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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. Cryo-electron microscopy is used to resolve the structure of human dynamin-related protein 1 co-assembled with its receptor mitochondrial dynamics protein of 49 kDa, along with an analysis of structure- and disease-based mutations.

Ban et al.  show that optic atrophy 1 (OPA1) and cardiolipin mediate mitochondrial fusion. In contrast, a homotypic trans -OPA1 interaction independent of cardiolipin mediates membrane tethering to form mitochondrial cristae. Mitochondria are highly dynamic organelles that undergo frequent fusion and fission. Optic atrophy 1 (OPA1) is an essential GTPase protein for both mitochondrial inner membrane (IM) fusion and cristae morphology 1 , 2 . Under mitochondria-stress conditions, membrane-anchored L-OPA1 is proteolytically cleaved to form peripheral S-OPA1, leading to the selection of damaged mitochondria for mitophagy 2 , 3 , 4 . However, molecular details of the selective mitochondrial fusion are less well understood. Here, we showed that L-OPA1 and cardiolipin (CL) cooperate in heterotypic mitochondrial IM fusion. We reconstituted an in vitro membrane fusion reaction using purified human L-OPA1 protein expressed in silkworm, and found that L-OPA1 on one side of the membrane and CL on the other side are sufficient for fusion. GTP-independent membrane tethering through L-OPA1 and CL primes the subsequent GTP-hydrolysis-dependent fusion, which can be modulated by the presence of S-OPA1. These results unveil the most minimal intracellular membrane fusion machinery. In contrast, independent of CL, a homotypic trans -OPA1 interaction mediates membrane tethering, thereby supporting the cristae structure. Thus, multiple OPA1 functions are modulated by local CL conditions for regulation of mitochondrial morphology and quality control.

Key Points Dynamin, the founding member of a family of dynamin-like proteins (DLPs) implicated in membrane remodelling, has a critical role in endocytic membrane fission events. The use of complementary approaches, including live-cell imaging, cell-free studies, X-ray crystallography and genetic studies in mice, has greatly advanced our understanding of the mechanisms by which dynamin acts. The mechanisms by which dynamin drives membrane fission have been the subject of intense debate. Recent crystallographic and cryo-electron microscopy studies of dynamin and DLPs support a model in which dynamin polymerization serves to bring two GTPase domains together, which allows GTP hydrolysis and the conformational changes in dynamin that are necessary for helix constriction and membrane fission. The role of dynamin is best defined during clathrin-dependent endocytosis and is essential only for a late step when membrane fission occurs. Gene-knockout studies in mice and the cells derived from them have provided numerous insights into dynamin function and the specific roles of the three dynamin isoforms. Dynamin 2 is ubiquitously expressed and has a housekeeping role in membrane dynamics. By contrast, dynamin 1 and dynamin 3 are specific to the nervous system and, although neither is essential for supporting a specific form of endocytosis at synapses, they may be important for allowing clathrin-mediated endocytosis to function over a very broad range of neuronal activities. Roles of abnormal dynamin function in genetic disease have begun to emerge. Whereas mutations in dynamin 2 show links to tissue-specific diseases, mutations in dynamin 1 specifically affect the nervous system. The dynamin GTPase mediates membrane remodelling during endocytosis. Through complementary approaches, including structural and genetic studies, the mechanisms by which dynamin regulates membrane fission events, and the unique physiological roles of its three isoforms, are becoming clear. Dynamin, the founding member of a family of dynamin-like proteins (DLPs) implicated in membrane remodelling, has a critical role in endocytic membrane fission events. The use of complementary approaches, including live-cell imaging, cell-free studies, X-ray crystallography and genetic studies in mice, has greatly advanced our understanding of the mechanisms by which dynamin acts, its essential roles in cell physiology and the specific function of different dynamin isoforms. In addition, several connections between dynamin and human disease have also emerged, highlighting specific contributions of this GTPase to the physiology of different tissues.

Dominant optic atrophy is one of the leading causes of childhood blindness. Around 60–80% of cases 1 are caused by mutations of the gene that encodes optic atrophy protein 1 (OPA1), a protein that has a key role in inner mitochondrial membrane fusion and remodelling of cristae and is crucial for the dynamic organization and regulation of mitochondria 2 . Mutations in OPA1 result in the dysregulation of the GTPase-mediated fusion process of the mitochondrial inner and outer membranes 3 . Here we used cryo-electron microscopy methods to solve helical structures of OPA1 assembled on lipid membrane tubes, in the presence and absence of nucleotide. These helical assemblies organize into densely packed protein rungs with minimal inter-rung connectivity, and exhibit nucleotide-dependent dimerization of the GTPase domains—a hallmark of the dynamin superfamily of proteins 4 . OPA1 also contains several unique secondary structures in the paddle domain that strengthen its membrane association, including membrane-inserting helices. The structural features identified in this study shed light on the effects of pathogenic point mutations on protein folding, inter-protein assembly and membrane interactions. Furthermore, mutations that disrupt the assembly interfaces and membrane binding of OPA1 cause mitochondrial fragmentation in cell-based assays, providing evidence of the biological relevance of these interactions. Cryo-electron microscopy structures of OPA1, mutations of which are associated with the disease dominant optic atrophy, provide insight into how structural features of OPA1 enable this protein to mediate mitochondrial-membrane fusion and remodelling.

In translation elongation, two translational guanosine triphosphatase (trGTPase) factors EF1A and EF2 alternately bind to the ribosome and promote polypeptide elongation. The ribosomal stalk is a multimeric ribosomal protein complex which plays an essential role in the recruitment of EF1A and EF2 to the ribosome and their GTP hydrolysis for efficient and accurate translation elongation. However, due to the flexible nature of the ribosomal stalk, its structural dynamics and mechanism of action remain unclear. Here, we applied high-speed atomic force microscopy (HS-AFM) to directly visualize the action of the archaeal ribosomal heptameric stalk complex, aP0•(aP1•aP1)₃ (P-stalk). HS-AFM movies clearly demonstrated the wobbling motion of the P-stalk on the large ribosomal subunit where the stalk base adopted two conformational states, a predicted canonical state, and a newly identified flipped state. Moreover, we showed that up to seven molecules of archaeal EF1A (aEF1A) and archaeal EF2 (aEF2) assembled around the ribosomal P-stalk, corresponding to the copy number of the common C-terminal factor-binding site of the P-stalk. These results provide visual evidence for the factor-pooling mechanism by the P-stalk within the ribosome and reveal that the ribosomal P-stalk promotes translation elongation by increasing the local concentration of translational GTPase factors.

Optogenetic and chemogenetic control of proteins has revealed otherwise inaccessible facets of signaling dynamics. Here, we use light- or ligand-sensitive domains to modulate the structural disorder of diverse proteins, thereby generating robust allosteric switches. Sensory domains were inserted into nonconserved, surface-exposed loops that were tight and identified computationally as allosterically coupled to active sites. Allosteric switches introduced into motility signaling proteins (kinases, guanosine triphosphatases, and guanine exchange factors) controlled conversion between conformations closely resembling natural active and inactive states, as well as modulated the morphodynamics of living cells. Our results illustrate a broadly applicable approach to design physiological protein switches.

Crystal structures of engineered human MFN1 in different stages of GTP hydrolysis provide insights into the GTP-induced conformational changes that promote MFN1 dimerization to bring about mitochondrial fusion. Mitofusin structure provides clues to mitochondrial fusion Mitochondrial fusion, which is essential for the functionality of these organelles, requires the activity of mitofusins, which are GTPases related to dynamin. Mitofusins are found in the mitochondrial outer membrane, so the oligomerization of mitofusins on different mitochondria, together with GTPase activity, results in organelle fusion. By resolving the crystal structure of truncated human mitofusin 1, Song Gao and colleagues provide insights into the GTP-induced conformational changes that promote MFN1 dimerization to bring about mitochondrial fusion. Their observations are relevant to disorders caused by mutations in mitofusin. Mitochondria are double-membraned organelles with variable shapes influenced by metabolic conditions, developmental stage, and environmental stimuli 1 , 2 , 3 , 4 . Their dynamic morphology is a result of regulated and balanced fusion and fission processes 5 , 6 . Fusion is crucial for the health and physiological functions of mitochondria, including complementation of damaged mitochondrial DNAs and the maintenance of membrane potential 6 , 7 , 8 . Mitofusins are dynamin-related GTPases that are essential for mitochondrial fusion 9 , 10 . They are embedded in the mitochondrial outer membrane and thought to fuse adjacent mitochondria via combined oligomerization and GTP hydrolysis 11 , 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 disorders 14 .

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. The crystal structure of DNM1L, a mediator of mitochondrial and peroxisomal fusion, suggests an assembly mode differing from dynamin and being uniquely adapted to accommodate mitochondria.

Mitochondria are dynamic organelles that exchange contents and undergo remodelling during cyclic fusion and fission. Genetic mutations in MFN2 (the gene encoding mitofusin 2) interrupt mitochondrial fusion and cause the untreatable neurodegenerative condition Charcot-Marie-Tooth disease type 2A (CMT2A). It has not yet been possible to directly modulate mitochondrial fusion, in part because the structural basis of mitofusin function is not completely understood. Here we show that mitofusins adopt either a fusion-constrained or a fusion-permissive molecular conformation, directed by specific intramolecular binding interactions, and demonstrate that mitofusin-dependent mitochondrial fusion can be regulated in mouse cells by targeting these conformational transitions. On the basis of this model, we engineered a cell-permeant minipeptide to destabilize the fusion-constrained conformation of mitofusin and promote the fusion-permissive conformation, reversing mitochondrial abnormalities in cultured fibroblasts and neurons that harbour CMT2A-associated genetic defects. The relationship between the conformational plasticity of mitofusin 2 and mitochondrial dynamism reveals a central mechanism that regulates mitochondrial fusion, the manipulation of which can correct mitochondrial pathology triggered by defective or imbalanced mitochondrial dynamics. Specific intramolecular interactions of mitofusin 2 amino acid sequences either constrain or permit mitochondrial fusion and the addition of short peptides matching these sequences stabilize the fusion-constrained or fusion-permissive form, thus inhibiting or promoting mitochondrial fusion. A new way to correct dysfunctional mitochondria Mitochondria continuously undergo fission and fusion to remodel and exchange content. Defects in these processes can cause disease. For instance, mutations in mitofusin 2—a protein that mediates mitochondrial fusion—cause the untreatable neurodegenerative condition Charcot–Marie–Tooth disease type 2A (CMT2A). Gerald Dorn and colleagues investigate the structural basis of mitofusin function and find that it adopts either a fusion-constrained or a fusion-permissive conformation. Using this knowledge, the authors engineer a cell-permeant minipeptide that destabilizes fusion-constrained mitofusin and promotes the fusion-permissive conformation. Intriguingly, introduction of this peptide into cultured fibroblasts and neurons with CMT2A-associated genetic defects reversed the associated mitochondrial abnormalities.

Mitochondrial membrane dynamics is a cellular rheostat that relates metabolic function and organelle morphology. Using an in vitro reconstitution system, we describe a mechanism for how mitochondrial inner-membrane fusion is regulated by the ratio of two forms of Opa1. We found that the long-form of Opa1 (l-Opa1) is sufficient for membrane docking, hemifusion and low levels of content release. However, stoichiometric levels of the processed, short form of Opa1 (s-Opa1) work together with l-Opa1 to mediate efficient and fast membrane pore opening. Additionally, we found that excess levels of s-Opa1 inhibit fusion activity, as seen under conditions of altered proteostasis. These observations describe a mechanism for gating membrane fusion.