To understand the steps involved in the evolution of translation, we used
, a ciliate with high coding capacity of the mitochondrial genome, as the model organism and characterized its mitochondrial ...ribosome (mitoribosome) using cryo-EM. The structure of the mitoribosome reveals an assembly of 94-ribosomal proteins and four-rRNAs with an additional protein mass of ~700 kDa on the small subunit, while the large subunit lacks 5S rRNA. The structure also shows that the small subunit head is constrained, tRNA binding sites are formed by mitochondria-specific protein elements, conserved protein bS1 is excluded, and bacterial RNA polymerase binding site is blocked. We provide evidence for anintrinsic protein targeting system through visualization of mitochondria-specific mL105 by the exit tunnel that would facilitate the recruitment of a nascent polypeptide. Functional protein uS3m is encoded by three complementary genes from the nucleus and mitochondrion, establishing a link between genetic drift and mitochondrial translation. Finally, we reannotated nine open reading frames in the mitochondrial genome that code for mitoribosomal proteins.
Mitochondrial ATP synthases form functional homodimers to induce cristae curvature that is a universal property of mitochondria. To expand on the understanding of this fundamental phenomenon, we ...characterized the unique type III mitochondrial ATP synthase in its dimeric and tetrameric form. The cryo-EM structure of a ciliate ATP synthase dimer reveals an unusual U-shaped assembly of 81 proteins, including a substoichiometrically bound ATPTT2, 40 lipids, and co-factors NAD and CoQ. A single copy of subunit ATPTT2 functions as a membrane anchor for the dimeric inhibitor IF
. Type III specific linker proteins stably tie the ATP synthase monomers in parallel to each other. The intricate dimer architecture is scaffolded by an extended subunit-a that provides a template for both intra- and inter-dimer interactions. The latter results in the formation of tetramer assemblies, the membrane part of which we determined to 3.1 Å resolution. The structure of the type III ATP synthase tetramer and its associated lipids suggests that it is the intact unit propagating the membrane curvature.
Mitoribosomes of green algae display a great structural divergence from their tracheophyte relatives, with fragmentation of both rRNA and proteins as a defining feature. Here, we report a 2.9 Å ...resolution structure of the mitoribosome from the alga Polytomella magna harbouring a reduced rRNA split into 13 fragments. We found that the rRNA contains a non-canonical reduced form of the 5S, as well as a permutation of the LSU domain I. The mt-5S rRNA is stabilised by mL40 that is also found in mitoribosomes lacking the 5S, which suggests an evolutionary pathway. Through comparison to other ribosomes with fragmented rRNAs, we observe that the pattern is shared across large evolutionary distances, and between cellular compartments, indicating an evolutionary convergence and supporting the concept of a primordial fragmented ribosome. On the protein level, eleven peripherally associated HEAT-repeat proteins are involved in the binding of 3' rRNA termini, and the structure features a prominent pseudo-trimer of one of them (mL116). Finally, in the exit tunnel, mL128 constricts the tunnel width of the vestibular area, and mL105, a homolog of a membrane targeting component mediates contacts with an inner membrane bound insertase. Together, the structural analysis provides insight into the evolution of the ribosomal machinery in mitochondria.
Abstract
Mitochondrial energy conversion requires an intricate architecture of the inner mitochondrial membrane
1
. Here we show that a supercomplex containing all four respiratory chain components ...contributes to membrane curvature induction in ciliates. 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-MDa assembly. Owing 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 tubulation of cristae. Our findings highlight how the evolution of protein subunits of respiratory complexes has led to the I–II–III
2
–IV
2
supercomplex that contributes to the shaping of the bioenergetic membrane, thereby enabling its functional specialization.
Endosomal sorting complexes required for transport (ESCRT) play key roles in protein sorting between membrane-bounded compartments of eukaryotic cells. Homologs of many ESCRT components are ...identifiable in various groups of archaea, especially in Asgardarchaeota, the archaeal phylum that is currently considered to include the closest relatives of eukaryotes, but not in bacteria. We performed a comprehensive search for ESCRT protein homologs in archaea and reconstructed ESCRT evolution using the phylogenetic tree of Vps4 ATPase (ESCRT IV) as a scaffold and using sensitive protein sequence analysis and comparison of structural models to identify previously unknown ESCRT proteins. Several distinct groups of ESCRT systems in archaea outside of Asgard were identified, including proteins structurally similar to ESCRT-I and ESCRT-II, and several other domains involved in protein sorting in eukaryotes, suggesting an early origin of these components. Additionally, distant homologs of CdvA proteins were identified in Thermoproteales which are likely components of the uncharacterized cell division system in these archaea. We propose an evolutionary scenario for the origin of eukaryotic and Asgard ESCRT complexes from ancestral building blocks, namely, the Vps4 ATPase, ESCRT-III components, wH (winged helix-turn-helix fold) and possibly also coiled-coil, and Vps28-like domains. The last archaeal common ancestor likely encompassed a complex ESCRT system that was involved in protein sorting. Subsequent evolution involved either simplification, as in the TACK superphylum, where ESCRT was co-opted for cell division, or complexification as in Asgardarchaeota. In Asgardarchaeota, the connection between ESCRT and the ubiquitin system that was previously considered a eukaryotic signature was already established.IMPORTANCEAll eukaryotic cells possess complex intracellular membrane organization. Endosomal sorting complexes required for transport (ESCRT) play a central role in membrane remodeling which is essential for cellular functionality in eukaryotes. Recently, it has been shown that Asgard archaea, the archaeal phylum that includes the closest known relatives of eukaryotes, encode homologs of many components of the ESCRT systems. We employed protein sequence and structure comparisons to reconstruct the evolution of ESCRT systems in archaea and identified several previously unknown homologs of ESCRT subunits, some of which can be predicted to participate in cell division. The results of this reconstruction indicate that the last archaeal common ancestor already encoded a complex ESCRT system that was involved in protein sorting. In Asgard archaea, ESCRT systems evolved toward greater complexity, and in particular, the connection between ESCRT and the ubiquitin system that was previously considered a eukaryotic signature was established.
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. Cryogenic electron microscopy identified that the absence of PsaH and Lhca2 gives rise to a head-to-head relative orientation of the PSI-light-harvesting complex I 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 light-harvesting complex II complexes in state transitions. We also define the most accurate available PSI-light-harvesting complex I model at 2.3 Å resolution, including a flexibly bound electron donor plastocyanin, and assign correct identities and orientations to all the pigments, as well as 621 water molecules that affect energy transfer pathways.
The topologies of α-helical membrane proteins are generally thought to be determined during their cotranslational insertion into the membrane. It is typically assumed that membrane topologies remain ...static after this process has ended. Recent findings, however, question this static view by suggesting that some parts of, or even the whole protein, can reorient in the membrane on a biologically relevant time scale. Here, we focus on antiparallel homo- or heterodimeric small multidrug resistance proteins and examine whether the individual monomers can undergo reversible topological inversion (flip flop) in the membrane until they are trapped in a fixed orientation by dimerization. By perturbing dimerization using various means, we show that the membrane orientation of a monomer is unaffected by the presence or absence of its dimerization partner. Thus, membrane-inserted monomers attain their final orientations independently of dimerization, suggesting that wholesale topological inversion is an unlikely event in vivo.
In structural biology, validation and verification of new atomic models are crucial and necessary steps which limit the production of reliable molecular models for publications and databases. An ...atomic model is the result of meticulous modeling and matching and is evaluated using a variety of metrics that provide clues to improve and refine the model so it fits our understanding of molecules and physical constraints. In cryo electron microscopy (cryo-EM) the validation is also part of an iterative modeling process in which there is a need to judge the quality of the model during the creation phase. A shortcoming is that the process and results of the validation are rarely communicated using visual metaphors. This work presents a visual framework for molecular validation. The framework was developed in close collaboration with domain experts in a participatory design process. Its core is a novel visual representation based on 2D heatmaps that shows all available validation metrics in a linear fashion, presenting a global overview of the atomic model and provide domain experts with interactive analysis tools. Additional information stemming from the underlying data, such as a variety of local quality measures, is used to guide the user's attention toward regions of higher relevance. Linked with the heatmap is a three-dimensional molecular visualization providing the spatial context of the structures and chosen metrics. Additional views of statistical properties of the structure are included in the visual framework. We demonstrate the utility of the framework and its visual guidance with examples from cryo-EM.
Mitoribosomes consist of ribosomal RNA and protein components, coordinated assembly of which is critical for function. We used mitoribosomes from Trypanosoma brucei with reduced RNA and increased ...protein mass to provide insights into the biogenesis of the mitoribosomal large subunit. Structural characterization of a stable assembly intermediate revealed 22 assembly factors, some of which have orthologues/counterparts/homologues in mammalian genomes. These assembly factors form a protein network that spans a distance of 180 Å, shielding the ribosomal RNA surface. The central protuberance and L7/L12 stalk are not assembled entirely and require removal of assembly 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, this extensive network of accessory factors stabilizes the immature sites and connects the functionally important regions of the mitoribosomal large subunit.
SYNOPSIS
The formation of the mitoribosome is an intricate process involving multiple protein factors that work in coordination. Here, structural characterisation of a T. brucei mitoribosomal large subunit assembly intermediate reveals the specific roles of an extensive assembly factor network.
Cryo‐EM of a mitoribosomal large subunit assembly intermediate reveals 22 associated assembly factors.
The 180 Å‐spanning network shields the ribosomal RNA surface and connects functionally important regions.
The central protuberance and the L7/L12 stalk are pre‐assembled in a non‐functional form
Conserved proteins GTPBP7 and mt‐EngA bind together at the subunit interface
A mitochondrial acyl‐carrier protein plays a role in docking the L1 stalk.
Assembly factors such as MRM and RPUSD4 represent catalytically‐inactivated enzymes serving as structural mediators.
Cryo‐EM structures of a Trypanosoma brucei mitoribosomal large subunit assembly intermediate reveals the specific roles of an extensive assembly factor network.
The neurotransmitter γ-aminobutyric acid (GABA) drives critical inhibitory processes in and beyond the nervous system, partly via ionotropic type-A receptors (GABAARs). Pharmacological properties of ...ρ-type GABAARs are particularly distinctive, yet the structural basis for their specialization remains unclear. Here, we present cryo-EM structures of a lipid-embedded human ρ1 GABAAR, including a partial intracellular domain, under apo, inhibited, and desensitized conditions. An apparent resting state, determined first in the absence of modulators, was recapitulated with the specific inhibitor (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid and blocker picrotoxin and provided a rationale for bicuculline insensitivity. Comparative structures, mutant recordings, and molecular simulations with and without GABA further explained the sensitized but slower activation of ρ1 relative to canonical subtypes. Combining GABA with picrotoxin also captured an apparent uncoupled intermediate state. This work reveals structural mechanisms of gating and modulation with applications to ρ-specific pharmaceutical design and to our biophysical understanding of ligand-gated ion channels.
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