Publications by authors named "Lukas Kater"

10 Publications

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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

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

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

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

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

Suppressor mutations in Rpf2-Rrs1 or Rpl5 bypass the Cgr1 function for pre-ribosomal 5S RNP-rotation.

Nat Commun 2018 10 5;9(1):4094. Epub 2018 Oct 5.

Biochemistry Centre, University of Heidelberg, Heidelberg, 69120, Germany.

During eukaryotic 60S biogenesis, the 5S RNP requires a large rotational movement to achieve its mature position. Cryo-EM of the Rix1-Rea1 pre-60S particle has revealed the post-rotation stage, in which a gently undulating α-helix corresponding to Cgr1 becomes wedged between Rsa4 and the relocated 5S RNP, but the purpose of this insertion was unknown. Here, we show that cgr1 deletion in yeast causes a slow-growth phenotype and reversion of the pre-60S particle to the pre-rotation stage. However, spontaneous extragenic suppressors could be isolated, which restore growth and pre-60S biogenesis in the absence of Cgr1. Whole-genome sequencing reveals that the suppressor mutations map in the Rpf2-Rrs1 module and Rpl5, which together stabilize the unrotated stage of the 5S RNP. Thus, mutations in factors stabilizing the pre-rotation stage facilitate 5S RNP relocation upon deletion of Cgr1, but Cgr1 itself could stabilize the post-rotation stage.
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http://dx.doi.org/10.1038/s41467-018-06660-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173701PMC
October 2018

The Protease ClpXP and the PAS Domain Protein DivL Regulate CtrA and Gene Transfer Agent Production in Rhodobacter capsulatus.

Appl Environ Microbiol 2018 06 17;84(11). Epub 2018 May 17.

Department of Microbiology and Immunology, The University of British Columbia, Vancouver, BC, Canada

Several members of the () produce a conserved horizontal gene transfer vector, called the gene transfer agent (GTA), that appears to have evolved from a bacteriophage. The model system used to study GTA biology is the GTA (RcGTA), a small, tailed bacteriophage-like particle produced by a subset of the cells in a culture. The response regulator CtrA is conserved in the and is an essential regulator of RcGTA production: it controls the production and maturation of the RcGTA particle and RcGTA release from cells. CtrA also controls the natural transformation-like system required for cells to receive RcGTA-donated DNA. Here, we report that dysregulation of the CckA-ChpT-CtrA phosphorelay either by the loss of the PAS domain protein DivL or by substitution of the autophosphorylation residue of the hybrid histidine kinase CckA decreased CtrA phosphorylation and greatly increased RcGTA protein production in We show that the loss of the ClpXP protease or the three C-terminal residues of CtrA results in increased CtrA levels in and identify ClpX(P) to be essential for the maturation of RcGTA particles. Furthermore, we show that CtrA phosphorylation is important for head spike production. Our results provide novel insight into the regulation of CtrA and GTAs in the Members of the are abundant in ocean and freshwater environments. The conserved GTA produced by many may have an important role in horizontal gene transfer (HGT) in aquatic environments and provide a significant contribution to their adaptation. GTA production is controlled by bacterial regulatory systems, including the conserved CckA-ChpT-CtrA phosphorelay; however, several questions about GTA regulation remain. Our identification that a short DivL homologue and ClpXP regulate CtrA in extends the model of CtrA regulation from to a member of the We found that the magnitude of RcGTA production greatly depends on DivL and CckA kinase activity, adding yet another layer of regulatory complexity to RcGTA. RcGTA is known to undergo CckA-dependent maturation, and we extend the understanding of this process by showing that the ClpX chaperone is required for formation of tailed, DNA-containing particles.
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http://dx.doi.org/10.1128/AEM.00275-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5960961PMC
June 2018

Visualizing the Assembly Pathway of Nucleolar Pre-60S Ribosomes.

Cell 2017 Dec;171(7):1599-1610.e14

Gene Center Munich and Center of Integrated Protein Science-Munich (CiPS-M), Department of Biochemistry, Feodor-Lynen-Str. 25, University of Munich, 81377 Munich, Germany. Electronic address:

Eukaryotic 60S ribosomal subunits are comprised of three rRNAs and ∼50 ribosomal proteins. The initial steps of their formation take place in the nucleolus, but, owing to a lack of structural information, this process is poorly understood. Using cryo-EM, we solved structures of early 60S biogenesis intermediates at 3.3 Å to 4.5 Å resolution, thereby providing insights into their sequential folding and assembly pathway. Besides revealing distinct immature rRNA conformations, we map 25 assembly factors in six different assembly states. Notably, the Nsa1-Rrp1-Rpf1-Mak16 module stabilizes the solvent side of the 60S subunit, and the Erb1-Ytm1-Nop7 complex organizes and connects through Erb1's meandering N-terminal extension, eight assembly factors, three ribosomal proteins, and three 25S rRNA domains. Our structural snapshots reveal the order of integration and compaction of the six major 60S domains within early nucleolar 60S particles developing stepwise from the solvent side around the exit tunnel to the central protuberance.
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http://dx.doi.org/10.1016/j.cell.2017.11.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5745149PMC
December 2017

Architecture of the Rix1-Rea1 checkpoint machinery during pre-60S-ribosome remodeling.

Nat Struct Mol Biol 2016 Jan 30;23(1):37-44. Epub 2015 Nov 30.

Heidelberg University Biochemistry Center, Heidelberg, Germany.

Ribosome synthesis is catalyzed by ∼200 assembly factors, which facilitate efficient production of mature ribosomes. Here, we determined the cryo-EM structure of a Saccharomyces cerevisiae nucleoplasmic pre-60S particle containing the dynein-related 550-kDa Rea1 AAA(+) ATPase and the Rix1 subcomplex. This particle differs from its preceding state, the early Arx1 particle, by two massive structural rearrangements: an ∼180° rotation of the 5S ribonucleoprotein complex and the central protuberance (CP) rRNA helices, and the removal of the 'foot' structure from the 3' end of the 5.8S rRNA. Progression from the Arx1 to the Rix1 particle was blocked by mutational perturbation of the Rix1-Rea1 interaction but not by a dominant-lethal Rea1 AAA(+) ATPase-ring mutant. After remodeling, the Rix1 subcomplex and Rea1 become suitably positioned to sense correct structural maturation of the CP, which allows unidirectional progression toward mature ribosomes.
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http://dx.doi.org/10.1038/nsmb.3132DOI Listing
January 2016

Generation of catalytic human Ago4 identifies structural elements important for RNA cleavage.

RNA 2014 Oct 11;20(10):1532-8. Epub 2014 Aug 11.

Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, 93053 Regensburg, Germany

Argonaute proteins bind small RNAs and mediate cleavage of complementary target RNAs. The human Argonaute protein Ago4 is catalytically inactive, although it is highly similar to catalytic Ago2. Here, we have generated Ago2-Ago4 chimeras and analyzed their cleavage activity in vitro. We identify several specific features that inactivate Ago4: the catalytic center, short sequence elements in the N-terminal domain, and an Ago4-specific insertion in the catalytic domain. In addition, we show that Ago2-mediated cleavage of the noncanonical miR-451 precursor can be carried out by any catalytic human Ago protein. Finally, phylogenetic analyses establish evolutionary distances between the Ago proteins. Interestingly, these distances do not fully correlate with the structural changes inactivating them, suggesting functional adaptations of individual human Ago proteins.
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http://dx.doi.org/10.1261/rna.045203.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4174435PMC
October 2014