Biology subjects

Amunts, A.

Publications and source records attributed to Amunts, A..

16 recordsLinked to original sources

bioRxiv2025

Human ribosome interactions reframe neomycin toxicity

Aminoglycosides such as neomycin are clinically limited by toxicity, commonly attributed to the inhibition of mitoribosomes. Here, we re-evaluate this mechanism by integrating Cryo-EM and functional assays to assess neomycins binding potential in vitro with its cellular engagement and functional consequences. Cryo-EM revealed extensive multi-site binding of neomycin on both human mitochondrial and cytosolic ribosomes in vitro, including conserved regions of the decoding center. In contrast, ribosomes isolated from neomycin-treated cells show markedly remodeled occupancy patterns, with no detectable neomycin bound to the mitoribosomes and reduced occupancy at a subset of cytosolic ribosome sites. However, neomycin remained functionally active in cells, inducing widespread stop-codon readthrough while having minimal effects on global mitochondrial and cytosolic protein synthesis. These findings reveal a disconnect between structural binding potential and cellular target engagement and identify disruption of translational fidelity, rather than global translational inhibition, as a major cellular consequence of neomycin exposure.

biochemistry↗

bioRxiv2025

Structure of Photosystem I with SOD reveals coupling between energy conversion and oxidative stress protection

In photosynthetic organisms, Photosystem I (PSI) not only converts energy into its chemical form but is also a major source of superoxide (O2-), which can damage cellular components. Its detoxification by superoxide dismutase (SOD) is essential for redox homeostasis. We report the structure of the PSI-SOD supercomplex from Chromera velia (C. velia), revealing a direct coupling between energy conversion and oxidative stress protection. The heterodimeric SOD comprises a catalytic SOD2 and a non-catalytic SOD1 anchoring it to the conserved stromal PSI surface near the ferredoxin-binding site with four contact areas. Structural comparison with plants and algae shows that SOD could occupy a similar position via interactions with PsaD. C. velia PSI displays several distinct features, including a split subunit PsaA linked by PsaV, a unique iso-fucoxanthin bound to PsaB, and an extended PsaF coordinating an extra chlorophyll in the membrane. The antenna includes five fucoxanthin-chlorophyll family proteins (FCPs) stabilized by adaptations unique to each antenna protein. These findings reveal the structural basis for the integration of a photoprotective mechanism with energy metabolism in photosynthetic membranes.

biochemistry↗

bioRxiv2025

Mettl15-Mettl17 modulates the transition from early to late pre-mitoribosome

The assembly of the mitoribosomal small subunit involves folding and modification of rRNA, and its association with mitoribosomal proteins. This process is assisted by a dynamic network of assembly factors. Conserved methyltransferases Mettl15 and Mettl17 act on the solvent-exposed surface of rRNA. Binding of Mettl17 is associated with the early assembly stage, whereas Mettl15 is involved in the late stage, but the mechanism of transition between the two was unclear. Here, we integrate structural data from Trypanosoma brucei with mammalian homologs and molecular dynamics simulations. We reveal how the interplay of Mettl15 and Mettl17 in intermediate steps links the distinct stages of small subunit assembly. The analysis suggests a model wherein Mettl17 acts as a platform for Mettl15 recruitment. Subsequent release of Mettl17 allows a conformational change of Mettl15 for substrate recognition. Upon methylation, Mettl15 adopts a loosely bound state which ultimately leads to its replacement by initiation factors, concluding the assembly. Together, our results indicate that assembly factors Mettl15 and Mettl17 cooperate to regulate the biogenesis process, and present a structural data resource for understanding molecular adaptations of assembly factors in mitoribosome.

molecular biology↗

bioRxiv2024

The mitochondrial methylation potential gates mitoribosome assembly

S-adenosylmethionine (SAM) is crucial for cellular processes, primarily serving as the principal methyl group donor of the cell and playing a key role in gene regulation and translation on the ribosome. Inside mitochondria, SAM-dependent methylations occur at several steps of gene expression, but their role and significance remain unclear. Using direct long-read RNA sequencing on mouse tissue and mouse embryonic fibroblasts, we demonstrate that the mitochondrial ribosomal gene cluster is not efficiently processed without mitochondrial SAM. This results in the accumulation of unprocessed ribosomal RNA precursors. Protein profiling of ribosome fractions revealed that these precursors are associated with processing and ribosome assembly factors, indicating stalling at an early stage. Structural analysis of the mitochondrial ribosome by cryogenic electron microscopy revealed that mitochondrial SAM is required during peptidyl transferase centre formation and mitochondrial ribosome assembly. Our data thus identify a critical role for methylation at two steps during mitochondrial gene expression.

molecular biology↗

bioRxiv2024

Improved automated model building for cryo-EM maps using CryFold

Constructing atomic models from cryogenic electron microscopy (cryo-EM) density maps is essential for interpreting molecular mechanisms. In this study, we present CryFold, an approach for de novo model building for cryo-EM maps, leveraging recent advancements in AlphaFold2 (1) to improve the state-of-the-art method ModelAngelo (2). To accommodate the cryo-EM map information, CryFold replaces the global attention mechanism in AlphaFold2 with local attention, which is further enhanced by a novel 3D rotary position embedding. CryFold produces more complete models, reduces the resolution requirement, and accelerates the modeling. The application of CryFold to three new maps with unknown structure demonstrates its ability to accurately distinguish between paralog sequences in noisy regions, detect previously uncharacterized proteins with unknown functions, precisely compartmentalize the map to isolate non-protein components, and improve the modeling of conformational changes. A particular case includes a 104-protein complex that has been modeled within a few hours, and a minor conformational change of a single protein domain has been detected at the periphery when models from two different maps were compared. CryFold stands as an accurate method currently available for model building of proteins in cryo-EM structure determination. The source code and model parameters are available at https://github.com/SBQ-1999/CryFold.

bioinformatics↗

bioRxiv2024

Structure of the II2-III2-IV2 mitochondrial supercomplex from the para-site Perkinsus marinus

Respiratory complexes have co-evolved into supercomplexes in different clades to sustain energy production at the basis of eukaryotic life. In this study, using cryogenic electron microscopy, we determined the 2.1 [A] resolution structure of a 104-subunit II2-III2-IV2 supercomplex from the parasite Perkinsus marinus, related to Apicomplexa, capable of complete electron transport from succinate to molecular oxygen. A feature of the parasite is the association of two copies of complex II via the apicomplexan subunit SDHG that interacts with both complexes III and IV and bridge the supercomplex. In the c1 state, we identified two protein factors, ISPR1 and ISPR2 bound on the surface of complex III, where Cytochrome c docks, acting as negative regulators. The acquisition of 15 specific subunits to complex IV results in its lateral offset, increasing the distance between the Cytochrome c electron donor and acceptor sites. The domain homologous to canonical mitochondria-encoded transmembrane subunit COX2 is made of three separate polypeptides encoded in the nucleus, and their correct assembly is a prerequisite for electron transport in the supercomplex. Subunits Cytochrome b and COX1 comprise a +2 frameshift introduced during protein synthesis by the mitoribosome. Among 114 modelled endogenous lipids, we detect a direct contribution to the formation of the divergent supercomplex and its functional sites, including assembly of CII and ubiquinone binding. Together, our findings expose the uniqueness of the principal components of bioenergetics in the mitochondria of parasites.

biochemistry↗

bioRxiv2023

Structure of mitoribosome reveals mechanism of mRNA binding, tRNA interactions with L1 stalk, roles of cofactors and rRNA modifications

The mitoribosome translates mitochondrial mRNAs and regulates energy conversion that is a signature of aerobic life forms. We present a 2.2 [A] resolution structure of human mitoribosome together with validated mitoribosomal RNA (rRNA) modifications, including aminoacylated CP-tRNAVal. The structure shows how mitoribosomal proteins stabilise binding of mRNA and tRNA helping to align it in the decoding center, whereas the GDP-bound mS29 stabilizes intersubunit communication. Comparison between different states, with respect to tRNA position, allowed to characterize a non-canonical L1 stalk, and molecular dynamics simulations revealed how it facilitates tRNA transition in a way that does not require interactions with rRNA. We also report functionally important polyamines that are depleted when cells are subjected to an antibiotic treatment. The structural, biochemical, and computational data illuminate the principal functional components of the translation mechanism in mitochondria and provide the most complete description so far of the structure and function of the human mitoribosome.

biochemistry↗

bioRxiv2022

dgram2dmap: Extraction, visualisation and formatting of distance constraints from AlphaFold distograms

Distograms are data structures output by AlphaFold, along with the predicted 3D coordinates of the target protein, that encode predictions about Euclidean distances between pairs of amino acids. Although distograms are often overlooked, they do provide information that is to some extent complementary to that of the final 3D model. Here, we introduce dgram2dmap, a simple tool to convert distograms into distance maps that can be visualised and used in external tools for further downstream analyses. dgram2dmap runs within seconds to minutes, is open source and available on GitHub: https://github.com/clami66/dgram2dmap.

bioinformatics↗

bioRxiv2022

METTL17 is an Fe-S cluster checkpoint for mitochondrial translation

AO_SCPLOWBSTRACTC_SCPLOWFriedreichs ataxia (FA) is the most common monogenic mitochondrial disease. FA is caused by a depletion of the mitochondrial protein frataxin (FXN), an iron-sulfur (Fe-S) cluster biogenesis factor. To better understand the cellular consequences of FA, we performed quantitative proteome profiling of human cells depleted for FXN. Nearly every known Fe-S cluster-containing protein was depleted in the absence of FXN, indicating that as a rule, cluster binding confers stability to Fe-S proteins. Proteomic and genetic interaction mapping identified impaired mitochondrial translation downstream of FXN loss, and specifically highlighted the methyltransferase-like protein METTL17 as a candidate effector. Using comparative sequence analysis, mutagenesis, biochemistry and cryogenic electron microscopy we show that METTL17 binds to the mitoribosomal small subunit during late assembly and harbors a previously unrecognized [Fe4S4]2+ cluster required for its stability on the mitoribosome. Notably, METTL17 overexpression rescued the mitochondrial translation and bioenergetic defects, but not the cellular growth, of FXN null cells. Our data suggest that METTL17 serves as an Fe-S cluster checkpoint: promoting the translation and assembly of Fe-S cluster rich OXPHOS proteins only when Fe-S cluster levels are replete.

cell biology↗

bioRxiv2022

Structural basis of mitochondrial membrane bending by I-II-III2-IV2 supercomplex

Mitochondrial energy conversion requires an intricate architecture of the inner mitochondrial membrane1. Here we show that in ciliates, the membrane curvature is provided by a supercomplex containing all four respiratory chain components. We report cryo-electron microscopy and cryo-tomography structures of the supercomplex that comprises 150 different proteins and 311 bound lipids, forming a stable 5.8-megadalton assembly. Due to subunit acquisition and extension, complex I associates with a complex IV dimer, generating a wedge-shaped gap that serves as a binding site for complex II. Together with a tilted complex III dimer association, it results in a curved membrane region. Using molecular dynamics simulations, we demonstrate that the divergent supercomplex actively contributes to the membrane curvature induction and cristae tubulation. Our findings explain how the architecture of the native I-II-III2-IV2 supercomplex reflects the functional specialization of bioenergetics by shaping the membrane.

biochemistry↗

bioRxiv2022

Activation mechanism of mitochondrial translation by LRPPRC-SLIRP

In mammalian mitochondria, mRNAs are co-transcriptionally stabilized by the protein factor LRPPRC. Here, we characterize LRPPRC as an mRNA delivery factor and report its cryo-EM structure in complex with SLIRP, mRNA and the mitoribosome. The structure shows that LRPPRC associates with the mitoribosomal proteins mS39 and the N-terminus of mS31 through recognition of the LRPPRC helical repeats. Together, the proteins form a corridor for hand-off the mRNA. The mRNA is directly bound to SLIRP, which also has a stabilizing function for LRPPRC. To delineate the effect of LRPPRC on individual mitochondrial transcripts, we used an RNAseq approach, metabolic labeling and mitoribosome profiling that showed a major influence on ND1, ND2, ATP6, COX1, COX2, and COX3 mRNA translation efficiency. Our data suggest that LRPPRC-SLIRP acts in recruitment of mitochondrial mRNAs to modulate their translation. Collectively, the data define LRPPRC-SLIRP as a regulator of the mitochondrial gene expression system.

biochemistry↗

bioRxiv2022

Structural basis of streptomycin off-target binding to human mitoribosome

The ribosome in mitochondria regulates cellular energy production, and its deactivation is associated with pathologies and ageing. Inhibition of human mitoribosome can be caused by antimicrobial off-target binding, which leads to clinical appearances. The anti-tuberculosis drug aminoglycoside streptomycin targets the small subunit and was shown to be coupled with a bilateral decreased visual acuity with central scotomas and an altered mitochondrial structure. Previously, we reported mitochondria-specific aspects of translation related to specialties of the human mitoribosome (Aibara et al., 2020). In this Research advance article, we report 2.23-[A] resolution structure of the human mitoribosomal small subunit in complex with streptomycin. The structural data reveals new details of the streptomycin interactions, including specific water molecules and metal ions involved in the coordination. The density for the streptose moiety reveals that previously modeled aldehyde group appears as a loosely bound density, and the hydroxyl group is not resolved. The density replacing the aldehyde group is within hydrogen bonding distance of four phosphate groups of rRNA, suggesting that the ribosome-bound streptomycin is likely to be in the hydrated gem-diol form rather than in the free aldehyde form. Since streptomycin is a widely used drug for treatment, the newly resolved fine features can serve as determinants for targeting.

molecular biology↗

bioRxiv2021

An ancestral interaction module promotes oligomerisation in divergent mitochondrial ATP synthases

Mitochondrial ATP synthase forms stable dimers arranged into oligomeric assemblies that generate the inner-membrane curvature essential for efficient energy conversion. Here, we report cryo-EM structures of the intact ATP synthase dimer from Trypanosoma brucei in ten different rotational states. The model consists of 25 subunits, including nine lineage-specific, as well as 36 lipids. The rotary mechanism is influenced by the divergent peripheral stalk, conferring a greater conformational flexibility. Proton transfer in the lumenal half-channel occurs via a chain of five ordered water molecules. The dimerization interface is formed by subunit-g that is critical for interactions but not for the catalytic activity. Although overall dimer architecture varies among eukaryotes, we find that subunit-g together with subunit-e form an ancestral oligomerization motif, which is shared between the trypanosomal and mammalian lineages. Therefore, our data defines the subunit-g/e module as a structural component determining ATP synthase oligomeric assemblies.

molecular biology↗

bioRxiv2021

Chloroplast photosystem I dimer and high resolution model of the complex with plastocyanin

Photosystem I (PSI) enables photo-electron transfer and regulates photosynthesis in the bioenergetic membranes of cyanobacteria and chloroplasts. Being a multi-subunit complex, its macromolecular organization affects the dynamics of photosynthetic membranes. Here, we reveal a chloroplast PSI from the green alga Chlamydomonas reinhardtii that is organized as a homodimer, comprising 40 protein subunits with 118 transmembrane helices that provide scaffold for 568 pigments. Our cryo-EM structure identifies that the absence of PsaH and Lhca2 gives rise to a head-to-head relative orientation of the PSI-LHCI monomers in a way that is essentially different from the oligomer formation in cyanobacteria. The light-harvesting protein Lhca9 is the key element for mediating this dimerization. The interface between the monomers is lacking PsaH, and thus partially overlaps with the surface area that would bind one of the LHCII complexes in state transitions. We also define the most accurate available PSI-LHCI model at 2.3 [A] resolution, including a flexibly bound electron donor plastocyanin, and assign correct identities and orientations of all the pigments, as well as 621 water molecules that affect energy transfer pathways.

plant biology↗

bioRxiv2020

Interconnected assembly factors regulate the biogenesis of mitoribosomal large subunit in trypanosomes

Mitoribosomes consist of ribosomal RNA and protein components, coordinated assembly of which is critical for function. We used mitoribosomes with reduced RNA and increased protein mass from Trypanosoma brucei, to provide insights into the biogenesis of mitoribosomal large subunit. Structural characterisation of a stable assembly intermediate revealed 22 assembly factors, some of which are also encoded in mammalian genomes. The assembly factors form a protein network that spans over 180 [A], shielding the ribosomal RNA surface. The entire central protuberance and L7/L12 stalk are not assembled, and require removal of the factors and remodeling of the mitoribosomal proteins to become functional. The conserved proteins GTPBP7 and mt-EngA are bound together at the subunit interface in proximity to the peptidyl transferase center. A mitochondrial acyl-carrier protein plays a role in docking the L1 stalk which needs to be repositioned during maturation. Additional enzymatically deactivated factors scaffold the assembly, while the exit tunnel is blocked. Together, the extensive network of the factors stabilizes the immature sites and connects the functionally important regions of the mitoribosomal large subunit.

evolutionary biology↗

bioRxiv2020

Analysis of translating mitoribosome reveals functional characteristics of protein synthesis in mitochondria of fungi

Mitoribosomes are specialized protein synthesis machineries in mitochondria. However, how mRNA binds to its dedicated channel, and tRNA moves as the mitoribosomal subunit rotate with respect to each other is not understood. We report models of the translating fungal mitoribosome with mRNA, tRNA and nascent polypeptide, as well as an assembly intermediate. Nicotinamide adenine dinucleotide (NAD) is found in the central protuberance of the large subunit, and the ATPase inhibitory factor 1 (IF1) in the small subunit. The models of the active mitoribosome explain how mRNA binds through a dedicated protein platform on the small subunit, tRNA is translocated with the help of the protein mL108, bridging it with L1 stalk on the large subunit, and nascent polypeptide paths through a newly shaped exit tunnel involving a series of structural rearrangements. An assembly intermediate is modeled with the maturation factor Atp25, providing insight in to the biogenesis of the mitoribosomal large subunit and translation regulation.

biochemistry↗