Multimodal binding and inhibition of bacterial ribosomes by the antimicrobial peptides Api137 and Api88.

Proline-rich antimicrobial peptides (PrAMPs) inhibit bacterial protein biosynthesis by binding to the polypeptide exit tunnel (PET) near the peptidyl transferase center. Api137, an optimized derivative of honeybee PrAMP apidaecin, inhibits protein expression by trapping release factors (RFs), which interact with stop codons on ribosomes to terminate translation. This study uses cryo-EM, functional assays and molecular dynamic (MD) simulations to show that Api137 additionally occupies a second binding site near the exit of the PET and can repress translation independently of RF-trapping. Api88, a C-terminally amidated (-CONH2) analog of Api137 (-COOH), binds to the same sites, occupies a third binding pocket and interferes with the translation process presumably without RF-trapping. In conclusion, apidaecin-derived PrAMPs inhibit bacterial ribosomes by multimodal mechanisms caused by minor structural changes and thus represent a promising pool for drug development efforts.

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.

Intramolecular activity regulation of adhesion GPCRs in light of recent structural and evolutionary information.

The class B2 of GPCRs known as adhesion G protein-coupled receptors (aGPCRs) has come under increasing academic and nonacademic research focus over the past decade due to their physiological importance as mechano-sensors in cell-cell and cell-matrix contexts. A major advance in understanding signal transduction of aGPCRs was achieved by the identification of the so-called Stachel sequence, which acts as an intramolecular agonist at the interface between the N terminus (Nt) and the seven-transmembrane helix domain (7TMD). Distinct extracellular signals received by the Nt are integrated at the Stachel into structural changes of the 7TMD towards an active state conformation. Until recently, little information was available on how the activation process of aGPCRs is realized at the molecular level. In the past three years several structures of the 7TMD plus the Stachel in complex with G proteins have been determined, which provide new insights into the architecture and molecular function of this receptor class. Herein, we review this structural information to extract common and distinct aGPCR features with particular focus on the Stachel binding site within the 7TMD. Our analysis extends the current view of aGPCR activation and exposes similarities and differences not only between diverse aGPCR members, but also compared to other GPCR classes.

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.

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.

A critical period of translational control during brain development at codon resolution.

Translation modulates the timing and amplification of gene expression after transcription. Brain development requires uniquely complex gene expression patterns, but large-scale measurements of translation directly in the prenatal brain are lacking. We measure the reactants, synthesis and products of mRNA translation spanning mouse neocortex neurogenesis, and discover a transient window of dynamic regulation at mid-gestation. Timed translation upregulation of chromatin-binding proteins like Satb2, which is essential for neuronal subtype differentiation, restricts protein expression in neuronal lineages despite broad transcriptional priming in progenitors. In contrast, translation downregulation of ribosomal proteins sharply decreases ribosome biogenesis, coinciding with a major shift in protein synthesis dynamics at mid-gestation. Changing activity of eIF4EBP1, a direct inhibitor of ribosome biogenesis, is concurrent with ribosome downregulation and affects neurogenesis of the Satb2 lineage. Thus, the molecular logic of brain development includes the refinement of transcriptional programs by translation. Modeling of the developmental neocortex translatome is provided as an open-source searchable resource at https://shiny.mdc-berlin.de/cortexomics .

Cryo-electron tomography reveals structural insights into the membrane remodeling mode of dynamin-like EHD filaments.

Eps15-homology domain containing proteins (EHDs) are eukaryotic, dynamin-related ATPases involved in cellular membrane trafficking. They oligomerize on membranes into filaments that induce membrane tubulation. While EHD crystal structures in open and closed conformations were previously reported, little structural information is available for the membrane-bound oligomeric form. Consequently, mechanistic insights into the membrane remodeling mechanism have remained sparse. Here, by using cryo-electron tomography and subtomogram averaging, we determined structures of nucleotide-bound EHD4 filaments on membrane tubes of various diameters at an average resolution of 7.6 Å. Assembly of EHD4 is mediated via interfaces in the G-domain and the helical domain. The oligomerized EHD4 structure resembles the closed conformation, where the tips of the helical domains protrude into the membrane. The variation in filament geometry and tube radius suggests a spontaneous filament curvature of approximately 1/70 nm-1. Combining the available structural and functional data, we suggest a model for EHD-mediated membrane remodeling.

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.

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.

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.

Structural basis of early translocation events on the ribosome.

Peptide-chain elongation during protein synthesis entails sequential aminoacyl-tRNA selection and translocation reactions that proceed rapidly (2-20 per second) and with a low error rate (around 10-3 to 10-5 at each step) over thousands of cycles1. The cadence and fidelity of ribosome transit through mRNA templates in discrete codon increments is a paradigm for movement in biological systems that must hold for diverse mRNA and tRNA substrates across domains of life. Here we use single-molecule fluorescence methods to guide the capture of structures of early translocation events on the bacterial ribosome. Our findings reveal that the bacterial GTPase elongation factor G specifically engages spontaneously achieved ribosome conformations while in an active, GTP-bound conformation to unlock and initiate peptidyl-tRNA translocation. These findings suggest that processes intrinsic to the pre-translocation ribosome complex can regulate the rate of protein synthesis, and that energy expenditure is used later in the translocation mechanism than previously proposed.

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.

Divergent architecture of the heterotrimeric NatC complex explains N-terminal acetylation of cognate substrates.

The heterotrimeric NatC complex, comprising the catalytic Naa30 and the two auxiliary subunits Naa35 and Naa38, co-translationally acetylates the N-termini of numerous eukaryotic target proteins. Despite its unique subunit composition, its essential role for many aspects of cellular function and its suggested involvement in disease, structure and mechanism of NatC have remained unknown. Here, we present the crystal structure of the Saccharomyces cerevisiae NatC complex, which exhibits a strikingly different architecture compared to previously described N-terminal acetyltransferase (NAT) complexes. Cofactor and ligand-bound structures reveal how the first four amino acids of cognate substrates are recognized at the Naa30-Naa35 interface. A sequence-specific, ligand-induced conformational change in Naa30 enables efficient acetylation. Based on detailed structure-function studies, we suggest a catalytic mechanism and identify a ribosome-binding patch in an elongated tip region of NatC. Our study reveals how NAT machineries have divergently evolved to N-terminally acetylate specific subsets of target 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.

Multifaceted Stoichiometry Control of Bacterial Operons Revealed by Deep Proteome Quantification.

More than half of the protein-coding genes in bacteria are organized in polycistronic operons composed of two or more genes. It remains under debate whether the operon organization maintains the stoichiometric expression of the genes within an operon. In this study, we performed a label-free data-independent acquisition hyper reaction monitoring mass-spectrometry (HRM-MS) experiment to quantify the Escherichia coli proteome in exponential phase and quantified 93.6% of the cytosolic proteins, covering 67.9% and 56.0% of the translating polycistronic operons in BW25113 and MG1655 strains, respectively. We found that the translational regulation contributes largely to the proteome complexity: the shorter operons tend to be more tightly controlled for stoichiometry than longer operons; the operons which mainly code for complexes is more tightly controlled for stoichiometry than the operons which mainly code for metabolic pathways. The gene interval (distance between adjacent genes in one operon) may serve as a regulatory factor for stoichiometry. The catalytic efficiency might be a driving force for differential expression of enzymes encoded in one operon. These results illustrated the multifaceted nature of the operon regulation: the operon unified transcriptional level and gene-specific translational level. This multi-level regulation benefits the host by optimizing the efficiency of the productivity of metabolic pathways and maintenance of different types of protein complexes.

Alanine Tails Signal Proteolysis in Bacterial Ribosome-Associated Quality Control.

In ribosome-associated quality control (RQC), Rqc2/NEMF closely supports the E3 ligase Ltn1/listerin in promoting ubiquitylation and degradation of aberrant nascent-chains obstructing large (60S) ribosomal subunits-products of ribosome stalling during translation. However, while Ltn1 is eukaryote-specific, Rqc2 homologs are also found in bacteria and archaea; whether prokaryotic Rqc2 has an RQC-related function has remained unknown. Here, we show that, as in eukaryotes, a bacterial Rqc2 homolog (RqcH) recognizes obstructed 50S subunits and promotes nascent-chain proteolysis. Unexpectedly, RqcH marks nascent-chains for degradation in a direct manner, by appending C-terminal poly-alanine tails that act as degrons recognized by the ClpXP protease. Furthermore, RqcH acts redundantly with tmRNA/ssrA and protects cells against translational and environmental stresses. Our results uncover a proteolytic-tagging mechanism with implications toward the function of related modifications in eukaryotes and suggest that RQC was already active in the last universal common ancestor (LUCA) to help cope with incomplete translation.

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.

Translatomics: The Global View of Translation.

In all kingdoms of life, proteins are synthesized by ribosomes in a process referred to as translation. The amplitude of translational regulation exceeds the sum of transcription, mRNA degradation and protein degradation. Therefore, it is essential to investigate translation in a global scale. Like the other "omics"-methods, translatomics investigates the totality of the components in the translation process, including but not limited to translating mRNAs, ribosomes, tRNAs, regulatory RNAs and nascent polypeptide chains. Technical advances in recent years have brought breakthroughs in the investigation of these components at global scale, both for their composition and dynamics. These methods have been applied in a rapidly increasing number of studies to reveal multifaceted aspects of translation control. The process of translation is not restricted to the conversion of mRNA coding sequences into polypeptide chains, it also controls the composition of the proteome in a delicate and responsive way. Therefore, translatomics has extended its unique and innovative power to many fields including proteomics, cancer research, bacterial stress response, biological rhythmicity and plant biology. Rational design in translation can enhance recombinant protein production for thousands of times. This brief review summarizes the main state-of-the-art methods of translatomics, highlights recent discoveries made in this field and introduces applications of translatomics on basic biological and biomedical research.

tRNA Translocation by the Eukaryotic 80S Ribosome and the Impact of GTP Hydrolysis.

Translocation moves the tRNA2⋅mRNA module directionally through the ribosome during the elongation phase of protein synthesis. Although translocation is known to entail large conformational changes within both the ribosome and tRNA substrates, the orchestrated events that ensure the speed and fidelity of this critical aspect of the protein synthesis mechanism have not been fully elucidated. Here, we present three high-resolution structures of intermediates of translocation on the mammalian ribosome where, in contrast to bacteria, ribosomal complexes containing the translocase eEF2 and the complete tRNA2⋅mRNA module are trapped by the non-hydrolyzable GTP analog GMPPNP. Consistent with the observed structures, single-molecule imaging revealed that GTP hydrolysis principally facilitates rate-limiting, final steps of translocation, which are required for factor dissociation and which are differentially regulated in bacterial and mammalian systems by the rates of deacyl-tRNA dissociation from the E site.