Publications by authors named "Robert Buschauer"

11 Publications

  • Page 1 of 1

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

The In Situ Structure of Parkinson's Disease-Linked LRRK2.

Cell 2020 09 11;182(6):1508-1518.e16. Epub 2020 Aug 11.

Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address:

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial Parkinson's disease. LRRK2 is a multi-domain protein containing a kinase and GTPase. Using correlative light and electron microscopy, in situ cryo-electron tomography, and subtomogram analysis, we reveal a 14-Å structure of LRRK2 bearing a pathogenic mutation that oligomerizes as a right-handed double helix around microtubules, which are left-handed. Using integrative modeling, we determine the architecture of LRRK2, showing that the GTPase and kinase are in close proximity, with the GTPase closer to the microtubule surface, whereas the kinase is exposed to the cytoplasm. We identify two oligomerization interfaces mediated by non-catalytic domains. Mutation of one of these abolishes LRRK2 microtubule-association. Our work demonstrates the power of cryo-electron tomography to generate models of previously unsolved structures in their cellular environment.
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http://dx.doi.org/10.1016/j.cell.2020.08.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7869717PMC
September 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

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

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 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

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

Assembly of a nucleus-like structure during viral replication in bacteria.

Science 2017 01;355(6321):194-197

Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093, USA.

We observed the assembly of a nucleus-like structure in bacteria during viral infection. Using fluorescence microscopy and cryo-electron tomography, we showed that Pseudomonas chlororaphis phage 201φ2-1 assembled a compartment that separated viral DNA from the cytoplasm. The phage compartment was centered by a bipolar tubulin-based spindle, and it segregated phage and bacterial proteins according to function. Proteins involved in DNA replication and transcription localized inside the compartment, whereas proteins involved in translation and nucleotide synthesis localized outside. Later during infection, viral capsids assembled on the cytoplasmic membrane and moved to the surface of the compartment for DNA packaging. Ultimately, viral particles were released from the compartment and the cell lysed. These results demonstrate that phages have evolved a specialized structure to compartmentalize viral replication.
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http://dx.doi.org/10.1126/science.aal2130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028185PMC
January 2017

Structural Dynamics of the YidC:Ribosome Complex during Membrane Protein Biogenesis.

Cell Rep 2016 12;17(11):2943-2954

Gene Center Munich, Department of Biochemistry, Ludwig-Maximilians-University Munich, Feodor-Lynen-Strasse 25, Munich 81377, Germany; Center for Integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-University, Butenandtstrasse 5-13, Munich 81377, Germany. Electronic address:

Members of the YidC/Oxa1/Alb3 family universally facilitate membrane protein biogenesis, via mechanisms that have thus far remained unclear. Here, we investigated two crucial functional aspects: the interaction of YidC with ribosome:nascent chain complexes (RNCs) and the structural dynamics of RNC-bound YidC in nanodiscs. We observed that a fully exposed nascent transmembrane domain (TMD) is required for high-affinity YidC:RNC interactions, while weaker binding may already occur at earlier stages of translation. YidC efficiently catalyzed the membrane insertion of nascent TMDs in both fluid and gel phase membranes. Cryo-electron microscopy and fluorescence analysis revealed a conformational change in YidC upon nascent chain insertion: the essential TMDs 2 and 3 of YidC were tilted, while the amphipathic helix EH1 relocated into the hydrophobic core of the membrane. We suggest that EH1 serves as a mechanical lever, facilitating a coordinated movement of YidC TMDs to trigger the release of nascent chains into the membrane.
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http://dx.doi.org/10.1016/j.celrep.2016.11.059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5186731PMC
December 2016