Principles of mitoribosomal small subunit assembly in eukaryotes.

Mitochondrial ribosomes (mitoribosomes) synthesize proteins encoded within the mitochondrial genome that are assembled into oxidative phosphorylation complexes. Thus, mitoribosome biogenesis is essential for ATP production and cellular metabolism1. Here we used cryo-electron microscopy to determine nine structures of native yeast and human mitoribosomal small subunit assembly intermediates, illuminating the mechanistic basis for how GTPases are used to control early steps of decoding centre formation, how initial rRNA folding and processing events are mediated, and how mitoribosomal proteins have active roles during assembly. Furthermore, this series of intermediates from two species with divergent mitoribosomal architecture uncovers both conserved principles and species-specific adaptations that govern the maturation of mitoribosomal small subunits in eukaryotes. By revealing the dynamic interplay between assembly factors, mitoribosomal proteins and rRNA that are required to generate functional subunits, our structural analysis provides a vignette for how molecular complexity and diversity can evolve in large ribonucleoprotein assemblies.

Structural insight and characterization of human Twinkle helicase in mitochondrial disease.

Twinkle is the mammalian helicase vital for replication and integrity of mitochondrial DNA. Over 90 Twinkle helicase disease variants have been linked to progressive external ophthalmoplegia and ataxia neuropathies among other mitochondrial diseases. Despite the biological and clinical importance, Twinkle represents the only remaining component of the human minimal mitochondrial replisome that has yet to be structurally characterized. Here, we present 3-dimensional structures of human Twinkle W315L. Employing cryo-electron microscopy (cryo-EM), we characterize the oligomeric assemblies of human full-length Twinkle W315L, define its multimeric interface, and map clinical variants associated with Twinkle in inherited mitochondrial disease. Cryo-EM, crosslinking-mass spectrometry, and molecular dynamics simulations provide insight into the dynamic movement and molecular consequences of the W315L clinical variant. Collectively, this ensemble of structures outlines a framework for studying Twinkle function in mitochondrial DNA replication and associated disease states.

Molecular Basis of Multiple Mitochondrial Dysfunctions Syndrome 2 Caused by CYS59TYR BOLA3 Mutation.

Multiple mitochondrial dysfunctions syndrome (MMDS) is a rare neurodegenerative disorder associated with mutations in genes with a vital role in the biogenesis of mitochondrial [4Fe-4S] proteins. Mutations in one of these genes encoding for BOLA3 protein lead to MMDS type 2 (MMDS2). Recently, a novel phenotype for MMDS2 with complete clinical recovery was observed in a patient containing a novel variant (c.176G > A, p.Cys59Tyr) in compound heterozygosity. In this work, we aimed to rationalize this unique phenotype observed in MMDS2. To do so, we first investigated the structural impact of the Cys59Tyr mutation on BOLA3 by NMR, and then we analyzed how the mutation affects both the formation of a hetero-complex between BOLA3 and its protein partner GLRX5 and the iron-sulfur cluster-binding properties of the hetero-complex by various spectroscopic techniques and by experimentally driven molecular docking. We show that (1) the mutation structurally perturbed the iron-sulfur cluster-binding region of BOLA3, but without abolishing [2Fe-2S]2+ cluster-binding on the hetero-complex; (2) tyrosine 59 did not replace cysteine 59 as iron-sulfur cluster ligand; and (3) the mutation promoted the formation of an aberrant apo C59Y BOLA3-GLRX5 complex. All these aspects allowed us to rationalize the unique phenotype observed in MMDS2 caused by Cys59Tyr mutation.

Composition and stage dynamics of mitochondrial complexes in Plasmodium falciparum.

Our current understanding of mitochondrial functioning is largely restricted to traditional model organisms, which only represent a fraction of eukaryotic diversity. The unusual mitochondrion of malaria parasites is a validated drug target but remains poorly understood. Here, we apply complexome profiling to map the inventory of protein complexes across the pathogenic asexual blood stages and the transmissible gametocyte stages of Plasmodium falciparum. We identify remarkably divergent composition and clade-specific additions of all respiratory chain complexes. Furthermore, we show that respiratory chain complex components and linked metabolic pathways are up to 40-fold more prevalent in gametocytes, while glycolytic enzymes are substantially reduced. Underlining this functional switch, we find that cristae are exclusively present in gametocytes. Leveraging these divergent properties and stage dynamics for drug development presents an attractive opportunity to discover novel classes of antimalarials and increase our repertoire of gametocytocidal drugs.

Structures of the human LONP1 protease reveal regulatory steps involved in protease activation.

The human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain activity, and mitochondrial transcription. As such, genetic or aging-associated imbalances in LONP1 activity are implicated in pathologic mitochondrial dysfunction associated with numerous human diseases. Despite this importance, the molecular basis for LONP1-dependent proteolytic activity remains poorly defined. Here, we solved cryo-electron microscopy structures of human LONP1 to reveal the underlying molecular mechanisms governing substrate proteolysis. We show that, like bacterial Lon, human LONP1 adopts both an open and closed spiral staircase orientation dictated by the presence of substrate and nucleotide. Unlike bacterial Lon, human LONP1 contains a second spiral staircase within its ATPase domain that engages substrate as it is translocated toward the proteolytic chamber. Intriguingly, and in contrast to its bacterial ortholog, substrate binding within the central ATPase channel of LONP1 alone is insufficient to induce the activated conformation of the protease domains. To successfully induce the active protease conformation in substrate-bound LONP1, substrate binding within the protease active site is necessary, which we demonstrate by adding bortezomib, a peptidomimetic active site inhibitor of LONP1. These results suggest LONP1 can decouple ATPase and protease activities depending on whether AAA+ or both AAA+ and protease domains bind substrate. Importantly, our structures provide a molecular framework to define the critical importance of LONP1 in regulating mitochondrial proteostasis in health and disease.

Assembly and Cryo-EM structure determination of yeast mitochondrial RNA polymerase initiation complex intermediates.

In yeast mitochondria, transcription initiation requires assembly of mitochondrial RNA polymerase and transcription initiation factor MTF1 at the DNA promoter initiation site. This protocol describes the purification of the component proteins and assembly of partially melted and fully melted initiation complex states. Both states co-exist in equilibrium in the same sample as seen by cryoelectron microscopy (cryo-EM) and allow elucidation of MTF1's structural roles in controlling the transition into elongation. We further outline how analysis of the complex by light scattering, thermal shift assay, and ultrafiltration assay exhibits reproducible results. For complete details on the use and execution of this protocol, please refer to De Wijngaert et al. (2021).

Bisphenol-A inhibits mitochondrial biogenesis via impairment of GFER mediated mitochondrial protein import in the rat brain hippocampus.

Mitochondrial biogenesis relies on different protein import machinery, as mitochondrial proteins are imported from the cytosol. The mitochondrial intermembrane space assembly (MIA) pathway consists of GFER/ALR and CHCHD4/Mia40, responsible for importing proteins and their oxidative folding inside the mitochondria. The MIA pathway plays an essential role in complex IV (COX IV) biogenesis via importing copper chaperone COX17, associated with the respiratory chain. BPA, an environmental toxicant, found in consumable plastics, causes neurotoxicity via impairment in mitochondrial dynamics, neurogenesis, and cognitive functions. We studied the levels of key regulatory proteins of mitochondrial import pathways and mitochondrial biogenesis after BPA exposure in the rat hippocampus. BPA caused a significant reduction in the levels of mitochondrial biogenesis proteins (PGC1α, and TFAM) and mitochondrial import protein (GFER). Immunohistochemical analysis showed reduced co-localization of NeuN with GFER, PGC-1α, and TFAM suggesting impaired mitochondrial biogenesis and protein import. BPA exposure resulted in damaged mitochondria with distorted cristae in neurons and caused a significant reduction in GFER localization inside IMS as depicted by immunogold electron microscopy. The reduced levels of GFER resulted in defective COX17 import. The translocation of cytochrome c into the cytosol and increased cleaved caspase-3 levels triggered apoptosis due to BPA toxicity. Overall, our study implicates GFER as a potential target for impaired mitochondrial protein machinery, biogenesis, and apoptosis against BPA neurotoxicity in the rat hippocampus.

A conserved arginine residue is critical for stabilizing the N2 FeS cluster in mitochondrial complex I.

Respiratory complex I (NADH:ubiquinone oxidoreductase), the first enzyme of the electron-transport chain, captures the free energy released by NADH oxidation and ubiquinone reduction to translocate protons across an energy-transducing membrane and drive ATP synthesis during oxidative phosphorylation. The cofactor that transfers the electrons directly to ubiquinone is an iron-sulfur cluster (N2) located in the NDUFS2/NUCM subunit. A nearby arginine residue (R121), which forms part of the second coordination sphere of the N2 cluster, is known to be posttranslationally dimethylated but its functional and structural significance are not known. Here, we show that mutations of this arginine residue (R121M/K) abolish the quinone-reductase activity, concomitant with disappearance of the N2 signature from the electron paramagnetic resonance (EPR) spectrum. Analysis of the cryo-EM structure of NDUFS2-R121M complex I at 3.7 Å resolution identified the absence of the cubane N2 cluster as the cause of the dysfunction, within an otherwise intact enzyme. The mutation further induced localized disorder in nearby elements of the quinone-binding site, consistent with the close connections between the cluster and substrate-binding regions. Our results demonstrate that R121 is required for the formation and/or stability of the N2 cluster and highlight the importance of structural analyses for mechanistic interpretation of biochemical and spectroscopic data on complex I variants.

Mitochondrial sorting and assembly machinery operates by ß-barrel switching.

The mitochondrial outer membrane contains so-called ß-barrel proteins, which allow communication between the cytosol and the mitochondrial interior1-3. Insertion of ß-barrel proteins into the outer membrane is mediated by the multisubunit mitochondrial sorting and assembly machinery (SAM, also known as TOB)4-6. Here we use cryo-electron microscopy to determine the structures of two different forms of the yeast SAM complex at a resolution of 2.8-3.2 Å. The dimeric complex contains two copies of the ß-barrel channel protein Sam50-Sam50a and Sam50b-with partially open lateral gates. The peripheral membrane proteins Sam35 and Sam37 cap the Sam50 channels from the cytosolic side, and are crucial for the structural and functional integrity of the dimeric complex. In the second complex, Sam50b is replaced by the ß-barrel protein Mdm10. In cooperation with Sam50a, Sam37 recruits and traps Mdm10 by penetrating the interior of its laterally closed ß-barrel from the cytosolic side. The substrate-loaded SAM complex contains one each of Sam50, Sam35 and Sam37, but neither Mdm10 nor a second Sam50, suggesting that Mdm10 and Sam50b function as placeholders for a ß-barrel substrate released from Sam50a. Our proposed mechanism for dynamic switching of ß-barrel subunits and substrate explains how entire precursor proteins can fold in association with the mitochondrial machinery for ß-barrel assembly.

Structural snapshot of the mitochondrial protein import gate.

The translocase of the outer mitochondrial membrane (TOM) complex is the main entry gate for most mitochondrial proteins. The TOM complex is a multisubunit membrane protein complex consisting of a ß-barrel protein Tom40 and six α-helical transmembrane (TM) proteins, receptor subunits Tom20, Tom22, and Tom70, and regulatory subunits Tom5, Tom6, and Tom7. Although nearly 30 years have passed since the main components of the TOM complex were identified and characterized, the structural details of the TOM complex remained poorly understood until recently. Thanks to the rapid development of the cryoelectron microscopy (EM) technology, high-resolution structures of the yeast TOM complex have become available. The identified structures showed a symmetric dimer containing five different subunits including Tom22. Biochemical and mutational analyses based on the TOM complex structure revealed the presence of different translocation paths within the Tom40 import channel for different classes of translocating precursor proteins. Previous studies including our cross-linking analyses indicated that the TOM complex in intact mitochondria is present as a mixture of the trimeric complex containing Tom22. Furthermore, the dimeric complex lacking Tom22, and the trimer and dimer may handle different sets of mitochondrial precursor proteins for translocation across the outer membrane. In this Structural Snapshot, we will discuss possible rearrangement of the subunit interactions upon dynamic conversion of the TOM complex between the different subunit assembly states, the Tom22-containing core dimer and trimer.

Structural snapshots of the cellular folded protein translocation machinery Bcs1.

Proteins destined to various intra- and extra-cellular locations must traverse membranes most frequently in an unfolded form. When the proteins being translocated need to remain in a folded state, specialized cellular transport machinery is used. One such machine is the membrane-bound AAA protein Bcs1 (Bcs1), which assists the iron-sulfur protein, an essential subunit of the respiratory Complex III, across the mitochondrial inner membrane. Recent structure determinations of mouse and yeast Bcs1 in three different nucleotide states reveal its homo-heptameric association and at least two dramatically different conformations. The apo and ADP-bound structures are similar, both containing a large substrate-binding cavity accessible to the mitochondrial matrix space, which contracts by concerted motion of the ATPase domains upon ATP binding, suggesting that bound substrate could then be pushed across the membrane. ATP hydrolysis drives substrate release and resets Bcs1 conformation back to the apo/ADP form. These structures shed new light on the mechanism of folded protein translocation across a membrane, provide better understanding on the assembly process of the respiratory Complex III, and correlate clinical presentations of disease-associated mutations with their locations in the 3D structure.

Exome sequencing identifies novel missense and deletion variants in RTN4IP1 associated with optic atrophy, global developmental delay, epilepsy, ataxia, and choreoathetosis.

Inherited optic neuropathies (IONs) are neurodegenerative disorders characterized by optic atrophy with or without extraocular manifestations. Optic atrophy-10 (OPA10) is an autosomal recessive ION recently reported to be caused by mutations in RTN4IP1, which encodes reticulon 4 interacting protein 1 (RTN4IP1), a mitochondrial ubiquinol oxydo-reductase. Here we report novel compound heterozygous mutations in RTN4IP1 in a male proband with developmental delay, epilepsy, optic atrophy, ataxia, and choreoathetosis. Workup was notable for transiently elevated lactate and lactate-to-pyruvate ratio, brain magnetic resonance imaging with optic atrophy and T2 signal abnormalities, and a nondiagnostic initial genetic workup, including chromosomal microarray and mitochondrial panel testing. Exome sequencing identified a paternally inherited missense variant (c.263T>G, p.Val88Gly) predicted to be deleterious and a maternally inherited deletion encompassing RTN4IP1. To our knowledge, this is the first report of a non-single nucleotide pathogenic variant associated with OPA10. This case highlights the expanding phenotypic spectrum of OPA10, the association between "syndromic" cases and severe RTN4IP1 mutations, and the importance of nonbiased genetic testing, such as ES, to analyze multiple genes and variants types, in patients suspected of having genetic disease.

Need for Speed: Examining Protein Behavior during CryoEM Grid Preparation at Different Timescales.

A host of new technologies are under development to improve the quality and reproducibility of cryoelectron microscopy (cryoEM) grid preparation. Here we have systematically investigated the preparation of three macromolecular complexes using three different vitrification devices (Vitrobot, chameleon, and a time-resolved cryoEM device) on various timescales, including grids made within 6 ms (the fastest reported to date), to interrogate particle behavior at the air-water interface for different timepoints. Results demonstrate that different macromolecular complexes can respond to the thin-film environment formed during cryoEM sample preparation in highly variable ways, shedding light on why cryoEM sample preparation can be difficult to optimize. We demonstrate that reducing time between sample application and vitrification is just one tool to improve cryoEM grid quality, but that it is unlikely to be a generic "silver bullet" for improving the quality of every cryoEM sample preparation.

Cryo-EM structures of S-OPA1 reveal its interactions with membrane and changes upon nucleotide binding.

Mammalian mitochondrial inner membrane fusion is mediated by optic atrophy 1 (OPA1). Under physiological conditions, OPA1 undergoes proteolytic processing to form a membrane-anchored long isoform (L-OPA1) and a soluble short isoform (S-OPA1). A combination of L-OPA1 and S-OPA1 is essential for efficient membrane fusion; however, the relevant mechanism is not well understood. In this study, we investigate the cryo-electron microscopic structures of S-OPA1-coated liposomes in nucleotide-free and GTPγS-bound states. S-OPA1 exhibits a general dynamin-like structure and can assemble onto membranes in a helical array with a dimer building block. We reveal that hydrophobic residues in its extended membrane-binding domain are critical for its tubulation activity. The binding of GTPγS triggers a conformational change and results in a rearrangement of the helical lattice and tube expansion similar to that of S-Mgm1. These observations indicate that S-OPA1 adopts a dynamin-like power stroke membrane remodeling mechanism during mitochondrial inner membrane fusion.

A pathway for assembling [4Fe-4S]2+ clusters in mitochondrial iron-sulfur protein biogenesis.

During its late steps, the mitochondrial iron-sulfur cluster (ISC) assembly machinery leads to the formation of [4Fe-4S] clusters. In vivo studies revealed that several proteins are implicated in the biosynthesis and trafficking of [4Fe-4S] clusters in mitochondria. However, they do not provide a clear picture into how these proteins cooperate. Here, we showed that three late-acting components of the mitochondrial ISC assembly machinery (GLRX5, BOLA3, and NFU1) are part of a ISC assembly pathway leading to the synthesis of a [4Fe-4S]2+ cluster on NFU1. We showed that the [2Fe-2S]2+ GLRX5-BOLA3 complex transfers its cluster to monomeric apo NFU1 to form, in the presence of a reductant, a [4Fe-4S]2+ cluster bound to dimeric NFU1. The cluster formation on NFU1 does not occur with [2Fe-2S]2+ GLRX5, and thus, the [4Fe-4S] cluster assembly pathway is activated only in the presence of BOLA3. These results define NFU1 as an 'assembler' of [4Fe-4S] clusters, that is, a protein able of converting two [2Fe-2S]2+ clusters into a [4Fe-4S]2+ cluster. Finally, we found that the [4Fe-4S]2+ cluster bound to NFU1 has a coordination site which is easily accessible to sulfur-containing ligands, as is typically observed in metallochaperones. This finding supports a role for NFU1 in promoting rapid and controlled cluster-exchange reaction.

Coupling of import and assembly pathways in mitochondrial protein biogenesis.

Biogenesis and function of mitochondria depend on the import of about 1000 precursor proteins that are produced on cytosolic ribosomes. The translocase of the outer membrane (TOM) forms the entry gate for most proteins. After passage through the TOM channel, dedicated preprotein translocases sort the precursor proteins into the mitochondrial subcompartments. Many proteins have to be assembled into oligomeric membrane-integrated complexes in order to perform their functions. In this review, we discuss a dual role of mitochondrial preprotein translocases in protein translocation and oligomeric assembly, focusing on the biogenesis of the TOM complex and the respiratory chain. The sorting and assembly machinery (SAM) of the outer mitochondrial membrane forms a dynamic platform for coupling transport and assembly of TOM subunits. The biogenesis of the cytochrome c oxidase of the inner membrane involves a molecular circuit to adjust translation of mitochondrial-encoded core subunits to the availability of nuclear-encoded partner proteins. Thus, mitochondrial protein translocases not only import precursor proteins but can also support their assembly into functional complexes.

Mitoribosomal small subunit biogenesis in trypanosomes involves an extensive assembly machinery.

Mitochondrial ribosomes (mitoribosomes) are large ribonucleoprotein complexes that synthesize proteins encoded by the mitochondrial genome. An extensive cellular machinery responsible for ribosome assembly has been described only for eukaryotic cytosolic ribosomes. Here we report that the assembly of the small mitoribosomal subunit in Trypanosoma brucei involves a large number of factors and proceeds through the formation of assembly intermediates, which we analyzed by using cryo-electron microscopy. One of them is a 4-megadalton complex, referred to as the small subunit assemblosome, in which we identified 34 factors that interact with immature ribosomal RNA (rRNA) and recognize its functionally important regions. The assembly proceeds through large-scale conformational changes in rRNA coupled with successive incorporation of mitoribosomal proteins, providing an example for the complexity of the ribosomal assembly process in mitochondria.

Structure and assembly of the mitochondrial membrane remodelling GTPase Mgm1.

Balanced fusion and fission are key for the proper function and physiology of mitochondria1,2. Remodelling of the mitochondrial inner membrane is mediated by the dynamin-like protein mitochondrial genome maintenance 1 (Mgm1) in fungi or the related protein optic atrophy 1 (OPA1) in animals3-5. Mgm1 is required for the preservation of mitochondrial DNA in yeast6, whereas mutations in the OPA1 gene in humans are a common cause of autosomal dominant optic atrophy-a genetic disorder that affects the optic nerve7,8. Mgm1 and OPA1 are present in mitochondria as a membrane-integral long form and a short form that is soluble in the intermembrane space. Yeast strains that express temperature-sensitive mutants of Mgm19,10 or mammalian cells that lack OPA1 display fragmented mitochondria11,12, which suggests that Mgm1 and OPA1 have an important role in inner-membrane fusion. Consistently, only the mitochondrial outer membrane-not the inner membrane-fuses in the absence of functional Mgm113. Mgm1 and OPA1 have also been shown to maintain proper cristae architecture10,14; for example, OPA1 prevents the release of pro-apoptotic factors by tightening crista junctions15. Finally, the short form of OPA1 localizes to mitochondrial constriction sites, where it presumably promotes mitochondrial fission16. How Mgm1 and OPA1 perform their diverse functions in membrane fusion, scission and cristae organization is at present unknown. Here we present crystal and electron cryo-tomography structures of Mgm1 from Chaetomium thermophilum. Mgm1 consists of a GTPase (G) domain, a bundle signalling element domain, a stalk, and a paddle domain that contains a membrane-binding site. Biochemical and cell-based experiments demonstrate that the Mgm1 stalk mediates the assembly of bent tetramers into helical filaments. Electron cryo-tomography studies of Mgm1-decorated lipid tubes and fluorescence microscopy experiments on reconstituted membrane tubes indicate how the tetramers assemble on positively or negatively curved membranes. Our findings convey how Mgm1 and OPA1 filaments dynamically remodel the mitochondrial inner membrane.

Structural basis for adPEO-causing mutations in the mitochondrial TWINKLE helicase.

TWINKLE is the helicase involved in replication and maintenance of mitochondrial DNA (mtDNA) in mammalian cells. Structurally, TWINKLE is closely related to the bacteriophage T7 gp4 protein and comprises a helicase and primase domain joined by a flexible linker region. Mutations in and around this linker region are responsible for autosomal dominant progressive external ophthalmoplegia (adPEO), a neuromuscular disorder associated with deletions in mtDNA. The underlying molecular basis of adPEO-causing mutations remains unclear, but defects in TWINKLE oligomerization are thought to play a major role. In this study, we have characterized these disease variants by single-particle electron microscopy and can link the diminished activities of the TWINKLE variants to altered oligomeric properties. Our results suggest that the mutations can be divided into those that (i) destroy the flexibility of the linker region, (ii) inhibit ring closure and (iii) change the number of subunits within a helicase ring. Furthermore, we demonstrate that wild-type TWINKLE undergoes large-scale conformational changes upon nucleoside triphosphate binding and that this ability is lost in the disease-causing variants. This represents a substantial advancement in the understanding of the molecular basis of adPEO and related pathologies and may aid in the development of future therapeutic strategies.