Polypeptide Exit Tunnel Research Articles

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.

Tetracenomycin X sequesters peptidyl-tRNA during translation of QK motifs.

As antimicrobial resistance threatens our ability to treat common bacterial infections, new antibiotics with limited cross-resistance are urgently needed. In this regard, natural products that target the bacterial ribosome have the potential to be developed into potent drugs through structure-guided design, provided their mechanisms of action are well understood. Here we use inverse toeprinting coupled to next-generation sequencing to show that the aromatic polyketide tetracenomycin X primarily inhibits peptide bond formation between an incoming aminoacyl-tRNA and a terminal Gln-Lys (QK) motif in the nascent polypeptide. Using cryogenic electron microscopy, we reveal that translation inhibition at QK motifs occurs via an unusual mechanism involving sequestration of the 3' adenosine of peptidyl-tRNALys in the drug-occupied nascent polypeptide exit tunnel of the ribosome. Our study provides mechanistic insights into the mode of action of tetracenomycin X on the bacterial ribosome and suggests a path forward for the development of novel aromatic polyketide antibiotics.

Structural basis for translation inhibition by the glycosylated drosocin peptide

The proline-rich antimicrobial peptide (PrAMP) drosocin is produced by Drosophila species to combat bacterial infection. Unlike many PrAMPs, drosocin is O-glycosylated at threonine 11, a post-translation modification that enhances its antimicrobial activity. Here we demonstrate that the O-glycosylation not only influences cellular uptake of the peptide but also interacts with its intracellular target, the ribosome. Cryogenic electron microscopy structures of glycosylated drosocin on the ribosome at 2.0–2.8-Å resolution reveal that the peptide interferes with translation termination by binding within the polypeptide exit tunnel and trapping RF1 on the ribosome, reminiscent of that reported for the PrAMP apidaecin. The glycosylation of drosocin enables multiple interactions with U2609 of the 23S rRNA, leading to conformational changes that break the canonical base pair with A752. Collectively, our study reveals novel molecular insights into the interaction of O-glycosylated drosocin with the ribosome, which provide a structural basis for future development of this class of antimicrobials.

A molecular network of conserved factors keeps ribosomes dormant in the egg.

Ribosomes are produced in large quantities during oogenesis and are stored in the egg. However, the egg and early embryo are translationally repressed1-4. Here, using mass spectrometry and cryo-electron microscopy analyses of ribosomes isolated from zebrafish (Danio rerio) and Xenopus laevis eggs and embryos, we provide molecular evidence that ribosomes transition from a dormant state to an active state during the first hours of embryogenesis. Dormant ribosomes are associated with four conserved factors that form two modules, consisting of Habp4-eEF2 and death associated protein 1b (Dap1b) or Dap in complex with eIF5a. Both modules occupy functionally important sites and act together to stabilize ribosomes and repress translation. Dap1b (also known as Dapl1in mammals) is a newly discovered translational inhibitor that stably inserts into the polypeptide exit tunnel. Addition of recombinant zebrafish Dap1b protein is sufficient to block translation and reconstitute the dormant egg ribosome state in a mammalian translation extract in vitro. Thus, a developmentally programmed, conserved ribosome state has a key role in ribosome storage and translational repression in the egg.

The human ribosome-associated complex suppresses prion formation in yeast.

Many human diseases are associated with the misfolding of amyloidogenic proteins. Understanding the mechanisms cells employ to ensure the integrity of the proteome is therefore a crucial step in the development of potential therapeutic interventions. Yeast cells possess numerous prion-forming proteins capable of adopting amyloid conformations, possibly as an epigenetic mechanism to cope with changing environmental conditions. The ribosome-associated complex (RAC), which docks near the ribosomal polypeptide exit tunnel and recruits the Hsp70 Ssb to chaperone nascent chains, can moderate the acquisition of these amyloid conformations in yeast. Here we examine the ability of the human RAC chaperone proteins Mpp11 and Hsp70L1 to function in place of their yeast RAC orthologues Zuo1 and Ssz1 in yeast lacking endogenous RAC and investigate the extent to which the human orthologues can perform RAC chaperone activities in yeast. We found that the Mpp11/Hsp70L1 complex can partially correct the growth defect seen in RAC-deficient yeast cells, although yeast/human hetero species complexes were variable in this ability. The proportion of cells in which the Sup35 protein undergoes spontaneous conversion to a [PSI+ ] prion conformation, which is increased in the absence of RAC, was reduced by the presence of the human RAC complex. However, the toxicity in yeast from expression of a pathogenically expanded polyQ protein was unable to be countered by the human RAC chaperones. This yeast system can serve as a facile model for studying the extent to which the human RAC chaperones contribute to combating cotranslational misfolding of other mammalian disease-associated proteins.

A male germ-cell-specific ribosome controls male fertility.

Ribosomes are highly sophisticated translation machines that have been demonstrated to be heterogeneous in the regulation of protein synthesis1,2. Male germ cell development involves complex translational regulation during sperm formation3. However, it remains unclear whether translation during sperm formation is performed by a specific ribosome. Here we report a ribosome with a specialized nascent polypeptide exit tunnel, RibosomeST, that is assembled with the male germ-cell-specific protein RPL39L, the paralogue of core ribosome (RibosomeCore) protein RPL39. Deletion of RibosomeST in mice causes defective sperm formation, resulting in substantially reduced fertility. Our comparison of single-particle cryo-electron microscopy structures of ribosomes from mouse kidneys and testes indicates that RibosomeST features a ribosomal polypeptide exit tunnel of distinct size and charge states compared with RibosomeCore. RibosomeST predominantly cotranslationally regulates the folding of a subset of male germ-cell-specific proteins that are essential for the formation of sperm. Moreover, we found that specialized functions of RibosomeST were not replaceable by RibosomeCore. Taken together, identification of this sperm-specific ribosome should greatly expand our understanding of ribosome function and tissue-specific regulation of protein expression pattern in mammals.

Translation initiation factor eIF4G1 modulates assembly of the polypeptide exit tunnel region in yeast ribosome biogenesis

eIF4G is an important eukaryotic translation initiation factor. In this study, eIF4G1, one of the eIF4G isoforms, was shown to directly participate in biogenesis of the large (60S) ribosomal subunit in Saccharomyces cerevisiae cells. Mutation of eIF4G1 decreased the amount 60S ribosomal subunits significantly. The C-terminal fragment of eIF4G1 could complement the function in 60S biogenesis. Analyses of its purified complex with mass spectrometry indicated that eIF4G1 associated with the pre-60S form directly. Strong genetic and direct protein-protein interactions were observed between eIF4G1 and Ssf1 protein. Upon deletion of eIF4G1, Ssf1, Rrp15, Rrp14 and Mak16 were abnormally retained on the pre-60S complex. This purturbed the loading of Arx1 and eL31 at the polypeptide exit tunnel (PET) site and the transition to a Nog2 complex. Our data indicate that eIF4G1 is important in facilitating PET maturation and 27S processing correctly. This article has an associated First Person interview with the first author of the paper.

First person – Yun-Ting Tseng

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Yun-Ting Tseng is first author on ‘ Translation initiation factor eIF4G1 modulates assembly of the polypeptide exit tunnel region in yeast ribosome biogenesis’, published in JCS. Yun-Ting is a PhD student in the lab of Kai-Yin Lo at the Department of Agricultural Chemistry, College of Bioresources and Agriculture, National Taiwan University, working within the field of ribosome biogenesis, exploring how ribosomes are assembled and matured correctly.

Ribosomal protein eL39 is important for maturation of the nascent polypeptide exit tunnel and proper protein folding during translation.

During translation, nascent polypeptide chains travel from the peptidyl transferase center through the nascent polypeptide exit tunnel (NPET) to emerge from 60S subunits. The NPET includes portions of five of the six 25S/5.8S rRNA domains and ribosomal proteins uL4, uL22, and eL39. Internal loops of uL4 and uL22 form the constriction sites of the NPET and are important for both assembly and function of ribosomes. Here, we investigated the roles of eL39 in tunnel construction, 60S biogenesis, and protein synthesis. We show that eL39 is important for proper protein folding during translation. Consistent with a delay in processing of 27S and 7S pre-rRNAs, eL39 functions in pre-60S assembly during middle nucleolar stages. Our biochemical assays suggest the presence of eL39 in particles at these stages, although it is not visualized in them by cryo-electron microscopy. This indicates that eL39 takes part in assembly even when it is not fully accommodated into the body of pre-60S particles. eL39 is also important for later steps of assembly, rotation of the 5S ribonucleoprotein complex, likely through long range rRNA interactions. Finally, our data strongly suggest the presence of alternative pathways of ribosome assembly, previously observed in the biogenesis of bacterial ribosomal subunits.

Cryo-EM structure of Mycobacterium tuberculosis 50S ribosomal subunit bound with clarithromycin reveals dynamic and specific interactions with macrolides

ABSTRACT Tuberculosis (TB) is the leading infectious disease caused by Mycobacterium tuberculosis (Mtb). Clarithromycin (CTY), an analog of erythromycin (ERY), is more potent against multidrug-resistance (MDR) TB. ERY and CTY were previously reported to bind to the nascent polypeptide exit tunnel (NPET) near peptidyl transferase center (PTC), but the only available CTY structure in complex with D. radiodurans (Dra) ribosome could be misinterpreted due to resolution limitation. To date, the mechanism of specificity and efficacy of CTY for Mtb remains elusive since the Mtb ribosome-CTY complex structure is still unknown. Here, we employed new sample preparation methods and solved the Mtb ribosome-CTY complex structure at 3.3Å with cryo-EM technique, where the crucial gate site A2062 (E. coli numbering) is located at the CTY binding site within NPET. Two alternative conformations of A2062, a novel syn-conformation as well as a swayed conformation bound with water molecule at interface, may play a role in coordinating the binding of specific drug molecules. The previously overlooked C–H hydrogen bond (H-bond) and π interaction may collectively contribute to the enhanced binding affinity. Together, our structure data provide a structural basis for the dynamic binding as well as the specificity of CTY and explain of how a single methyl group in CTY improves its potency, which provides new evidence to reveal previously unclear mechanism of translational modulation for future drug design and anti-TB therapy. Furthermore, our sample preparation method may facilitate drug discovery based on the complexes with low water solubility drugs by cryo-EM technique.

Ribosome exit tunnel electrostatics.

The impact of ribosome exit tunnel electrostatics on the protein elongation rate or on forces acting upon the nascent polypeptide chain are currently not fully elucidated. In the past, researchers have measured the electrostatic potential inside the ribosome polypeptide exit tunnel at a limited number of spatial points, at least in rabbit reticulocytes. Here we present a basic electrostatic model of the exit tunnel of the ribosome, providing a quantitative physical description of the tunnel interaction with the nascent proteins at all centro-axial points inside the tunnel. We show that a strong electrostatic screening is due to water molecules (not mobile ions) attracted to the ribosomal nucleic acid phosphate moieties buried in the immediate vicinity of the tunnel wall. We also show how the tunnel wall components and local ribosomal protein protrusions impact on the electrostatic potential profile and impede charged amino acid residues from progressing through the tunnel, affecting the elongation rate in a range of -40% to +85% when compared to the average elongation rate. The time spent by the ribosome to decode the genetic encrypted message is constrained accordingly. We quantitatively derive, at single-residue resolution, the axial forces acting on the nascent peptide from its particular sequence embedded in the tunnel. The model sheds light on how the experimental data point measurements of the potential are linked to the local structural chemistry of the inner wall, shape, and size of the tunnel. The model consistently connects experimental observations coming from different fields in molecular biology, x-ray crystallography, physical chemistry, biomechanics, and synthetic and multiomics biology. Our model should be a valuable tool to gain insight into protein synthesis dynamics, translational control, and the role of the ribosome's mechanochemistry in the cotranslational protein folding.

Spatially Enriched Paralog Rearrangements Argue Functionally Diverse Ribosomes Arise during Cold Acclimation in Arabidopsis.

Ribosome biogenesis is essential for plants to successfully acclimate to low temperature. Without dedicated steps supervising the 60S large subunits (LSUs) maturation in the cytosol, e.g., Rei-like (REIL) factors, plants fail to accumulate dry weight and fail to grow at suboptimal low temperatures. Around REIL, the final 60S cytosolic maturation steps include proofreading and assembly of functional ribosomal centers such as the polypeptide exit tunnel and the P-Stalk, respectively. In consequence, these ribosomal substructures and their assembly, especially during low temperatures, might be changed and provoke the need for dedicated quality controls. To test this, we blocked ribosome maturation during cold acclimation using two independent reil double mutant genotypes and tested changes in their ribosomal proteomes. Additionally, we normalized our mutant datasets using as a blank the cold responsiveness of a wild-type Arabidopsis genotype. This allowed us to neglect any reil-specific effects that may happen due to the presence or absence of the factor during LSU cytosolic maturation, thus allowing us to test for cold-induced changes that happen in the early nucleolar biogenesis. As a result, we report that cold acclimation triggers a reprogramming in the structural ribosomal proteome. The reprogramming alters the abundance of specific RP families and/or paralogs in non-translational LSU and translational polysome fractions, a phenomenon known as substoichiometry. Next, we tested whether the cold-substoichiometry was spatially confined to specific regions of the complex. In terms of RP proteoforms, we report that remodeling of ribosomes after a cold stimulus is significantly constrained to the polypeptide exit tunnel (PET), i.e., REIL factor binding and functional site. In terms of RP transcripts, cold acclimation induces changes in RP families or paralogs that are significantly constrained to the P-Stalk and the ribosomal head. The three modulated substructures represent possible targets of mechanisms that may constrain translation by controlled ribosome heterogeneity. We propose that non-random ribosome heterogeneity controlled by specialized biogenesis mechanisms may contribute to a preferential or ultimately even rigorous selection of transcripts needed for rapid proteome shifts and successful acclimation.

A role for the ribosome-associated complex in activation of the IRE1 branch of UPR.

The ubiquitous ribosome-associated complex (RAC) is a chaperone that spans ribosomes, making contacts near both the polypeptide exit tunnel and the decoding center, a position prime for sensing and coordinating translation and folding. Loss of RAC is known to result in growth defects and sensitization to translational and osmotic stresses. However, the physiological substrates of RAC and the mechanism(s) by which RAC is involved in responding to specific stresses in higher eukaryotes remain obscure. The data presented here uncover an essential function of mammalian RAC in the unfolded protein response (UPR). Knockdown of RAC sensitizes mammalian cells to endoplasmic reticulum (ER) stress and selectively interferes with IRE1 branch activation. Higher-order oligomerization of the inositol-requiring enzyme 1α (IRE1α) kinase/endoribonuclease depends upon RAC. These results reveal a surveillance function for RAC in the UPR, as follows: modulating IRE1α clustering as required for endonuclease activation and splicing of the substrate Xbp1 mRNA.

Mechanism of membrane-tethered mitochondrial protein synthesis.

Mitochondrial ribosomes (mitoribosomes) are tethered to the mitochondrial inner membrane to facilitate the cotranslational membrane insertion of the synthesized proteins. We report cryo-electron microscopy structures of human mitoribosomes with nascent polypeptide, bound to the insertase oxidase assembly 1-like (OXA1L) through three distinct contact sites. OXA1L binding is correlated with a series of conformational changes in the mitoribosomal large subunit that catalyze the delivery of newly synthesized polypeptides. The mechanism relies on the folding of mL45 inside the exit tunnel, forming two specific constriction sites that would limit helix formation of the nascent chain. A gap is formed between the exit and the membrane, making the newly synthesized proteins accessible. Our data elucidate the basis by which mitoribosomes interact with the OXA1L insertase to couple protein synthesis and membrane delivery.

Making the energy makers

Mitoribosome Within a mitochondrion, the powerhouse of eukaryotic cells, synthesis of the specialized transmembrane proteins of the electron transport chain is performed by dedicated mitoribosomes. The mechanism by which mitoribosomes couple protein synthesis with membrane insertion is poorly understood. Itoh et al. determined structures of the human mitoribosome during nascent chain synthesis while bound to its membrane insertase. These structures revealed a series of coordinated conformational changes within the polypeptide exit tunnel. The gating mechanism offers a fundamental molecular insight into how membrane proteins are synthesized in human mitochondria. Science , this issue p. [846][1] [1]: /lookup/doi/10.1126/science.abe0763

Structural insights into assembly of the ribosomal nascent polypeptide exit tunnel

The nascent polypeptide exit tunnel (NPET) is a major functional center of 60S ribosomal subunits. However, little is known about how the NPET is constructed during ribosome assembly. We utilized molecular genetics, biochemistry, and cryo-electron microscopy (cryo-EM) to investigate the functions of two NPET-associated proteins, ribosomal protein uL4 and assembly factor Nog1, in NPET assembly. Structures of mutant pre-ribosomes lacking the tunnel domain of uL4 reveal a misassembled NPET, including an aberrantly flexible ribosomal RNA helix 74, resulting in at least three different blocks in 60S assembly. Structures of pre-ribosomes lacking the C-terminal extension of Nog1 demonstrate that this extension scaffolds the tunnel domain of uL4 in the NPET to help maintain stability in the core of pre-60S subunits. Our data reveal that uL4 and Nog1 work together in the maturation of ribosomal RNA helix 74, which is required to ensure proper construction of the NPET and 60S ribosomal subunits.

1016 – REGULATION OF RIBOSOME BIOGENESIS IN HEMATOPOIETIC STEM CELLS

Dysregulation of ribosome biogenesis is the underlying etiology in a group of bone marrow failure syndromes called ribosomopathies. As prominently illustrated by one type of ribosomopathies, Shwachman-Diamond Syndrome (SDS), germline biallelic mutations in three assembly factors essential for the maturation of the 60S ribosome subunit result in progressive hematopoietic stem and progenitor cell (HSPC) failure, suggesting that HSPCs are sensitive to perturbations in ribosome assembly. However, how ribosome assembly is regulated in HSPCs remains poorly understood, as are approaches to restore in hematopoietic stem cell (HSC) function in the context of defective ribosome biogenesis. Here we uncover a novel role for the E3 ubiquitin ligase, HectD1, in regulating HSC function via ribosome biogenesis. HSCs from hematopoietic-specific <i>Hectd1</i> knockout mice exhibit a striking defect in transplantation ability and self-renewal. <i>Hectd1</i>-deficient HSCs fail to be maintained ex vivo, concomitant with a reduction in global protein synthesis and growth rate. The mechanism underlying HSC dysfunction upon <i>Hectd1</i> deficiency is directly linked to aberrant ribosome assembly. We demonstrate that HectD1 ubiquitinates and degrades an assembly factor for the polypeptide exit tunnel of the 60S ribosome in the cytoplasm. Depletion of <i>HectD1</i> led to an accumulation of the anti-association factor eIF6 on the 60S and decreased 80S to 60S ratio, consistent with a subunit joining defect associated with SDS-like diseases. Importantly, we found that restoration of ribosome assembly in <i>HectD1</i>-deficient cells promoted protein synthesis and enhanced HSC reconstitution capacity. These findings highlight the importance of ribosome assembly in HSC regeneration and represent a rare <i>in vivo</i> example of genetic suppression of HSC defects associated with dysfunctional ribosome biogenesis.

Coupling of 5S RNP rotation with maturation of functional centers during large ribosomal subunit assembly

The protein composition and structure of assembling 60S ribosomal subunits undergo numerous changes as pre-ribosomes transition from the nucleolus to the nucleoplasm. This includes stable anchoring of the Rpf2 subcomplex containing 5S rRNA, rpL5, rpL11, Rpf2 and Rrs1, which initially docks onto the flexible domain V of rRNA at earlier stages of assembly. In this work, we tested the function of the C-terminal domain (CTD) of Rpf2 during these anchoring steps, by truncating this extension and assaying effects on middle stages of subunit maturation. The rpf2Δ255-344 mutation affects proper folding of rRNA helices H68-70 during anchoring of the Rpf2 subcomplex. In addition, several assembly factors (AFs) are absent from pre-ribosomes or in altered conformations. Consequently, major remodeling events fail to occur: rotation of the 5S RNP, maturation of the peptidyl transferase center (PTC) and the nascent polypeptide exit tunnel (NPET), and export of assembling subunits to the cytoplasm.

Tetracenomycin X inhibits translation by binding within the ribosomal exit tunnel.

The increase in multi-drug resistant pathogenic bacteria is making our current arsenal of clinically used antibiotics obsolete, highlighting the urgent need for new lead compounds with distinct target binding sites to avoid cross-resistance. Here we report that the aromatic polyketide antibiotic tetracenomycin (TcmX) is a potent inhibitor of protein synthesis, and does not induce DNA damage as previously thought. Despite the structural similarity to the well-known translation inhibitor tetracycline, we show that TcmX does not interact with the small ribosomal subunit, but rather binds to the large subunit, within the polypeptide exit tunnel. This previously unappreciated binding site is located adjacent to the macrolide-binding site, where TcmX stacks on the noncanonical basepair formed by U1782 and U2586 of the 23S ribosomal RNA. Although the binding site is distinct from the macrolide antibiotics, our results indicate that like macrolides, TcmX allows translation of short oligopeptides before further translation is blocked.

Cotranslational Folding of Protein Domains on the Ribosome

Protein domains start to fold co-translationally, while they are being synthesized on the ribosome. Co-translational folding is vectorial; it starts in the confined space of the polypeptide exit tunnel of the ribosome and is modulated by the speed of translation. Defects in protein folding cause many human diseases; thus, understanding the co-translational folding is of eminent importance. We investigated co-translational folding of protein domains of different architecture in real-time as the protein is being synthesized by the ribosome. We showed that folding of the nascent protein proceeds through a several dynamic conformations that forms within and outside the peptide tunnel of the ribosome before exiting from the ribosome. The rate of translation defines the pathway and timing of folding. These results show how different protein domains fold co-translationally.