Publications by authors named "Thomas Monecke"

14 Publications

  • Page 1 of 1

OpenPepXL: An Open-Source Tool for Sensitive Identification of Cross-Linked Peptides in XL-MS.

Mol Cell Proteomics 2020 12 16;19(12):2157-2168. Epub 2020 Oct 16.

Biomolecular Interactions, Max Planck Institute for Developmental Biology, Tübingen, Germany; Institute for Bioinformatics and Medical Informatics, University of Tübingen, Tübingen, Germany; Applied Bioinformatics, Dept. of Computer Science, University of Tübingen, Tübingen, Germany; Institute for Translational Bioinformatics, University Hospital Tübingen, Tübingen, Germany; Quantitative Biology Center, University of Tübingen, Tübingen, Germany. Electronic address:

Cross-linking MS (XL-MS) has been recognized as an effective source of information about protein structures and interactions. In contrast to regular peptide identification, XL-MS has to deal with a quadratic search space, where peptides from every protein could potentially be cross-linked to any other protein. To cope with this search space, most tools apply different heuristics for search space reduction. We introduce a new open-source XL-MS database search algorithm, OpenPepXL, which offers increased sensitivity compared with other tools. OpenPepXL searches the full search space of an XL-MS experiment without using heuristics to reduce it. Because of efficient data structures and built-in parallelization OpenPepXL achieves excellent runtimes and can also be deployed on large compute clusters and cloud services while maintaining a slim memory footprint. We compared OpenPepXL to several other commonly used tools for identification of noncleavable labeled and label-free cross-linkers on a diverse set of XL-MS experiments. In our first comparison, we used a data set from a fraction of a cell lysate with a protein database of 128 targets and 128 decoys. At 5% FDR, OpenPepXL finds from 7% to over 50% more unique residue pairs (URPs) than other tools. On data sets with available high-resolution structures for cross-link validation OpenPepXL reports from 7% to over 40% more structurally validated URPs than other tools. Additionally, we used a synthetic peptide data set that allows objective validation of cross-links without relying on structural information and found that OpenPepXL reports at least 12% more validated URPs than other tools. It has been built as part of the OpenMS suite of tools and supports Windows, macOS, and Linux operating systems. OpenPepXL also supports the MzIdentML 1.2 format for XL-MS identification results. It is freely available under a three-clause BSD license at https://openms.org/openpepxl.
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http://dx.doi.org/10.1074/mcp.TIR120.002186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710140PMC
December 2020

Combining dehydration, construct optimization and improved data collection to solve the crystal structure of a CRM1-RanGTP-SPN1-Nup214 quaternary nuclear export complex.

Acta Crystallogr F Struct Biol Commun 2015 Dec 18;71(Pt 12):1481-7. Epub 2015 Nov 18.

Abteilung für Molekulare Strukturbiologie, Institut für Mikrobiologie und Genetik, Göttinger Zentrum für Molekulare Biowissenschaften (GZMB), Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany.

High conformational flexibility is an intrinsic and indispensable property of nuclear transport receptors, which makes crystallization and structure determination of macromolecular complexes containing exportins or importins particularly challenging. Here, the crystallization and structure determination of a quaternary nuclear export complex consisting of the exportin CRM1, the small GTPase Ran in its GTP-bound form, the export cargo SPN1 and an FG repeat-containing fragment of the nuclear pore complex component nucleoporin Nup214 fused to maltose-binding protein is reported. Optimization of constructs, seeding and the development of a sophisticated protocol including successive PEG-mediated crystal dehydration as well as additional post-mounting steps were essential to obtain well diffracting crystals.
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http://dx.doi.org/10.1107/S2053230X15021524DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4666476PMC
December 2015

Structural and Functional Characterization of CRM1-Nup214 Interactions Reveals Multiple FG-Binding Sites Involved in Nuclear Export.

Cell Rep 2015 Oct 17;13(4):690-702. Epub 2015 Oct 17.

Department of Molecular Biology, Faculty of Medicine, GZMB, Georg-August-University Göttingen, Humboldtallee 23, 37073 Göttingen, Germany. Electronic address:

CRM1 is the major nuclear export receptor. During translocation through the nuclear pore, transport complexes transiently interact with phenylalanine-glycine (FG) repeats of multiple nucleoporins. On the cytoplasmic side of the nuclear pore, CRM1 tightly interacts with the nucleoporin Nup214. Here, we present the crystal structure of a 117-amino-acid FG-repeat-containing fragment of Nup214, in complex with CRM1, Snurportin 1, and RanGTP at 2.85 Å resolution. The structure reveals eight binding sites for Nup214 FG motifs on CRM1, with intervening stretches that are loosely attached to the transport receptor. Nup214 binds to N- and C-terminal regions of CRM1, thereby clamping CRM1 in a closed conformation and stabilizing the export complex. The role of conserved hydrophobic pockets for the recognition of FG motifs was analyzed in biochemical and cell-based assays. Comparative studies with RanBP3 and Nup62 shed light on specificities of CRM1-nucleoporin binding, which serves as a paradigm for transport receptor-nucleoporin interactions.
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http://dx.doi.org/10.1016/j.celrep.2015.09.042DOI Listing
October 2015

Structural Basis of Targeting the Exportin CRM1 in Cancer.

Cells 2015 Sep 21;4(3):538-68. Epub 2015 Sep 21.

Abteilung für Molekulare Strukturbiologie, Institut für Mikrobiologie und Genetik, GZMB, Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, Göttingen 37077, Germany.

Recent studies have demonstrated the interference of nucleocytoplasmic trafficking with the establishment and maintenance of various cancers. Nucleocytoplasmic transport is highly regulated and coordinated, involving different nuclear transport factors or receptors, importins and exportins, that mediate cargo transport from the cytoplasm into the nucleus or the other way round, respectively. The exportin CRM1 (Chromosome region maintenance 1) exports a plethora of different protein cargoes and ribonucleoprotein complexes. Structural and biochemical analyses have enabled the deduction of individual steps of the CRM1 transport cycle. In addition, CRM1 turned out to be a valid target for anticancer drugs as it exports numerous proto-oncoproteins and tumor suppressors. Clearly, detailed understanding of the flexibility, regulatory features and cooperative binding properties of CRM1 for Ran and cargo is a prerequisite for the design of highly effective drugs. The first compound found to inhibit CRM1-dependent nuclear export was the natural drug Leptomycin B (LMB), which blocks export by competitively interacting with a highly conserved cleft on CRM1 required for nuclear export signal recognition. Clinical studies revealed serious side effects of LMB, leading to a search for alternative natural and synthetic drugs and hence a multitude of novel therapeutics. The present review examines recent progress in understanding the binding mode of natural and synthetic compounds and their inhibitory effects.
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http://dx.doi.org/10.3390/cells4030538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4588050PMC
September 2015

Allosteric control of the exportin CRM1 unraveled by crystal structure analysis.

FEBS J 2014 Sep 6;281(18):4179-94. Epub 2014 Jun 6.

Abteilung für Molekulare Strukturbiologie, Institut für Mikrobiologie und Genetik, Göttinger Zentrum für Molekulare Biowissenschaften, Georg-August-Universität Göttingen, Germany.

Nucleocytoplasmic trafficking in eukaryotic cells is a highly regulated and coordinated process which involves an increasing variety of soluble nuclear transport receptors. Generally, transport receptors specifically bind their cargo and facilitate its transition through nuclear pore complexes, aqueous channels connecting the two compartments. Directionality of such transport events by receptors of the importin β superfamily requires the interaction with the small GTPase Ras-related nuclear antigen (Ran). While importins need RanGTP to release their cargo in the nucleus and thus to terminate import, exportins recruit cargo in the RanGTP-bound state. The exportin chromosome region maintenance 1 (CRM1) is a highly versatile transport receptor that exports a plethora of different protein and RNP cargoes. Moreover, binding of RanGTP and of cargo to CRM1 are highly cooperative events despite the fact that cargo and RanGTP do not interact directly in crystal structures of assembled export complexes. Integrative approaches have recently unraveled the individual steps of the CRM1 transport cycle at a structural level and explained how the HEAT-repeat architecture of CRM1 provides a framework for the key elements to mediate allosteric interactions with RanGTP, Ran binding proteins and cargo. Moreover, during the last decade, CRM1 has become a more and more appreciated target for anti-cancer drugs. Hence, detailed understanding of the flexibility, the regulatory features and the positive binding cooperativity between CRM1, Ran and cargo is a prerequisite for the development of highly effective drugs. Here we review recent structural advances in the characterization of CRM1 and CRM1-containing complexes with a special emphasis on X-ray crystallographic studies.
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http://dx.doi.org/10.1111/febs.12842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4231977PMC
September 2014

Translation initiation factor eIF3b contains a nine-bladed β-propeller and interacts with the 40S ribosomal subunit.

Structure 2014 Jun 24;22(6):923-30. Epub 2014 Apr 24.

Department of Molecular Structural Biology, Institute for Microbiology and Genetics, GZMB, Georg-August-University Göttingen, 37077 Göttingen, Germany. Electronic address:

The multisubunit eukaryotic translation initiation factor 3, among which the subunit b (eIF3b) is a major scaffold protein, plays essential roles in protein synthesis. Here, we report the crystal structure of the WD40 domain of Chaetomium thermophilum eIF3b, revealing a nine-bladed β-propeller fold. Sequence analysis indicates that this propeller architecture is common to all eIF3b orthologs. Revisiting the cryoelectron microscopy (cryo-EM) map of the 43S preinitiation complex suggests an interaction of the eIF3b with the 40S ribosomal subunit involving the ribosomal protein S9e and the 18S rRNA. This model is strongly supported by the direct binding of eIF3b to 40S ribosomes and to the isolated ribosomal protein rpS9e in vitro.
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http://dx.doi.org/10.1016/j.str.2014.03.010DOI Listing
June 2014

Crystal structures of the novel cytosolic 5'-nucleotidase IIIB explain its preference for m7GMP.

PLoS One 2014 6;9(3):e90915. Epub 2014 Mar 6.

Abteilung für Molekulare Strukturbiologie, Institut für Mikrobiologie und Genetik, Göttinger Zentrum für Molekulare Biowissenschaften, Georg-August-Universität Göttingen, Göttingen, Germany.

5'-nucleotidases catalyze the hydrolytic dephosphorylation of nucleoside monophosphates. As catabolic enzymes they contribute significantly to the regulation of cellular nucleotide levels; misregulation of nucleotide metabolism and nucleotidase deficiencies are associated with a number of diseases. The seven human 5'-nucleotidases differ with respect to substrate specificity and cellular localization. Recently, the novel cytosolic 5'-nucleotidase III-like protein, or cN-IIIB, has been characterized in human and Drosophila. cN-IIIB exhibits a strong substrate preference for the modified nucleotide 7-methylguanosine monophosphate but the structural reason for this preference was unknown. Here, we present crystal structures of cN-IIIB from Drosophila melanogaster bound to the reaction products 7-methylguanosine or cytidine. The structural data reveal that the cytosine- and 7-methylguanine moieties of the products are stacked between two aromatic residues in a coplanar but off-centered position. 7-methylguanosine is specifically bound through π-π interactions and distinguished from unmodified guanosine by additional cation-π coulomb interactions between the aromatic side chains and the positively charged 7-methylguanine. Notably, the base is further stabilized by T-shaped edge-to-face stacking of an additional tryptophan packing perpendicularly against the purine ring and forming, together with the other aromates, an aromatic slot. The structural data in combination with site-directed mutagenesis experiments reveal the molecular basis for the broad substrate specificity of cN-IIIB but also explain the substrate preference for 7-methylguanosine monophosphate. Analyzing the substrate specificities of cN-IIIB and the main pyrimidine 5'-nucleotidase cN-IIIA by mutagenesis studies, we show that cN-IIIA dephosphorylates the purine m7GMP as well, hence redefining its substrate spectrum. Docking calculations with cN-IIIA and m7GMP as well as biochemical data reveal that Asn69 does not generally exclude the turnover of purine substrates thus correcting previous suggestions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0090915PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3946280PMC
February 2015

Structural determinants and mechanism of mammalian CRM1 allostery.

Structure 2013 Aug 11;21(8):1350-60. Epub 2013 Jul 11.

Abteilung für Theoretische und Computergestützte Biophysik, Max-Planck-Institut für Biophysikalische Chemie, Am Faßberg 11, 37077 Göttingen, Germany.

Proteins carrying nuclear export signals cooperatively assemble with the export factor CRM1 and the effector protein RanGTP. In lower eukaryotes, this cooperativity is coupled to CRM1 conformational changes; however, it is unknown if mammalian CRM1 maintains its compact conformation or shows similar structural flexibility. Here, combinations of small-angle X-ray solution scattering and electron microscopy experiments with molecular dynamics simulations reveal pronounced conformational flexibility in mammalian CRM1 and demonstrate that RanGTP binding induces association of its N- and C-terminal regions to form a toroid structure. The CRM1 toroid is stabilized mainly by local interactions between the terminal regions, rather than by global strain. The CRM1 acidic loop is key in transmitting the effect of this RanGTP-induced global conformational change to the NES-binding cleft by shifting its population to the open state, which displays enhanced cargo affinity. Cooperative CRM1 export complex assembly thus constitutes a highly dynamic process, encompassing an intricate interplay of global and local structural changes.
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http://dx.doi.org/10.1016/j.str.2013.05.015DOI Listing
August 2013

Structural basis for cooperativity of CRM1 export complex formation.

Proc Natl Acad Sci U S A 2013 Jan 31;110(3):960-5. Epub 2012 Dec 31.

Abteilung für Molekulare Strukturbiologie, Institut für Mikrobiologie und Genetik, Göttinger Zentrum für Molekulare Biowissenschaften, Georg-August-Universität Göttingen, D-37077 Göttingen, Germany.

In eukaryotes, the nucleocytoplasmic transport of macromolecules is mainly mediated by soluble nuclear transport receptors of the karyopherin-β superfamily termed importins and exportins. The highly versatile exportin chromosome region maintenance 1 (CRM1) is essential for nuclear depletion of numerous structurally and functionally unrelated protein and ribonucleoprotein cargoes. CRM1 has been shown to adopt a toroidal structure in several functional transport complexes and was thought to maintain this conformation throughout the entire nucleocytoplasmic transport cycle. We solved crystal structures of free CRM1 from the thermophilic eukaryote Chaetomium thermophilum. Surprisingly, unbound CRM1 exhibits an overall extended and pitched superhelical conformation. The two regulatory regions, namely the acidic loop and the C-terminal α-helix, are dramatically repositioned in free CRM1 in comparison with the ternary CRM1-Ran-Snurportin1 export complex. Single-particle EM analysis demonstrates that, in a noncrystalline environment, free CRM1 exists in equilibrium between extended, superhelical and compact, ring-like conformations. Molecular dynamics simulations show that the C-terminal helix plays an important role in regulating the transition from an extended to a compact conformation and reveal how the binding site for nuclear export signals of cargoes is modulated by different CRM1 conformations. Combining these results, we propose a model for the cooperativity of CRM1 export complex assembly involving the long-range allosteric communication between the distant binding sites of GTP-bound Ran and cargo.
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http://dx.doi.org/10.1073/pnas.1215214110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3549083PMC
January 2013

NES consensus redefined by structures of PKI-type and Rev-type nuclear export signals bound to CRM1.

Nat Struct Mol Biol 2010 Nov 24;17(11):1367-76. Epub 2010 Oct 24.

Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Germany.

Classic nuclear export signals (NESs) confer CRM1-dependent nuclear export. Here we present crystal structures of the RanGTP-CRM1 complex alone and bound to the prototypic PKI or HIV-1 Rev NESs. These NESs differ markedly in the spacing of their key hydrophobic (Φ) residues, yet CRM1 recognizes them with the same rigid set of five Φ pockets. The different Φ spacings are compensated for by different conformations of the bound NESs: in the case of PKI, an α-helical conformation, and in the case of Rev, an extended conformation with a critical proline docking into a Φ pocket. NMR analyses of CRM1-bound and CRM1-free PKI NES suggest that CRM1 selects NES conformers that pre-exist in solution. Our data lead to a new structure-based NES consensus, and explain why NESs differ in their affinities for CRM1 and why supraphysiological NESs bind the exportin so tightly.
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http://dx.doi.org/10.1038/nsmb.1931DOI Listing
November 2010

Crystal structure of the nuclear export receptor CRM1 in complex with Snurportin1 and RanGTP.

Science 2009 May 23;324(5930):1087-91. Epub 2009 Apr 23.

Abteilung für Molekulare Strukturbiologie, Institut für Mikrobiologie und Genetik, GZMB, Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany.

CRM1 mediates nuclear export of numerous unrelated cargoes, which may carry a short leucine-rich nuclear export signal or export signatures that include folded domains. How CRM1 recognizes such a variety of cargoes has been unknown up to this point. Here we present the crystal structure of the SPN1.CRM1.RanGTP export complex at 2.5 angstrom resolution (where SPN1 is snurportin1 and RanGTP is guanosine 5' triphosphate-bound Ran). SPN1 is a nuclear import adapter for cytoplasmically assembled, m(3)G-capped spliceosomal U snRNPs (small nuclear ribonucleoproteins). The structure shows how CRM1 can specifically return the cargo-free form of SPN1 to the cytoplasm. The extensive contact area includes five hydrophobic residues at the SPN1 amino terminus that dock into a hydrophobic cleft of CRM1, as well as numerous hydrophilic contacts of CRM1 to m(3)G cap-binding domain and carboxyl-terminal residues of SPN1. The structure suggests that RanGTP promotes cargo-binding to CRM1 solely through long-range conformational changes in the exportin.
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http://dx.doi.org/10.1126/science.1173388DOI Listing
May 2009

Structural basis for m7G-cap hypermethylation of small nuclear, small nucleolar and telomerase RNA by the dimethyltransferase TGS1.

Nucleic Acids Res 2009 Jul 22;37(12):3865-77. Epub 2009 Apr 22.

Abteilung für Molekulare Strukturbiologie, Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, 37077 Göttingen, Germany.

The 5'-cap of spliceosomal small nuclear RNAs, some small nucleolar RNAs and of telomerase RNA was found to be hypermethylated in vivo. The Trimethylguanosine Synthase 1 (TGS1) mediates this conversion of the 7-methylguanosine-cap to the 2,2,7-trimethylguanosine (m(3)G)-cap during maturation of the RNPs. For mammalian UsnRNAs the generated m(2,2,7)G-cap is one part of a bipartite import signal mediating the transport of the UsnRNP-core complex into the nucleus. In order to understand the structural organization of human TGS1 as well as substrate binding and recognition we solved the crystal structure of the active TGS1 methyltransferase domain containing both, the minimal substrate m(7)GTP and the reaction product S-adenosyl-L-homocysteine (AdoHcy). The methyltransferase of human TGS1 harbors the canonical class 1 methyltransferase fold as well as an unique N-terminal, alpha-helical domain of 40 amino acids, which is essential for m(7)G-cap binding and catalysis. The crystal structure of the substrate bound methyltransferase domain as well as mutagenesis studies provide insight into the catalytic mechanism of TGS1.
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http://dx.doi.org/10.1093/nar/gkp249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2709555PMC
July 2009

Structure analysis of the conserved methyltransferase domain of human trimethylguanosine synthase TGS1.

Acta Crystallogr D Biol Crystallogr 2009 Apr 19;65(Pt 4):332-8. Epub 2009 Mar 19.

Department of Molecular Structural Biology, Institute of Microbiology and Genetics, Georg-August-University Göttingen, Göttingen, Germany.

Methyltransferases play an important role in the post-transcriptional maturation of most ribonucleic acids. The modification of spliceosomal UsnRNAs includes N2-dimethylation of the m(7)G cap catalyzed by trimethylguanosine synthase 1 (TGS1). This 5'-cap hypermethylation occurs during the biogenesis of UsnRNPs as it initiates the m(3)G cap-dependent nuclear import of UsnRNPs. The conserved methyltransferase domain of human TGS1 has been purified, crystallized and the crystal structure of this domain with bound substrate m(7)GpppA was solved by means of multiple-wavelength anomalous dispersion. Crystal structure analysis revealed that m(7)GpppA binds via its adenosine moiety to the structurally conserved adenosylmethionine-binding pocket, while the m(7) guanosine remains unbound. This unexpected binding only occurs in the absence of AdoMet and suggests an incomplete binding pocket for the m(7)G cap which is caused by the N-terminal truncation of the protein. These structural data are consistent with the finding that the crystallized fragment of human TGS1 is catalytically inactive, while a fragment that is 17 amino acids longer exhibits activity.
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http://dx.doi.org/10.1107/S0907444909003102DOI Listing
April 2009

Crystal structure of the RRM domain of poly(A)-specific ribonuclease reveals a novel m(7)G-cap-binding mode.

J Mol Biol 2008 Oct 31;382(4):827-34. Epub 2008 Jul 31.

Abteilung für Molekulare Strukturbiologie, Institut für Mikrobiologie und Genetik, GZMB, Georg-August-Universität Göttingen, 37077 Göttingen, Germany.

Poly(A)-specific ribonuclease (PARN) is a processive 3'-exoribonuclease involved in the decay of eukaryotic mRNAs. Interestingly, PARN interacts not only with the 3' end of the mRNA but also with its 5' end as PARN contains an RRM domain that specifically binds both the poly(A) tail and the 7-methylguanosine (m(7)G) cap. The interaction of PARN with the 5' cap of mRNAs stimulates the deadenylation activity and enhances the processivity of this reaction. We have determined the crystal structure of the PARN-RRM domain with a bound m(7)G triphosphate nucleotide, revealing a novel binding mode for the m(7)G cap. The structure of the m(7)G binding pocket is located outside of the canonical RNA-binding surface of the RRM domain and differs significantly from that of other m(7)G-cap-binding proteins. The crystal structure also shows a remarkable conformational flexibility of the RRM domain, leading to a perfect exchange of two alpha-helices with an adjacent protein molecule in the crystal lattice.
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http://dx.doi.org/10.1016/j.jmb.2008.07.073DOI Listing
October 2008