Structural snapshots of actively translating human ribosomes.

Macromolecular machines, such as the ribosome, undergo large-scale conformational changes during their functional cycles. Although their mode of action is often compared to that of mechanical machines, a crucial difference is that, at the molecular dimension, thermodynamic effects dominate functional cycles, with proteins fluctuating stochastically between functional states defined by energetic minima on an energy landscape. Here, we have used cryo-electron microscopy to image ex-vivo-derived human polysomes as a source of actively translating ribosomes. Multiparticle refinement and 3D variability analysis allowed us to visualize a variety of native translation intermediates. Significantly populated states include not only elongation cycle intermediates in pre- and post-translocational states, but also eEF1A-containing decoding and termination/recycling complexes. Focusing on the post-translocational state, we extended this assessment to the single-residue level, uncovering striking details of ribosome-ligand interactions and identifying both static and functionally important dynamic elements.

Snapshots of native pre-50S ribosomes reveal a biogenesis factor network and evolutionary specialization.

Ribosome biogenesis is a fundamental multi-step cellular process that culminates in the formation of ribosomal subunits, whose production and modification are regulated by numerous biogenesis factors. In this study, we analyze physiologic prokaryotic ribosome biogenesis by isolating bona fide pre-50S subunits from an Escherichia coli strain with the biogenesis factor ObgE, affinity tagged at its native gene locus. Our integrative structural approach reveals a network of interacting biogenesis factors consisting of YjgA, RluD, RsfS, and ObgE on the immature pre-50S subunit. In addition, our study provides mechanistic insight into how the GTPase ObgE, in concert with other biogenesis factors, facilitates the maturation of the 50S functional core and reveals both conserved and divergent evolutionary features of ribosome biogenesis between prokaryotes and eukaryotes.

Protein Synthesis in the Developing Neocortex at Near-Atomic Resolution Reveals Ebp1-Mediated Neuronal Proteostasis at the 60S Tunnel Exit.

Protein synthesis must be finely tuned in the developing nervous system as the final essential step of gene expression. This study investigates the architecture of ribosomes from the neocortex during neurogenesis, revealing Ebp1 as a high-occupancy 60S peptide tunnel exit (TE) factor during protein synthesis at near-atomic resolution by cryoelectron microscopy (cryo-EM). Ribosome profiling demonstrated Ebp1-60S binding is highest during start codon initiation and N-terminal peptide elongation, regulating ribosome occupancy of these codons. Membrane-targeting domains emerging from the 60S tunnel, which recruit SRP/Sec61 to the shared binding site, displace Ebp1. Ebp1 is particularly abundant in the early-born neural stem cell (NSC) lineage and regulates neuronal morphology. Ebp1 especially impacts the synthesis of membrane-targeted cell adhesion molecules (CAMs), measured by pulsed stable isotope labeling by amino acids in cell culture (pSILAC)/bioorthogonal noncanonical amino acid tagging (BONCAT) mass spectrometry (MS). Therefore, Ebp1 is a central component of protein synthesis, and the ribosome TE is a focal point of gene expression control in the molecular specification of neuronal morphology during development.

Structure-Based Mechanisms of a Molecular RNA Polymerase/Chaperone Machine Required for Ribosome Biosynthesis.

Bacterial ribosomal RNAs are synthesized by a dedicated, conserved transcription-elongation complex that transcribes at high rates, shields RNA polymerase from premature termination, and supports co-transcriptional RNA folding, modification, processing, and ribosomal subunit assembly by presently unknown mechanisms. We have determined cryo-electron microscopy structures of complete Escherichia coli ribosomal RNA transcription elongation complexes, comprising RNA polymerase; DNA; RNA bearing an N-utilization-site-like anti-termination element; Nus factors A, B, E, and G; inositol mono-phosphatase SuhB; and ribosomal protein S4. Our structures and structure-informed functional analyses show that fast transcription and anti-termination involve suppression of NusA-stabilized pausing, enhancement of NusG-mediated anti-backtracking, sequestration of the NusG C-terminal domain from termination factor ρ, and the ρ blockade. Strikingly, the factors form a composite RNA chaperone around the RNA polymerase RNA-exit tunnel, which supports co-transcriptional RNA folding and annealing of distal RNA regions. Our work reveals a polymerase/chaperone machine required for biosynthesis of functional ribosomes.

Structural mechanism of CRL4-instructed STAT2 degradation via a novel cytomegaloviral DCAF receptor.

Human cytomegalovirus (CMV) is a ubiquitously distributed pathogen whose rodent counterparts such as mouse and rat CMV serve as common infection models. Here, we conducted global proteome profiling of rat CMV-infected cells and uncovered a pronounced loss of the transcription factor STAT2, which is crucial for antiviral interferon signalling. Via deletion mutagenesis, we found that the viral protein E27 is required for CMV-induced STAT2 depletion. Cellular and in vitro analyses showed that E27 exploits host-cell Cullin4-RING ubiquitin ligase (CRL4) complexes to induce poly-ubiquitylation and proteasomal degradation of STAT2. Cryo-electron microscopy revealed how E27 mimics molecular surface properties of cellular CRL4 substrate receptors called DCAFs (DDB1- and Cullin4-associated factors), thereby displacing them from the catalytic core of CRL4. Moreover, structural analyses showed that E27 recruits STAT2 through a bipartite binding interface, which partially overlaps with the IRF9 binding site. Structure-based mutations in M27, the murine CMV homologue of E27, impair the interferon-suppressing capacity and virus replication in mouse models, supporting the conserved importance of DCAF mimicry for CMV immune evasion.

Structural Basis for the Action of an All-Purpose Transcription Anti-termination Factor.

Bacteriophage λN protein, a model anti-termination factor, binds nascent RNA and host Nus factors, rendering RNA polymerase resistant to all pause and termination signals. A 3.7-Å-resolution cryo-electron microscopy structure and structure-informed functional analyses reveal a multi-pronged strategy by which the intrinsically unstructured λN directly modifies RNA polymerase interactions with the nucleic acids and subverts essential functions of NusA, NusE, and NusG to reprogram the transcriptional apparatus. λN repositions NusA and remodels the ß subunit flap tip, which likely precludes folding of pause or termination RNA hairpins in the exit tunnel and disrupts termination-supporting interactions of the α subunit C-terminal domains. λN invades and traverses the RNA polymerase hybrid cavity, likely stabilizing the hybrid and impeding pause- or termination-related conformational changes of polymerase. λN also lines upstream DNA, seemingly reinforcing anti-backtracking and anti-swiveling by NusG. Moreover, λN-repositioned NusA and NusE sequester the NusG C-terminal domain, counteracting ρ-dependent termination. Other anti-terminators likely utilize similar mechanisms to enable processive transcription.

Structural Visualization of the Formation and Activation of the 50S Ribosomal Subunit during In Vitro Reconstitution.

The assembly of ribosomal subunits is an essential prerequisite for protein biosynthesis in all domains of life. Although biochemical and biophysical approaches have advanced our understanding of ribosome assembly, our mechanistic comprehension of this process is still limited. Here, we perform an in vitro reconstitution of the Escherichia coli 50S ribosomal subunit. Late reconstitution products were subjected to high-resolution cryo-electron microscopy and multiparticle refinement analysis to reconstruct five distinct precursors of the 50S subunit with 4.3-3.8 Å resolution. These assembly intermediates define a progressive maturation pathway culminating in a late assembly particle, whose structure is more than 96% identical to a mature 50S subunit. Our structures monitor the formation and stabilization of structural elements in a nascent particle in unprecedented detail and identify the maturation of the rRNA-based peptidyl transferase center as the final critical step along the 50S assembly pathway.

Structural insights into Cullin4-RING ubiquitin ligase remodelling by Vpr from simian immunodeficiency viruses.

Viruses have evolved means to manipulate the host's ubiquitin-proteasome system, in order to down-regulate antiviral host factors. The Vpx/Vpr family of lentiviral accessory proteins usurp the substrate receptor DCAF1 of host Cullin4-RING ligases (CRL4), a family of modular ubiquitin ligases involved in DNA replication, DNA repair and cell cycle regulation. CRL4DCAF1 specificity modulation by Vpx and Vpr from certain simian immunodeficiency viruses (SIV) leads to recruitment, poly-ubiquitylation and subsequent proteasomal degradation of the host restriction factor SAMHD1, resulting in enhanced virus replication in differentiated cells. To unravel the mechanism of SIV Vpr-induced SAMHD1 ubiquitylation, we conducted integrative biochemical and structural analyses of the Vpr protein from SIVs infecting Cercopithecus cephus (SIVmus). X-ray crystallography reveals commonalities between SIVmus Vpr and other members of the Vpx/Vpr family with regard to DCAF1 interaction, while cryo-electron microscopy and cross-linking mass spectrometry highlight a divergent molecular mechanism of SAMHD1 recruitment. In addition, these studies demonstrate how SIVmus Vpr exploits the dynamic architecture of the multi-subunit CRL4DCAF1 assembly to optimise SAMHD1 ubiquitylation. Together, the present work provides detailed molecular insight into variability and species-specificity of the evolutionary arms race between host SAMHD1 restriction and lentiviral counteraction through Vpx/Vpr proteins.

Cryo-EM structure of the Shigella type III needle complex.

The Type III Secretion Systems (T3SS) needle complex is a conserved syringe-shaped protein translocation nanomachine with a mass of about 3.5 MDa essential for the survival and virulence of many Gram-negative bacterial pathogens. This system is composed of a membrane-embedded basal body and an extracellular needle that deliver effector proteins into host cells. High-resolution structures of the T3SS from different organisms and infection stages are needed to understand the underlying molecular mechanisms of effector translocation. Here, we present the cryo-electron microscopy structure of the isolated Shigella T3SS needle complex. The inner membrane (IM) region of the basal body adopts 24-fold rotational symmetry and forms a channel system that connects the bacterial periplasm with the export apparatus cage. The secretin oligomer adopts a heterogeneous architecture with 16- and 15-fold cyclic symmetry in the periplasmic N-terminal connector and C-terminal outer membrane ring, respectively. Two out of three IM subunits bind the secretin connector via a ß-sheet augmentation. The cryo-EM map also reveals the helical architecture of the export apparatus core, the inner rod, the needle and their intervening interfaces.

Molecular architecture of the ribosome-bound Hepatitis C Virus internal ribosomal entry site RNA.

Internal ribosomal entry sites (IRESs) are structured cis-acting RNAs that drive an alternative, cap-independent translation initiation pathway. They are used by many viruses to hijack the translational machinery of the host cell. IRESs facilitate translation initiation by recruiting and actively manipulating the eukaryotic ribosome using only a subset of canonical initiation factor and IRES transacting factors. Here we present cryo-EM reconstructions of the ribosome 80S- and 40S-bound Hepatitis C Virus (HCV) IRES. The presence of four subpopulations for the 80S•HCV IRES complex reveals dynamic conformational modes of the complex. At a global resolution of 3.9 Šfor the most stable complex, a derived atomic model reveals a complex fold of the IRES RNA and molecular details of its interaction with the ribosome. The comparison of obtained structures explains how a modular architecture facilitates mRNA loading and tRNA binding to the P-site. This information provides the structural foundation for understanding the mechanism of HCV IRES RNA-driven translation initiation.

Architecture of polyglutamine-containing fibrils from time-resolved fluorescence decay.

The disease risk and age of onset of Huntington disease (HD) and nine other repeat disorders strongly depend on the expansion of CAG repeats encoding consecutive polyglutamines (polyQ) in the corresponding disease protein. PolyQ length-dependent misfolding and aggregation are the hallmarks of CAG pathologies. Despite intense effort, the overall structure of these aggregates remains poorly understood. Here, we used sensitive time-dependent fluorescent decay measurements to assess the architecture of mature fibrils of huntingtin (Htt) exon 1 implicated in HD pathology. Varying the position of the fluorescent labels in the Htt monomer with expanded 51Q (Htt51Q) and using structural models of putative fibril structures, we generated distance distributions between donors and acceptors covering all possible distances between the monomers or monomer dimensions within the polyQ amyloid fibril. Using Monte Carlo simulations, we systematically scanned all possible monomer conformations that fit the experimentally measured decay times. Monomers with four-stranded 51Q stretches organized into five-layered ß-sheets with alternating N termini of the monomers perpendicular to the fibril axis gave the best fit to our data. Alternatively, the core structure of the polyQ fibrils might also be a zipper layer with antiparallel four-stranded stretches as this structure showed the next best fit. All other remaining arrangements are clearly excluded by the data. Furthermore, the assessed dimensions of the polyQ stretch of each monomer provide structural evidence for the observed polyQ length threshold in HD pathology. Our approach can be used to validate the effect of pharmacological substances that inhibit or alter amyloid growth and structure.

Cryo-electron microscopic structure of SecA protein bound to the 70S ribosome.

SecA is an ATP-dependent molecular motor pumping secretory and outer membrane proteins across the cytoplasmic membrane in bacteria. SecA associates with the protein-conducting channel, the heterotrimeric SecYEG complex, in a so-called posttranslational manner. A recent study further showed binding of a monomeric state of SecA to the ribosome. However, the true oligomeric state of SecA remains controversial because SecA can also form functional dimers, and high-resolution crystal structures exist for both the monomer and the dimer. Here we present the cryo-electron microscopy structures of Escherichia coli SecA bound to the ribosome. We show that not only a monomeric SecA binds to the ribosome but also that two copies of SecA can be observed that form an elongated dimer. Two copies of SecA completely surround the tunnel exit, providing a unique environment to the nascent polypeptides emerging from the ribosome. We identified the N-terminal helix of SecA required for a stable association with the ribosome. The structures indicate a possible function of the dimeric form of SecA at the ribosome.

Transient disome complex formation in native polysomes during ongoing protein synthesis captured by cryo-EM.

Structural studies of translating ribosomes traditionally rely on in vitro assembly and stalling of ribosomes in defined states. To comprehensively visualize bacterial translation, we reactivated ex vivo-derived E. coli polysomes in the PURE in vitro translation system and analyzed the actively elongating polysomes by cryo-EM. We find that 31% of 70S ribosomes assemble into disome complexes that represent eight distinct functional states including decoding and termination intermediates, and a pre-nucleophilic attack state. The functional diversity of disome complexes together with RNase digest experiments suggests that paused disome complexes transiently form during ongoing elongation. Structural analysis revealed five disome interfaces between leading and queueing ribosomes that undergo rearrangements as the leading ribosome traverses through the elongation cycle. Our findings reveal at the molecular level how bL9's CTD obstructs the factor binding site of queueing ribosomes to thwart harmful collisions and illustrate how translation dynamics reshape inter-ribosomal contacts.

Cryo-EM captures early ribosome assembly in action.

Ribosome biogenesis is a fundamental multi-step cellular process in all domains of life that involves the production, processing, folding, and modification of ribosomal RNAs (rRNAs) and ribosomal proteins. To obtain insights into the still unexplored early assembly phase of the bacterial 50S subunit, we exploited a minimal in vitro reconstitution system using purified ribosomal components and scalable reaction conditions. Time-limited assembly assays combined with cryo-EM analysis visualizes the structurally complex assembly pathway starting with a particle consisting of ordered density for only ~500 nucleotides of 23S rRNA domain I and three ribosomal proteins. In addition, our structural analysis reveals that early 50S assembly occurs in a domain-wise fashion, while late 50S assembly proceeds incrementally. Furthermore, we find that both ribosomal proteins and folded rRNA helices, occupying surface exposed regions on pre-50S particles, induce, or stabilize rRNA folds within adjacent regions, thereby creating cooperativity.

Structure of the actively translating plant 80S ribosome at 2.2 Å resolution.

In plant cells, translation occurs in three compartments: the cytosol, the plastids and the mitochondria. While the structures of the (prokaryotic-type) ribosomes in plastids and mitochondria are well characterized, high-resolution structures of the eukaryotic 80S ribosomes in the cytosol have been lacking. Here the structure of translating tobacco (Nicotiana tabacum) 80S ribosomes was solved by cryo-electron microscopy with a global resolution of 2.2 Å. The ribosome structure includes two tRNAs, decoded mRNA and the nascent peptide chain, thus providing insights into the molecular underpinnings of the cytosolic translation process in plants. The map displays conserved and plant-specific rRNA modifications and the positions of numerous ionic cofactors, and it uncovers the role of monovalent ions in the decoding centre. The model of the plant 80S ribosome enables broad phylogenetic comparisons that reveal commonalities and differences in the ribosomes of plants and those of other eukaryotes, thus putting our knowledge about eukaryotic translation on a firmer footing.

A nanobody recognizes a unique conserved epitope and potently neutralizes SARS-CoV-2 omicron variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) Omicron variant sub-lineages spread rapidly worldwide, mostly due to their immune-evasive properties. This has put a significant part of the population at risk for severe disease and underscores the need for effective anti-SARS-CoV-2 agents against emergent strains in vulnerable patients. Camelid nanobodies are attractive therapeutic candidates due to their high stability, ease of large-scale production, and potential for delivery via inhalation. Here, we characterize the receptor binding domain (RBD)-specific nanobody W25 and show superior neutralization activity toward Omicron sub-lineages in comparison to all other SARS-CoV2 variants. Structure analysis of W25 in complex with the SARS-CoV2 spike glycoprotein shows that W25 engages an RBD epitope not covered by any of the antibodies previously approved for emergency use. In vivo evaluation of W25 prophylactic and therapeutic treatments across multiple SARS-CoV-2 variant infection models, together with W25 biodistribution analysis in mice, demonstrates favorable pre-clinical properties. Together, these data endorse W25 for further clinical development.

Structure of the mammalian ribosome as it decodes the selenocysteine UGA codon.

The elongation of eukaryotic selenoproteins relies on a poorly understood process of interpreting in-frame UGA stop codons as selenocysteine (Sec). We used cryo-electron microscopy to visualize Sec UGA recoding in mammals. A complex between the noncoding Sec-insertion sequence (SECIS), SECIS-binding protein 2 (SBP2), and 40S ribosomal subunit enables Sec-specific elongation factor eEFSec to deliver Sec. eEFSec and SBP2 do not interact directly but rather deploy their carboxyl-terminal domains to engage with the opposite ends of the SECIS. By using its Lys-rich and carboxyl-terminal segments, the ribosomal protein eS31 simultaneously interacts with Sec-specific transfer RNA (tRNASec) and SBP2, which further stabilizes the assembly. eEFSec is indiscriminate toward l-serine and facilitates its misincorporation at Sec UGA codons. Our results support a fundamentally distinct mechanism of Sec UGA recoding in eukaryotes from that in bacteria.

Steps toward translocation-independent RNA polymerase inactivation by terminator ATPase ρ.

Factor-dependent transcription termination mechanisms are poorly understood. We determined a series of cryo-electron microscopy structures portraying the hexameric adenosine triphosphatase (ATPase) ρ on a pathway to terminating NusA/NusG-modified elongation complexes. An open ρ ring contacts NusA, NusG, and multiple regions of RNA polymerase, trapping and locally unwinding proximal upstream DNA. NusA wedges into the ρ ring, initially sequestering RNA. Upon deflection of distal upstream DNA over the RNA polymerase zinc-binding domain, NusA rotates underneath one capping ρ subunit, which subsequently captures RNA. After detachment of NusG and clamp opening, RNA polymerase loses its grip on the RNA:DNA hybrid and is inactivated. Our structural and functional analyses suggest that ρ, and other termination factors across life, may use analogous strategies to allosterically trap transcription complexes in a moribund state.

Structures of active melanocortin-4 receptor-Gs-protein complexes with NDP-α-MSH and setmelanotide.

The melanocortin-4 receptor (MC4R), a hypothalamic master regulator of energy homeostasis and appetite, is a class A G-protein-coupled receptor and a prime target for the pharmacological treatment of obesity. Here, we present cryo-electron microscopy structures of MC4R-Gs-protein complexes with two drugs recently approved by the FDA, the peptide agonists NDP-α-MSH and setmelanotide, with 2.9 Å and 2.6 Å resolution. Together with signaling data from structure-derived MC4R mutants, the complex structures reveal the agonist-induced origin of transmembrane helix (TM) 6-regulated receptor activation. The ligand-binding modes of NDP-α-MSH, a high-affinity linear variant of the endogenous agonist α-MSH, and setmelanotide, a cyclic anti-obesity drug with biased signaling toward Gq/11, underline the key role of TM3 in ligand-specific interactions and of calcium ion as a ligand-adaptable cofactor. The agonist-specific TM3 interplay subsequently impacts receptor-Gs-protein interfaces at intracellular loop 2, which also regulates the G-protein coupling profile of this promiscuous receptor. Finally, our structures reveal mechanistic details of MC4R activation/inhibition, and provide important insights into the regulation of the receptor signaling profile which will facilitate the development of tailored anti-obesity drugs.

Cryo-EM structures reveal intricate Fe-S cluster arrangement and charging in Rhodobacter capsulatus formate dehydrogenase.

Metal-containing formate dehydrogenases (FDH) catalyse the reversible oxidation of formate to carbon dioxide at their molybdenum or tungsten active site. They display a diverse subunit and cofactor composition, but structural information on these enzymes is limited. Here we report the cryo-electron microscopic structures of the soluble Rhodobacter capsulatus FDH (RcFDH) as isolated and in the presence of reduced nicotinamide adenine dinucleotide (NADH). RcFDH assembles into a 360 kDa dimer of heterotetramers revealing a putative interconnection of electron pathway chains. In the presence of NADH, the RcFDH structure shows charging of cofactors, indicative of an increased electron load.