blue: MID49, Yellow on MID49: Dynamin Recruitment Region (DRR), Orange on G-domains: Exposed receptor interfaces. As the G-domains bind a GTP molecule (not shown), an allosteric relay propagates through hinge 1 (between the BSE and the stalk) and hinge 2 (between the G-domain and the BSE) until the G-domain turns ~90degrees and moves ~40Å relative to the stalk. In this nucleotide-bound state, linear copolymers are stabilized by MID49/51 receptor binding. GTP hydrolysis and exchange returns the hinges to the apo conformation and promotes receptor dissociation. Upon nucleotide rebinding in the absence of MID49/51 receptors, the hinges can access a second GTP-dependent conformation that promotes curling by inducing a ~30 degree bending between adjacent interface-2 DRP1 dimers. This is the conformation that leads to closed ring formation.

Video 3: A hypothetical path-dependent allosteric curling reaction to constrict mitochondria.

DRP1: Green, MID49: Blue, white dots: GTP, pink dots: GDP. MID49 recruits tetramers of DRP1GTP, stabilizing a ~linear arrangement of DRP1 stalks and copolymerization of a filament on the outer mitochondrial membrane (OMM). The variable domain, which is rendered as a loop-like region, may also interact with the lipids of the OMM. GTP hydrolysis and exchange returns the hinges to the apo conformation and promotes receptor dissociation. Subsequent rounds of GTP hydrolysis and exchange progressively shorten the filament and enable DRP1’s hinges to access a second, GTP-dependent conformation that promotes curling by inducing a ~30 degree bend between adjacent interface-2 DRP1 dimers. This is the conformation that promotes curling into closed rings as the filaments shorten to an average length of ~12 dimers.

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Kalia, R., Wang, R.YR., Yusuf, A. et al. Structural basis of mitochondrial receptor binding and constriction by DRP1. Nature 558, 401–405 (2018). https://doi.org/10.1038/s41586-018-0211-2

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  • DOI: https://doi.org/10.1038/s41586-018-0211-2

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