Publications by authors named "Roland Beckmann"

132 Publications

The SARS-unique domain (SUD) of SARS-CoV and SARS-CoV-2 interacts with human Paip1 to enhance viral RNA translation.

EMBO J 2021 Apr 20:e102277. Epub 2021 Apr 20.

Max-von-Pettenkofer Institute, Ludwig-Maximilians-University Munich, Munich, Germany.

The ongoing outbreak of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) demonstrates the continuous threat of emerging coronaviruses (CoVs) to public health. SARS-CoV-2 and SARS-CoV share an otherwise non-conserved part of non-structural protein 3 (Nsp3), therefore named as "SARS-unique domain" (SUD). We previously found a yeast-2-hybrid screen interaction of the SARS-CoV SUD with human poly(A)-binding protein (PABP)-interacting protein 1 (Paip1), a stimulator of protein translation. Here, we validate SARS-CoV SUD:Paip1 interaction by size-exclusion chromatography, split-yellow fluorescent protein, and co-immunoprecipitation assays, and confirm such interaction also between the corresponding domain of SARS-CoV-2 and Paip1. The three-dimensional structure of the N-terminal domain of SARS-CoV SUD ("macrodomain II", Mac2) in complex with the middle domain of Paip1, determined by X-ray crystallography and small-angle X-ray scattering, provides insights into the structural determinants of the complex formation. In cellulo, SUD enhances synthesis of viral but not host proteins via binding to Paip1 in pBAC-SARS-CoV replicon-transfected cells. We propose a possible mechanism for stimulation of viral translation by the SUD of SARS-CoV and SARS-CoV-2.
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http://dx.doi.org/10.15252/embj.2019102277DOI Listing
April 2021

Structure of Gcn1 bound to stalled and colliding 80S ribosomes.

Proc Natl Acad Sci U S A 2021 Apr;118(14)

Institute for Biochemistry and Molecular Biology, University of Hamburg, 20146 Hamburg, Germany;

The Gcn pathway is conserved in all eukaryotes, including mammals such as humans, where it is a crucial part of the integrated stress response (ISR). Gcn1 serves as an essential effector protein for the kinase Gcn2, which in turn is activated by stalled ribosomes, leading to phosphorylation of eIF2 and a subsequent global repression of translation. The fine-tuning of this adaptive response is performed by the Rbg2/Gir2 complex, a negative regulator of Gcn2. Despite the wealth of available biochemical data, information on structures of Gcn proteins on the ribosome has remained elusive. Here we present a cryo-electron microscopy structure of the yeast Gcn1 protein in complex with stalled and colliding 80S ribosomes. Gcn1 interacts with both 80S ribosomes within the disome, such that the Gcn1 HEAT repeats span from the P-stalk region on the colliding ribosome to the P-stalk and the A-site region of the lead ribosome. The lead ribosome is stalled in a nonrotated state with peptidyl-tRNA in the A-site, uncharged tRNA in the P-site, eIF5A in the E-site, and Rbg2/Gir2 in the A-site factor binding region. By contrast, the colliding ribosome adopts a rotated state with peptidyl-tRNA in a hybrid A/P-site, uncharged-tRNA in the P/E-site, and Mbf1 bound adjacent to the mRNA entry channel on the 40S subunit. Collectively, our findings reveal the interaction mode of the Gcn2-activating protein Gcn1 with colliding ribosomes and provide insight into the regulation of Gcn2 activation. The binding of Gcn1 to a disome has important implications not only for the Gcn2-activated ISR, but also for the general ribosome-associated quality control pathways.
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http://dx.doi.org/10.1073/pnas.2022756118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8040806PMC
April 2021

DutaFabs are engineered therapeutic Fab fragments that can bind two targets simultaneously.

Nat Commun 2021 01 29;12(1):708. Epub 2021 Jan 29.

Roche Pharma Research and Early Development, Roche Innovation Center Munich, Roche Diagnostics GmbH, Penzberg, Germany.

We report the development of a platform of dual targeting Fab (DutaFab) molecules, which comprise two spatially separated and independent binding sites within the human antibody CDR loops: the so-called H-side paratope encompassing HCDR1, HCDR3 and LCDR2, and the L-side paratope encompassing LCDR1, LCDR3 and HCDR2. Both paratopes can be independently selected and combined into the desired bispecific DutaFabs in a modular manner. X-ray crystal structures illustrate that DutaFabs are able to bind two target molecules simultaneously at the same Fv region comprising a VH-VL heterodimer. In the present study, this platform is applied to generate DutaFabs specific for VEGFA and PDGF-BB, which show high affinities, physico-chemical stability and solubility, as well as superior efficacy over anti-VEGF monotherapy in vivo. These molecules exemplify the usefulness of DutaFabs as a distinct class of antibody therapeutics, which is currently being evaluated in patients.
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http://dx.doi.org/10.1038/s41467-021-20949-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846786PMC
January 2021

SAP domain forms a flexible part of DNA aperture in Ku70/80.

FEBS J 2021 Jan 29. Epub 2021 Jan 29.

Department of Biochemistry, University of Cambridge, Cambridge, UK.

Nonhomologous end joining (NHEJ) is a DNA repair mechanism that religates double-strand DNA breaks to maintain genomic integrity during the entire cell cycle. The Ku70/80 complex recognizes DNA breaks and serves as an essential hub for recruitment of NHEJ components. Here, we describe intramolecular interactions of the Ku70 C-terminal domain, known as the SAP domain. Using single-particle cryo-electron microscopy, mass spectrometric analysis of intermolecular cross-linking and molecular modelling simulations, we captured variable positions of the SAP domain depending on DNA binding. The first position was localized at the DNA aperture in the Ku70/80 apo form but was not observed in the DNA-bound state. The second position, which was observed in both apo and DNA-bound states, was found below the DNA aperture, close to the helical arm of Ku70. The localization of the SAP domain in the DNA aperture suggests a function as a flexible entry gate for broken DNA. DATABASES: EM maps have been deposited in EMDB (EMD-11933). Coordinates have been deposited in Protein Data Bank (PDB 7AXZ). Other data are available from corresponding authors upon a request.
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http://dx.doi.org/10.1111/febs.15732DOI Listing
January 2021

Structure of the Maturing 90S Pre-ribosome in Association with the RNA Exosome.

Mol Cell 2021 01 15;81(2):293-303.e4. Epub 2020 Dec 15.

Heidelberg University Biochemistry Center (BZH), Im Neuenheimer Feld 328, 69120 Heidelberg, Germany. Electronic address:

Ribosome assembly is catalyzed by numerous trans-acting factors and coupled with irreversible pre-rRNA processing, driving the pathway toward mature ribosomal subunits. One decisive step early in this progression is removal of the 5' external transcribed spacer (5'-ETS), an RNA extension at the 18S rRNA that is integrated into the huge 90S pre-ribosome structure. Upon endo-nucleolytic cleavage at an internal site, A, the 5'-ETS is separated from the 18S rRNA and degraded. Here we present biochemical and cryo-electron microscopy analyses that depict the RNA exosome, a major 3'-5' exoribonuclease complex, in a super-complex with the 90S pre-ribosome. The exosome is docked to the 90S through its co-factor Mtr4 helicase, a processive RNA duplex-dismantling helicase, which strategically positions the exosome at the base of 5'-ETS helices H9-H9', which are dislodged in our 90S-exosome structures. These findings suggest a direct role of the exosome in structural remodeling of the 90S pre-ribosome to drive eukaryotic ribosome synthesis.
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http://dx.doi.org/10.1016/j.molcel.2020.11.009DOI Listing
January 2021

Architecture of the active post-translational Sec translocon.

EMBO J 2021 Feb 11;40(3):e105643. Epub 2020 Dec 11.

Gene Center Munich, Department of Biochemistry, University of Munich, Munich, Germany.

In eukaryotes, most secretory and membrane proteins are targeted by an N-terminal signal sequence to the endoplasmic reticulum, where the trimeric Sec61 complex serves as protein-conducting channel (PCC). In the post-translational mode, fully synthesized proteins are recognized by a specialized channel additionally containing the Sec62, Sec63, Sec71, and Sec72 subunits. Recent structures of this Sec complex in the idle state revealed the overall architecture in a pre-opened state. Here, we present a cryo-EM structure of the yeast Sec complex bound to a substrate, and a crystal structure of the Sec62 cytosolic domain. The signal sequence is inserted into the lateral gate of Sec61α similar to previous structures, yet, with the gate adopting an even more open conformation. The signal sequence is flanked by two Sec62 transmembrane helices, the cytoplasmic N-terminal domain of Sec62 is more rigidly positioned, and the plug domain is relocated. We crystallized the Sec62 domain and mapped its interaction with the C-terminus of Sec63. Together, we obtained a near-complete and integrated model of the active Sec complex.
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http://dx.doi.org/10.15252/embj.2020105643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7849165PMC
February 2021

A structural inventory of native ribosomal ABCE1-43S pre-initiation complexes.

EMBO J 2021 01 8;40(1):e105179. Epub 2020 Dec 8.

Gene Center and Center for Integrated Protein Science Munich, Department of Biochemistry, University of Munich, Munich, Germany.

In eukaryotic translation, termination and ribosome recycling phases are linked to subsequent initiation of a new round of translation by persistence of several factors at ribosomal sub-complexes. These comprise/include the large eIF3 complex, eIF3j (Hcr1 in yeast) and the ATP-binding cassette protein ABCE1 (Rli1 in yeast). The ATPase is mainly active as a recycling factor, but it can remain bound to the dissociated 40S subunit until formation of the next 43S pre-initiation complexes. However, its functional role and native architectural context remains largely enigmatic. Here, we present an architectural inventory of native yeast and human ABCE1-containing pre-initiation complexes by cryo-EM. We found that ABCE1 was mostly associated with early 43S, but also with later 48S phases of initiation. It adopted a novel hybrid conformation of its nucleotide-binding domains, while interacting with the N-terminus of eIF3j. Further, eIF3j occupied the mRNA entry channel via its ultimate C-terminus providing a structural explanation for its antagonistic role with respect to mRNA binding. Overall, the native human samples provide a near-complete molecular picture of the architecture and sophisticated interaction network of the 43S-bound eIF3 complex and the eIF2 ternary complex containing the initiator tRNA.
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http://dx.doi.org/10.15252/embj.2020105179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7780240PMC
January 2021

Structural basis for the final steps of human 40S ribosome maturation.

Nature 2020 11 18;587(7835):683-687. Epub 2020 Nov 18.

Gene Center Munich, Department of Biochemistry, University of Munich, Munich, Germany.

Eukaryotic ribosomes consist of a small 40S and a large 60S subunit that are assembled in a highly coordinated manner. More than 200 factors ensure correct modification, processing and folding of ribosomal RNA and the timely incorporation of ribosomal proteins. Small subunit maturation ends in the cytosol, when the final rRNA precursor, 18S-E, is cleaved at site 3 by the endonuclease NOB1. Previous structures of human 40S precursors have shown that NOB1 is kept in an inactive state by its partner PNO1. The final maturation events, including the activation of NOB1 for the decisive rRNA-cleavage step and the mechanisms driving the dissociation of the last biogenesis factors have, however, remained unresolved. Here we report five cryo-electron microscopy structures of human 40S subunit precursors, which describe the compositional and conformational progression during the final steps of 40S assembly. Our structures explain the central role of RIOK1 in the displacement and dissociation of PNO1, which in turn allows conformational changes and activation of the endonuclease NOB1. In addition, we observe two factors, eukaryotic translation initiation factor 1A domain-containing protein (EIF1AD) and leucine-rich repeat-containing protein 47 (LRRC47), which bind to late pre-40S particles near RIOK1 and the central rRNA helix 44. Finally, functional data shows that EIF1AD is required for efficient assembly factor recycling and 18S-E processing. Our results thus enable a detailed understanding of the last steps in 40S formation in human cells and, in addition, provide evidence for principal differences in small ribosomal subunit formation between humans and the model organism Saccharomyces cerevisiae.
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http://dx.doi.org/10.1038/s41586-020-2929-xDOI Listing
November 2020

90 pre-ribosome transformation into the primordial 40 subunit.

Science 2020 09;369(6510):1470-1476

Gene Center, Department of Biochemistry, University of Munich, 81377 Munich, Germany.

Production of small ribosomal subunits initially requires the formation of a 90 precursor followed by an enigmatic process of restructuring into the primordial pre-40 subunit. We elucidate this process by biochemical and cryo-electron microscopy analysis of intermediates along this pathway in yeast. First, the remodeling RNA helicase Dhr1 engages the 90 pre-ribosome, followed by Utp24 endonuclease-driven RNA cleavage at site A, thereby separating the 5'-external transcribed spacer (ETS) from 18 ribosomal RNA. Next, the 5'-ETS and 90 assembly factors become dislodged, but this occurs sequentially, not en bloc. Eventually, the primordial pre-40 emerges, still retaining some 90 factors including Dhr1, now ready to unwind the final small nucleolar U3-18 RNA hybrid. Our data shed light on the elusive 90 to pre-40 transition and clarify the principles of assembly and remodeling of large ribonucleoproteins.
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http://dx.doi.org/10.1126/science.abb4119DOI Listing
September 2020

An ATP-dependent partner switch links flagellar C-ring assembly with gene expression.

Proc Natl Acad Sci U S A 2020 08 11;117(34):20826-20835. Epub 2020 Aug 11.

Center for Synthetic Microbiology (SYNMIKRO), Philipps-University Marburg, 35043 Marburg, Germany;

Bacterial flagella differ in their number and spatial arrangement. In many species, the MinD-type ATPase FlhG (also YlxH/FleN) is central to the numerical control of bacterial flagella, and its deletion in polarly flagellated bacteria typically leads to hyperflagellation. The molecular mechanism underlying this numerical control, however, remains enigmatic. Using the model species , we show that FlhG links assembly of the flagellar C ring with the action of the master transcriptional regulator FlrA (named FleQ in other species). While FlrA and the flagellar C-ring protein FliM have an overlapping binding site on FlhG, their binding depends on the ATP-dependent dimerization state of FlhG. FliM interacts with FlhG independent of nucleotide binding, while FlrA exclusively interacts with the ATP-dependent FlhG dimer and stimulates FlhG ATPase activity. Our in vivo analysis of FlhG partner switching between FliM and FlrA reveals its mechanism in the numerical restriction of flagella, in which the transcriptional activity of FlrA is down-regulated through a negative feedback loop. Our study demonstrates another level of regulatory complexity underlying the spationumerical regulation of flagellar biogenesis and implies that flagellar assembly transcriptionally regulates the production of more initial building blocks.
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http://dx.doi.org/10.1073/pnas.2006470117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456076PMC
August 2020

EDF1 coordinates cellular responses to ribosome collisions.

Elife 2020 08 3;9. Epub 2020 Aug 3.

Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, United States.

Translation of aberrant mRNAs induces ribosomal collisions, thereby triggering pathways for mRNA and nascent peptide degradation and ribosomal rescue. Here we use sucrose gradient fractionation combined with quantitative proteomics to systematically identify proteins associated with collided ribosomes. This approach identified Endothelial differentiation-related factor 1 (EDF1) as a novel protein recruited to collided ribosomes during translational distress. Cryo-electron microscopic analyses of EDF1 and its yeast homolog Mbf1 revealed a conserved 40S ribosomal subunit binding site at the mRNA entry channel near the collision interface. EDF1 recruits the translational repressors GIGYF2 and EIF4E2 to collided ribosomes to initiate a negative-feedback loop that prevents new ribosomes from translating defective mRNAs. Further, EDF1 regulates an immediate-early transcriptional response to ribosomal collisions. Our results uncover mechanisms through which EDF1 coordinates multiple responses of the ribosome-mediated quality control pathway and provide novel insights into the intersection of ribosome-mediated quality control with global transcriptional regulation.
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http://dx.doi.org/10.7554/eLife.58828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486125PMC
August 2020

Structure and function of yeast Lso2 and human CCDC124 bound to hibernating ribosomes.

PLoS Biol 2020 07 20;18(7):e3000780. Epub 2020 Jul 20.

Gene Center and Center for Integrated Protein Science Munich, Department of Biochemistry, University of Munich, Munich, Germany.

Cells adjust to nutrient deprivation by reversible translational shutdown. This is accompanied by maintaining inactive ribosomes in a hibernation state, in which they are bound by proteins with inhibitory and protective functions. In eukaryotes, such a function was attributed to suppressor of target of Myb protein 1 (Stm1; SERPINE1 mRNA-binding protein 1 [SERBP1] in mammals), and recently, late-annotated short open reading frame 2 (Lso2; coiled-coil domain containing short open reading frame 124 [CCDC124] in mammals) was found to be involved in translational recovery after starvation from stationary phase. Here, we present cryo-electron microscopy (cryo-EM) structures of translationally inactive yeast and human ribosomes. We found Lso2/CCDC124 accumulating on idle ribosomes in the nonrotated state, in contrast to Stm1/SERBP1-bound ribosomes, which display a rotated state. Lso2/CCDC124 bridges the decoding sites of the small with the GTPase activating center (GAC) of the large subunit. This position allows accommodation of the duplication of multilocus region 34 protein (Dom34)-dependent ribosome recycling system, which splits Lso2-containing, but not Stm1-containing, ribosomes. We propose a model in which Lso2 facilitates rapid translation reactivation by stabilizing the recycling-competent state of inactive ribosomes.
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http://dx.doi.org/10.1371/journal.pbio.3000780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7392345PMC
July 2020

Structural basis for translational shutdown and immune evasion by the Nsp1 protein of SARS-CoV-2.

Science 2020 09 17;369(6508):1249-1255. Epub 2020 Jul 17.

Gene Center Munich, Department of Biochemistry, University of Munich, Munich, Germany.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. A major virulence factor of SARS-CoVs is the nonstructural protein 1 (Nsp1), which suppresses host gene expression by ribosome association. Here, we show that Nsp1 from SARS-CoV-2 binds to the 40 ribosomal subunit, resulting in shutdown of messenger RNA (mRNA) translation both in vitro and in cells. Structural analysis by cryo-electron microscopy of in vitro-reconstituted Nsp1-40 and various native Nsp1-40 and -80 complexes revealed that the Nsp1 C terminus binds to and obstructs the mRNA entry tunnel. Thereby, Nsp1 effectively blocks retinoic acid-inducible gene I-dependent innate immune responses that would otherwise facilitate clearance of the infection. Thus, the structural characterization of the inhibitory mechanism of Nsp1 may aid structure-based drug design against SARS-CoV-2.
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http://dx.doi.org/10.1126/science.abc8665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402621PMC
September 2020

Construction of the Central Protuberance and L1 Stalk during 60S Subunit Biogenesis.

Mol Cell 2020 08 14;79(4):615-628.e5. Epub 2020 Jul 14.

Biochemie-Zentrum der Universität Heidelberg, 69120 Heidelberg, Germany. Electronic address:

Ribosome assembly is driven by numerous assembly factors, including the Rix1 complex and the AAA ATPase Rea1. These two assembly factors catalyze 60S maturation at two distinct states, triggering poorly understood large-scale structural transitions that we analyzed by cryo-electron microscopy. Two nuclear pre-60S intermediates were discovered that represent previously unknown states after Rea1-mediated removal of the Ytm1-Erb1 complex and reveal how the L1 stalk develops from a pre-mature nucleolar to a mature-like nucleoplasmic state. A later pre-60S intermediate shows how the central protuberance arises, assisted by the nearby Rix1-Rea1 machinery, which was solved in its pre-ribosomal context to molecular resolution. This revealed a Rix1-Ipi3 tetramer anchored to the pre-60S via Ipi1, strategically positioned to monitor this decisive remodeling. These results are consistent with a general underlying principle that temporarily stabilized immature RNA domains are successively remodeled by assembly factors, thereby ensuring failsafe assembly progression.
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http://dx.doi.org/10.1016/j.molcel.2020.06.032DOI Listing
August 2020

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

Nat Chem Biol 2020 10 29;16(10):1071-1077. Epub 2020 Jun 29.

Center of Life Sciences, Skolkovo Institute of Science and Technology, Skolkovo, Russia.

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.
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http://dx.doi.org/10.1038/s41589-020-0578-xDOI Listing
October 2020

Author Correction: 3.2-Å-resolution structure of the 90S preribosome before A1 pre-rRNA cleavage.

Nat Struct Mol Biol 2020 Jul;27(7):683

Department of Biochemistry, Gene Center Munich and Center of Integrated Protein Science-Munich (CiPS-M), University of Munich, Munich, Germany.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41594-020-0456-yDOI Listing
July 2020

Novel bioanalytical method for the characterization of the immune response directed against a bispecific F(ab) fragment.

Bioanalysis 2020 Apr 30;12(8):509-517. Epub 2020 Apr 30.

Roche Pharma Research & Early Development, Pharmaceutical Sciences, Roche Innovation Center Munich, Im Nonnenwald 2, 82377 Penzberg, Germany.

The work was aimed at developing a bioanalytical approach to identify immunogenic parts of a bispecific F(ab) fragment and to characterize the immune response seen in a preclinical study. The bioanalytical method consists of a set of domain detection assays that use germlined variants of the drug. The method demonstrated that anti-drug antibodies (ADAs) were predominantly directed against both antigen-binding sites of the drug. The method was capable to discriminate between ADAs directed against one of the antigen-binding sites, both sites or the constant domain, allowing for an estimation of the relative binding prevalence for these subunits. The developed approach provides a practical and robust solution for exploratory characterization of ADAs against multidomain biotherapeutics.
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http://dx.doi.org/10.4155/bio-2020-0064DOI Listing
April 2020

The Ccr4-Not complex monitors the translating ribosome for codon optimality.

Science 2020 04;368(6488)

Gene Center and Department of Biochemistry, University of Munich, 81377 Munich, Germany.

Control of messenger RNA (mRNA) decay rate is intimately connected to translation elongation, but the spatial coordination of these events is poorly understood. The Ccr4-Not complex initiates mRNA decay through deadenylation and activation of decapping. We used a combination of cryo-electron microscopy, ribosome profiling, and mRNA stability assays to examine the recruitment of Ccr4-Not to the ribosome via specific interaction of the Not5 subunit with the ribosomal E-site in This interaction occurred when the ribosome lacked accommodated A-site transfer RNA, indicative of low codon optimality. Loss of the interaction resulted in the inability of the mRNA degradation machinery to sense codon optimality. Our findings elucidate a physical link between the Ccr4-Not complex and the ribosome and provide mechanistic insight into the coupling of decoding efficiency with mRNA stability.
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http://dx.doi.org/10.1126/science.aay6912DOI Listing
April 2020

RQT complex dissociates ribosomes collided on endogenous RQC substrate SDD1.

Nat Struct Mol Biol 2020 04 23;27(4):323-332. Epub 2020 Mar 23.

Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.

Ribosome-associated quality control (RQC) represents a rescue pathway in eukaryotic cells that is triggered upon translational stalling. Collided ribosomes are recognized for subsequent dissociation followed by degradation of nascent peptides. However, endogenous RQC-inducing sequences and the mechanism underlying the ubiquitin-dependent ribosome dissociation remain poorly understood. Here, we identified SDD1 messenger RNA from Saccharomyces cerevisiae as an endogenous RQC substrate and reveal the mechanism of its mRNA-dependent and nascent peptide-dependent translational stalling. In vitro translation of SDD1 mRNA enabled the reconstitution of Hel2-dependent polyubiquitination of collided disomes and, preferentially, trisomes. The distinct trisome architecture, visualized using cryo-EM, provides the structural basis for the more-efficient recognition by Hel2 compared with that of disomes. Subsequently, the Slh1 helicase subunit of the RQC trigger (RQT) complex preferentially dissociates the first stalled polyubiquitinated ribosome in an ATP-dependent manner. Together, these findings provide fundamental mechanistic insights into RQC and its physiological role in maintaining cellular protein homeostasis.
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http://dx.doi.org/10.1038/s41594-020-0393-9DOI Listing
April 2020

Molecular analysis of the ribosome recycling factor ABCE1 bound to the 30S post-splitting complex.

EMBO J 2020 05 17;39(9):e103788. Epub 2020 Feb 17.

Department of Biochemistry, Gene Center, Ludwig-Maximilians University Munich, München, Germany.

Ribosome recycling by the twin-ATPase ABCE1 is a key regulatory process in mRNA translation and surveillance and in ribosome-associated protein quality control in Eukarya and Archaea. Here, we captured the archaeal 30S ribosome post-splitting complex at 2.8 Å resolution by cryo-electron microscopy. The structure reveals the dynamic behavior of structural motifs unique to ABCE1, which ultimately leads to ribosome splitting. More specifically, we provide molecular details on how conformational rearrangements of the iron-sulfur cluster domain and hinge regions of ABCE1 are linked to closure of its nucleotide-binding sites. The combination of mutational and functional analyses uncovers an intricate allosteric network between the ribosome, regulatory domains of ABCE1, and its two structurally and functionally asymmetric ATP-binding sites. Based on these data, we propose a refined model of how signals from the ribosome are integrated into the ATPase cycle of ABCE1 to orchestrate ribosome recycling.
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http://dx.doi.org/10.15252/embj.2019103788DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7196836PMC
May 2020

Structure of the Bcs1 AAA-ATPase suggests an airlock-like translocation mechanism for folded proteins.

Nat Struct Mol Biol 2020 02 27;27(2):142-149. Epub 2020 Jan 27.

Gene Center and Center for Integrated Protein Science Munich, Department of Biochemistry, University of Munich, Munich, Germany.

Some proteins require completion of folding before translocation across a membrane into another cellular compartment. Yet the permeability barrier of the membrane should not be compromised and mechanisms have remained mostly elusive. Here, we present the structure of Saccharomyces cerevisiae Bcs1, an AAA-ATPase of the inner mitochondrial membrane. Bcs1 facilitates the translocation of the Rieske protein, Rip1, which requires folding and incorporation of a 2Fe-2S cluster before translocation and subsequent integration into the bc1 complex. Surprisingly, Bcs1 assembles into exclusively heptameric homo-oligomers, with each protomer consisting of an amphipathic transmembrane helix, a middle domain and an ATPase domain. Together they form two aqueous vestibules, the first being accessible from the mitochondrial matrix and the second positioned in the inner membrane, with both separated by the seal-forming middle domain. On the basis of this unique architecture, we propose an airlock-like translocation mechanism for folded Rip1.
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http://dx.doi.org/10.1038/s41594-019-0364-1DOI Listing
February 2020

Molecular mechanism of translational stalling by inhibitory codon combinations and poly(A) tracts.

EMBO J 2020 02 20;39(3):e103365. Epub 2019 Dec 20.

Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Inhibitory codon pairs and poly(A) tracts within the translated mRNA cause ribosome stalling and reduce protein output. The molecular mechanisms that drive these stalling events, however, are still unknown. Here, we use a combination of in vitro biochemistry, ribosome profiling, and cryo-EM to define molecular mechanisms that lead to these ribosome stalls. First, we use an in vitro reconstituted yeast translation system to demonstrate that inhibitory codon pairs slow elongation rates which are partially rescued by increased tRNA concentration or by an artificial tRNA not dependent on wobble base-pairing. Ribosome profiling data extend these observations by revealing that paused ribosomes with empty A sites are enriched on these sequences. Cryo-EM structures of stalled ribosomes provide a structural explanation for the observed effects by showing decoding-incompatible conformations of mRNA in the A sites of all studied stall- and collision-inducing sequences. Interestingly, in the case of poly(A) tracts, the inhibitory conformation of the mRNA in the A site involves a nucleotide stacking array. Together, these data demonstrate a novel mRNA-induced mechanisms of translational stalling in eukaryotic ribosomes.
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http://dx.doi.org/10.15252/embj.2019103365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996574PMC
February 2020

Partially inserted nascent chain unzips the lateral gate of the Sec translocon.

EMBO Rep 2019 10 5;20(10):e48191. Epub 2019 Aug 5.

Gene Center Munich, Ludwig-Maximilian-University, Munich, Germany.

The Sec translocon provides the lipid bilayer entry for ribosome-bound nascent chains and thus facilitates membrane protein biogenesis. Despite the appreciated role of the native environment in the translocon:ribosome assembly, structural information on the complex in the lipid membrane is scarce. Here, we present a cryo-electron microscopy-based structure of bacterial translocon SecYEG in lipid nanodiscs and elucidate an early intermediate state upon insertion of the FtsQ anchor domain. Insertion of the short nascent chain causes initial displacements within the lateral gate of the translocon, where α-helices 2b, 7, and 8 tilt within the membrane core to "unzip" the gate at the cytoplasmic side. Molecular dynamics simulations demonstrate that the conformational change is reversed in the absence of the ribosome, and suggest that the accessory α-helices of SecE subunit modulate the lateral gate conformation. Site-specific cross-linking validates that the FtsQ nascent chain passes the lateral gate upon insertion. The structure and the biochemical data suggest that the partially inserted nascent chain remains highly flexible until it acquires the transmembrane topology.
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http://dx.doi.org/10.15252/embr.201948191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6776908PMC
October 2019

Thermophile 90S Pre-ribosome Structures Reveal the Reverse Order of Co-transcriptional 18S rRNA Subdomain Integration.

Mol Cell 2019 09 1;75(6):1256-1269.e7. Epub 2019 Aug 1.

Heidelberg University Biochemistry Center (BZH), Im Neuenheimer Feld 328, 69120 Heidelberg, Germany. Electronic address:

Eukaryotic ribosome biogenesis involves RNA folding and processing that depend on assembly factors and small nucleolar RNAs (snoRNAs). The 90S (SSU-processome) is the earliest pre-ribosome structurally analyzed, which was suggested to assemble stepwise along the growing pre-rRNA from 5' > 3', but this directionality may not be accurate. Here, by analyzing the structure of a series of 90S assembly intermediates from Chaetomium thermophilum, we discover a reverse order of 18S rRNA subdomain incorporation. Large parts of the 18S rRNA 3' and central domains assemble first into the 90S before the 5' domain is integrated. This final incorporation depends on a contact between a heterotrimer Enp2-Bfr2-Lcp5 recruited to the flexible 5' domain and Kre33, which reconstitutes the Kre33-Enp-Brf2-Lcp5 module on the compacted 90S. Keeping the 5' domain temporarily segregated from the 90S scaffold could provide extra time to complete the multifaceted 5' domain folding, which depends on a distinct set of snoRNAs and processing factors.
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http://dx.doi.org/10.1016/j.molcel.2019.06.032DOI Listing
September 2019

Structural and mutational analysis of the ribosome-arresting human XBP1u.

Elife 2019 06 27;8. Epub 2019 Jun 27.

Gene Center, Department of Biochemistry, Center for integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität München, Munich, Germany.

XBP1u, a central component of the unfolded protein response (UPR), is a mammalian protein containing a functionally critical translational arrest peptide (AP). Here, we present a 3 Å cryo-EM structure of the stalled human XBP1u AP. It forms a unique turn in the ribosomal exit tunnel proximal to the peptidyl transferase center where it causes a subtle distortion, thereby explaining the temporary translational arrest induced by XBP1u. During ribosomal pausing the hydrophobic region 2 (HR2) of XBP1u is recognized by SRP, but fails to efficiently gate the Sec61 translocon. An exhaustive mutagenesis scan of the XBP1u AP revealed that only 8 out of 20 mutagenized positions are optimal; in the remaining 12 positions, we identify 55 different mutations increase the level of translational arrest. Thus, the wildtype XBP1u AP induces only an intermediate level of translational arrest, allowing efficient targeting by SRP without activating the Sec61 channel.
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http://dx.doi.org/10.7554/eLife.46267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6624018PMC
June 2019

Publisher Correction: Structure and function of Vms1 and Arb1 in RQC and mitochondrial proteome homeostasis.

Nature 2019 Jul;571(7764):E4

Gene Center and Center for Integrated Protein Science Munich, Department of Biochemistry, University of Munich, Munich, Germany.

Change history: In this Letter, the bottom blot in Fig. 2g (for 'IB: Myc') was missing. This has been corrected online.
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http://dx.doi.org/10.1038/s41586-019-1360-7DOI Listing
July 2019

Structure and function of Vms1 and Arb1 in RQC and mitochondrial proteome homeostasis.

Nature 2019 06 12;570(7762):538-542. Epub 2019 Jun 12.

Gene Center and Center for Integrated Protein Science Munich, Department of Biochemistry, University of Munich, Munich, Germany.

Ribosome-associated quality control (RQC) provides a rescue pathway for eukaryotic cells to process faulty proteins after translational stalling of cytoplasmic ribosomes. After dissociation of ribosomes, the stalled tRNA-bound peptide remains associated with the 60S subunit and extended by Rqc2 by addition of C-terminal alanyl and threonyl residues (CAT tails), whereas Vms1 catalyses cleavage and release of the peptidyl-tRNA before or after addition of CAT tails. In doing so, Vms1 counteracts CAT-tailing of nuclear-encoded mitochondrial proteins that otherwise drive aggregation and compromise mitochondrial and cellular homeostasis. Here we present structural and functional insights into the interaction of Saccharomyces cerevisiae Vms1 with 60S subunits in pre- and post-peptidyl-tRNA cleavage states. Vms1 binds to 60S subunits with its Vms1-like release factor 1 (VLRF1), zinc finger and ankyrin domains. VLRF1 overlaps with the Rqc2 A-tRNA position and interacts with the ribosomal A-site, projecting its catalytic GSQ motif towards the CCA end of the tRNA, its Y285 residue dislodging the tRNA A73 for nucleolytic cleavage. Moreover, in the pre-state, we found the ABCF-type ATPase Arb1 in the ribosomal E-site, which stabilizes the delocalized A73 of the peptidyl-tRNA and stimulates Vms1-dependent tRNA cleavage. Our structural analysis provides mechanistic insights into the interplay of the RQC factors Vms1, Rqc2 and Arb1 and their role in the protection of mitochondria from the aggregation of toxic proteins.
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http://dx.doi.org/10.1038/s41586-019-1307-zDOI Listing
June 2019

Structure of the 80S ribosome-Xrn1 nuclease complex.

Nat Struct Mol Biol 2019 04 25;26(4):275-280. Epub 2019 Mar 25.

Gene Center and Center for Integrated Protein Science Munich, Department of Biochemistry, University of Munich, Munich, Germany.

Messenger RNA (mRNA) homeostasis represents an essential part of gene expression, in which the generation of mRNA by RNA polymerase is counter-balanced by its degradation by nucleases. The conserved 5'-to-3' exoribonuclease Xrn1 has a crucial role in eukaryotic mRNA homeostasis by degrading decapped or cleaved mRNAs post-translationally and, more surprisingly, also co-translationally. Here we report that active Xrn1 can directly and specifically interact with the translation machinery. A cryo-electron microscopy structure of a programmed Saccharomyces cerevisiae 80S ribosome-Xrn1 nuclease complex reveals how the conserved core of Xrn1 enables binding at the mRNA exit site of the ribosome. This interface provides a conduit for channelling of the mRNA from the ribosomal decoding site directly into the active center of the nuclease, thus separating mRNA decoding from degradation by only 17 ± 1 nucleotides. These findings explain how rapid 5'-to-3' mRNA degradation is coupled efficiently to its final round of mRNA translation.
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http://dx.doi.org/10.1038/s41594-019-0202-5DOI Listing
April 2019

Reconstitution of the human SRP system and quantitative and systematic analysis of its ribosome interactions.

Nucleic Acids Res 2019 04;47(6):3184-3196

Heidelberg University Biochemistry Center (BZH), INF 328, D-69120 Heidelberg, Germany.

Co-translational protein targeting to membranes depends on the regulated interaction of two ribonucleoprotein particles (RNPs): the ribosome and the signal recognition particle (SRP). Human SRP is composed of an SRP RNA and six proteins with the SRP GTPase SRP54 forming the targeting complex with the heterodimeric SRP receptor (SRαβ) at the endoplasmic reticulum membrane. While detailed structural and functional data are available especially for the bacterial homologs, the analysis of human SRP was impeded by the unavailability of recombinant SRP. Here, we describe the large-scale production of all human SRP components and the reconstitution of homogeneous SRP and SR complexes. Binding to human ribosomes is determined by microscale thermophoresis for individual components, assembly intermediates and entire SRP, and binding affinities are correlated with structural information available for all ribosomal contacts. We show that SRP RNA does not bind to the ribosome, while SRP binds with nanomolar affinity involving a two-step mechanism of the key-player SRP54. Ultrasensitive binding of SRP68/72 indicates avidity by multiple binding sites that are dominated by the C-terminus of SRP72. Our data extend the experimental basis to understand the mechanistic principles of co-translational targeting in mammals and may guide analyses of complex RNP-RNP interactions in general.
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http://dx.doi.org/10.1093/nar/gky1324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451106PMC
April 2019

Collided ribosomes form a unique structural interface to induce Hel2-driven quality control pathways.

EMBO J 2019 03 4;38(5). Epub 2019 Jan 4.

Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan

Ribosome stalling triggers quality control pathways targeting the mRNA (NGD: no-go decay) and the nascent polypeptide (RQC: ribosome-associated quality control). RQC requires Hel2-dependent uS10 ubiquitination and the RQT complex in yeast. Here, we report that Hel2-dependent uS10 ubiquitination and Slh1/Rqt2 are crucial for RQC and NGD induction within a di-ribosome (disome) unit, which consists of the leading stalled ribosome and the following colliding ribosome. Hel2 preferentially ubiquitinated a disome over a monosome on a quality control inducing reporter mRNA in an translation reaction. Cryo-EM analysis of the disome unit revealed a distinct structural arrangement suitable for recognition and modification by Hel2. The absence of the RQT complex or uS10 ubiquitination resulted in the elimination of NGD within the disome unit. Instead, we observed Hel2-mediated cleavages upstream of the disome, governed by initial Not4-mediated monoubiquitination of eS7 and followed by Hel2-mediated K63-linked polyubiquitination. We propose that Hel2-mediated ribosome ubiquitination is required both for canonical NGD (NGD) and RQC coupled to the disome and that RQC-uncoupled NGD outside the disome (NGD) can occur in a Not4-dependent manner.
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http://dx.doi.org/10.15252/embj.2018100276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6396155PMC
March 2019