2024-03-29T01:32:05Zhttp://repository.helmholtz-hzi.de/oai/requestoai:repository.helmholtz-hzi.de:10033/5591542019-08-30T11:32:17Zcom_10033_311308col_10033_621053
Simon, Bernd
Masiewicz, Pawel
Ephrussi, Anne
Carlomagno, Teresa
Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany.
2015-07-07T13:33:47Z
2015-07-07T13:33:47Z
2015-06-18
The structure of the SOLE element of oskar mRNA. 2015: RNA
1469-9001
26089324
10.1261/rna.049601.115
http://hdl.handle.net/10033/559154
RNA (New York, N.Y.)
mRNA localization by active transport is a regulated process that requires association of mRNPs with protein motors for transport along either the microtubule or the actin cytoskeleton. oskar mRNA localization at the posterior pole of the Drosophila oocyte requires a specific mRNA sequence, termed the SOLE, which comprises nucleotides of both exon 1 and exon 2 and is assembled upon splicing. The SOLE folds into a stem-loop structure. Both SOLE RNA and the exon junction complex (EJC) are required for oskar mRNA transport along the microtubules by kinesin. The SOLE RNA likely constitutes a recognition element for a yet unknown protein, which either belongs to the EJC or functions as a bridge between the EJC and the mRNA. Here, we determine the solution structure of the SOLE RNA by Nuclear Magnetic Resonance spectroscopy. We show that the SOLE forms a continuous helical structure, including a few noncanonical base pairs, capped by a pentanucleotide loop. The helix displays a widened major groove, which could accommodate a protein partner. In addition, the apical helical segment undergoes complex dynamics, with potential functional significance.
ENG
The structure of the SOLE element of oskar mRNA.
Article2018-06-12T17:36:48ZmRNA localization by active transport is a regulated process that requires association of mRNPs with protein motors for transport along either the microtubule or the actin cytoskeleton. oskar mRNA localization at the posterior pole of the Drosophila oocyte requires a specific mRNA sequence, termed the SOLE, which comprises nucleotides of both exon 1 and exon 2 and is assembled upon splicing. The SOLE folds into a stem-loop structure. Both SOLE RNA and the exon junction complex (EJC) are required for oskar mRNA transport along the microtubules by kinesin. The SOLE RNA likely constitutes a recognition element for a yet unknown protein, which either belongs to the EJC or functions as a bridge between the EJC and the mRNA. Here, we determine the solution structure of the SOLE RNA by Nuclear Magnetic Resonance spectroscopy. We show that the SOLE forms a continuous helical structure, including a few noncanonical base pairs, capped by a pentanucleotide loop. The helix displays a widened major groove, which could accommodate a protein partner. In addition, the apical helical segment undergoes complex dynamics, with potential functional significance.oai:repository.helmholtz-hzi.de:10033/6123012019-08-30T11:34:22Zcom_10033_311308col_10033_621053
Marchanka, Alexander
Simon, Bernd
Althoff-Ospelt, Gerhard
Carlomagno, Teresa
Helmholtzzentrum für Infektionsforschung, 38124 Braunschweig
2016-06-09T10:47:51Z
2016-06-09T10:47:51Z
2015
RNA structure determination by solid-state NMR spectroscopy. 2015, 6:7024 Nat Commun
2041-1723
25960310
10.1038/ncomms8024
http://hdl.handle.net/10033/612301
Nature communications
Knowledge of the RNA three-dimensional structure, either in isolation or as part of RNP complexes, is fundamental to understand the mechanism of numerous cellular processes. Because of its flexibility, RNA represents a challenge for crystallization, while the large size of cellular complexes brings solution-state NMR to its limits. Here, we demonstrate an alternative approach on the basis of solid-state NMR spectroscopy. We develop a suite of experiments and RNA labeling schemes and demonstrate for the first time that ssNMR can yield a RNA structure at high-resolution. This methodology allows structural analysis of segmentally labelled RNA stretches in high-molecular weight cellular machines—independent of their ability to crystallize—and opens the way to mechanistic studies of currently difficult-to-access RNA-protein assemblies.
en
Magnetic Resonance Spectroscopy
Models, Molecular
Nucleic Acid Conformation
RNA
Ribonucleoproteins
RNA structure determination by solid-state NMR spectroscopy.
Article2018-06-12T22:18:17ZKnowledge of the RNA three-dimensional structure, either in isolation or as part of RNP complexes, is fundamental to understand the mechanism of numerous cellular processes. Because of its flexibility, RNA represents a challenge for crystallization, while the large size of cellular complexes brings solution-state NMR to its limits. Here, we demonstrate an alternative approach on the basis of solid-state NMR spectroscopy. We develop a suite of experiments and RNA labeling schemes and demonstrate for the first time that ssNMR can yield a RNA structure at high-resolution. This methodology allows structural analysis of segmentally labelled RNA stretches in high-molecular weight cellular machines—independent of their ability to crystallize—and opens the way to mechanistic studies of currently difficult-to-access RNA-protein assemblies.oai:repository.helmholtz-hzi.de:10033/6138592019-08-30T11:33:05Zcom_10033_311308col_10033_621053
Bowman, Andrew
Lercher, Lukas
Singh, Hari R
Zinne, Daria
Timinszky, Gyula
Carlomagno, Teresa
Ladurner, Andreas G
Biomedical Center, Faculty of Medicine, Ludwig-Maximilians-Universit ̈ at München, Großhaderner Strasse 9..
2016-06-21T09:28:36Z
2016-06-21T09:28:36Z
2016-04-20
The histone chaperone sNASP binds a conserved peptide motif within the globular core of histone H3 through its TPR repeats. 2016, 44 (7):3105-17 Nucleic Acids Res.
1362-4962
26673727
10.1093/nar/gkv1372
http://hdl.handle.net/10033/613859
Nucleic acids research
Eukaryotic chromatin is a complex yet dynamic structure, which is regulated in part by the assembly and disassembly of nucleosomes. Key to this process is a group of proteins termed histone chaperones that guide the thermodynamic assembly of nucleosomes by interacting with soluble histones. Here we investigate the interaction between the histone chaperone sNASP and its histone H3 substrate. We find that sNASP binds with nanomolar affinity to a conserved heptapeptide motif in the globular domain of H3, close to the C-terminus. Through functional analysis of sNASP homologues we identified point mutations in surface residues within the TPR domain of sNASP that disrupt H3 peptide interaction, but do not completely disrupt binding to full length H3 in cells, suggesting that sNASP interacts with H3 through additional contacts. Furthermore, chemical shift perturbations from(1)H-(15)N HSQC experiments show that H3 peptide binding maps to the helical groove formed by the stacked TPR motifs of sNASP. Our findings reveal a new mode of interaction between a TPR repeat domain and an evolutionarily conserved peptide motif found in canonical H3 and in all histone H3 variants, including CenpA and have implications for the mechanism of histone chaperoning within the cell.
en
The histone chaperone sNASP binds a conserved peptide motif within the globular core of histone H3 through its TPR repeats.
Article2018-06-13T07:39:59ZEukaryotic chromatin is a complex yet dynamic structure, which is regulated in part by the assembly and disassembly of nucleosomes. Key to this process is a group of proteins termed histone chaperones that guide the thermodynamic assembly of nucleosomes by interacting with soluble histones. Here we investigate the interaction between the histone chaperone sNASP and its histone H3 substrate. We find that sNASP binds with nanomolar affinity to a conserved heptapeptide motif in the globular domain of H3, close to the C-terminus. Through functional analysis of sNASP homologues we identified point mutations in surface residues within the TPR domain of sNASP that disrupt H3 peptide interaction, but do not completely disrupt binding to full length H3 in cells, suggesting that sNASP interacts with H3 through additional contacts. Furthermore, chemical shift perturbations from(1)H-(15)N HSQC experiments show that H3 peptide binding maps to the helical groove formed by the stacked TPR motifs of sNASP. Our findings reveal a new mode of interaction between a TPR repeat domain and an evolutionarily conserved peptide motif found in canonical H3 and in all histone H3 variants, including CenpA and have implications for the mechanism of histone chaperoning within the cell.oai:repository.helmholtz-hzi.de:10033/6206782019-08-30T11:27:46Zcom_10033_311308col_10033_621053
Miller, Thomas C R
Simon, Bernd
Rybin, Vladimir
Grötsch, Helga
Curtet, Sandrine
Khochbin, Saadi
Carlomagno, Teresa
Müller, Christoph W
Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
2017-01-05T09:27:01Z
2017-01-05T09:27:01Z
2016-12-19
A bromodomain-DNA interaction facilitates acetylation-dependent bivalent nucleosome recognition by the BET protein BRDT. 2016, 7:13855 Nat Commun
2041-1723
27991587
10.1038/ncomms13855
http://hdl.handle.net/10033/620678
Nature communications
Bromodomains are critical components of many chromatin modifying/remodelling proteins and are emerging therapeutic targets, yet how they interact with nucleosomes, rather than acetylated peptides, remains unclear. Using BRDT as a model, we characterized how the BET family of bromodomains interacts with site-specifically acetylated nucleosomes. Here we report that BRDT interacts with nucleosomes through its first (BD1), but not second (BD2) bromodomain, and that acetylated histone recognition by BD1 is complemented by a bromodomain-DNA interaction. Simultaneous DNA and histone recognition enhances BRDT's nucleosome binding affinity and specificity, and its ability to localize to acetylated chromatin in cells. Conservation of DNA binding in bromodomains of BRD2, BRD3 and BRD4, indicates that bivalent nucleosome recognition is a key feature of these bromodomains and possibly others. Our results elucidate the molecular mechanism of BRDT association with nucleosomes and identify structural features of the BET bromodomains that may be targeted for therapeutic inhibition.
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
A bromodomain-DNA interaction facilitates acetylation-dependent bivalent nucleosome recognition by the BET protein BRDT.
Article2018-06-13T01:08:52ZBromodomains are critical components of many chromatin modifying/remodelling proteins and are emerging therapeutic targets, yet how they interact with nucleosomes, rather than acetylated peptides, remains unclear. Using BRDT as a model, we characterized how the BET family of bromodomains interacts with site-specifically acetylated nucleosomes. Here we report that BRDT interacts with nucleosomes through its first (BD1), but not second (BD2) bromodomain, and that acetylated histone recognition by BD1 is complemented by a bromodomain-DNA interaction. Simultaneous DNA and histone recognition enhances BRDT's nucleosome binding affinity and specificity, and its ability to localize to acetylated chromatin in cells. Conservation of DNA binding in bromodomains of BRD2, BRD3 and BRD4, indicates that bivalent nucleosome recognition is a key feature of these bromodomains and possibly others. Our results elucidate the molecular mechanism of BRDT association with nucleosomes and identify structural features of the BET bromodomains that may be targeted for therapeutic inhibition.oai:repository.helmholtz-hzi.de:10033/6208952019-08-30T11:32:16Zcom_10033_620636com_10033_620626com_10033_311308col_10033_620665col_10033_621053col_10033_620629
Suwandi, Abdulhadi
Bargen, Imke
Pils, Marina C
Krey, Martina
Zur Lage, Susanne
Singh, Anurag K
Basler, Tina
Falk, Christine S
Seidler, Ursula
Hornef, Mathias W
Goethe, Ralph
Weiss, Siegfried
Helmholtz Centre for infection research, Inhoffenstr.7, 38124 Braunschweig, Germany.
2017-04-12T14:43:10Z
2017-04-12T14:43:10Z
2017
CD4 T Cell Dependent Colitis Exacerbation Following Re-Exposure of Mycobacterium avium ssp. paratuberculosis. 2017, 7:75 Front Cell Infect Microbiol
2235-2988
28361039
110.1097/MIB.0000000000000157
http://hdl.handle.net/10033/620895
Frontiers in cellular and infection microbiology
Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD), a chronic inflammatory bowel disease of cattle characterized by intermittent to chronic diarrhea. In addition, MAP has been isolated from Crohn's disease (CD) patients. The impact of MAP on severity of clinical symptoms in JD as well as its role in CD are yet unknown. We have previously shown that MAP is able to colonize inflamed enteric tissue and to exacerbate the inflammatory tissue response (Suwandi et al., 2014). In the present study, we analyzed how repeated MAP administration influences the course of dextran sulfate sodium (DSS)-induced colitis. In comparison to mice exposed to DSS or MAP only, repeated exposure of DSS-treated mice to MAP (DSS/MAP) revealed a significantly enhanced clinical score, reduction of colon length as well as severe CD4(+) T cell infiltration into the colonic lamina propria. Functional analysis identified a critical role of CD4(+) T cells in the MAP-induced disease exacerbation. Additionally, altered immune responses were observed when closely related mycobacteria species such as M. avium ssp. avium and M. avium ssp. hominissuis were administered. These data reveal the specific ability of MAP to aggravate intestinal inflammation and clinical symptoms. Overall, this phenotype is compatible with similar disease promoting capabilites of MAP in JD and CD.
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
CD4 T Cell Dependent Colitis Exacerbation Following Re-Exposure of Mycobacterium avium ssp. paratuberculosis.
Article2018-06-13T00:12:59ZMycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD), a chronic inflammatory bowel disease of cattle characterized by intermittent to chronic diarrhea. In addition, MAP has been isolated from Crohn's disease (CD) patients. The impact of MAP on severity of clinical symptoms in JD as well as its role in CD are yet unknown. We have previously shown that MAP is able to colonize inflamed enteric tissue and to exacerbate the inflammatory tissue response (Suwandi et al., 2014). In the present study, we analyzed how repeated MAP administration influences the course of dextran sulfate sodium (DSS)-induced colitis. In comparison to mice exposed to DSS or MAP only, repeated exposure of DSS-treated mice to MAP (DSS/MAP) revealed a significantly enhanced clinical score, reduction of colon length as well as severe CD4(+) T cell infiltration into the colonic lamina propria. Functional analysis identified a critical role of CD4(+) T cells in the MAP-induced disease exacerbation. Additionally, altered immune responses were observed when closely related mycobacteria species such as M. avium ssp. avium and M. avium ssp. hominissuis were administered. These data reveal the specific ability of MAP to aggravate intestinal inflammation and clinical symptoms. Overall, this phenotype is compatible with similar disease promoting capabilites of MAP in JD and CD.oai:repository.helmholtz-hzi.de:10033/6212422019-08-30T11:35:39Zcom_10033_311308col_10033_621053
Lercher, Lukas
Danilenko, Nataliya
Kirkpatrick, John
Carlomagno, Teresa
Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany.
2018-01-18T15:21:31Z
2018-01-18T15:21:31Z
2017-12-29
Structural characterization of the Asf1-Rtt109 interaction and its role in histone acetylation. 2017 Nucleic Acids Res.
1362-4962
29300933
10.1093/nar/gkx1283
http://hdl.handle.net/10033/621242
Nucleic acids research
Acetylation of histone H3 at lysine-56 by the histone acetyltransferase Rtt109 in lower eukaryotes is important for maintaining genomic integrity and is required for C. albicans pathogenicity. Rtt109 is activated by association with two different histone chaperones, Vps75 and Asf1, through an unknown mechanism. Here, we reveal that the Rtt109 C-terminus interacts directly with Asf1 and elucidate the structural basis of this interaction. In addition, we find that the H3 N-terminus can interact via the same interface on Asf1, leading to a competition between the two interaction partners. This, together with the recruitment and position of the substrate, provides an explanation of the role of the Rtt109 C-terminus in Asf1-dependent Rtt109 activation.
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Structural characterization of the Asf1-Rtt109 interaction and its role in histone acetylation.
Article2018-06-13T00:07:17ZAcetylation of histone H3 at lysine-56 by the histone acetyltransferase Rtt109 in lower eukaryotes is important for maintaining genomic integrity and is required for C. albicans pathogenicity. Rtt109 is activated by association with two different histone chaperones, Vps75 and Asf1, through an unknown mechanism. Here, we reveal that the Rtt109 C-terminus interacts directly with Asf1 and elucidate the structural basis of this interaction. In addition, we find that the H3 N-terminus can interact via the same interface on Asf1, leading to a competition between the two interaction partners. This, together with the recruitment and position of the substrate, provides an explanation of the role of the Rtt109 C-terminus in Asf1-dependent Rtt109 activation.oai:repository.helmholtz-hzi.de:10033/6212802019-08-30T11:28:51Zcom_10033_311308col_10033_621053
Kozak, Sandra
Lercher, Lukas
Karanth, Megha N
Meijers, Rob
Carlomagno, Teresa
Boivin, Stephane
Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
2018-02-15T13:50:18Z
2018-02-15T13:50:18Z
2016
Optimization of protein samples for NMR using thermal shift assays. 2016, 64 (4):281-9 J. Biomol. NMR
1573-5001
26984476
10.1007/s10858-016-0027-z
http://hdl.handle.net/10033/621280
Journal of biomolecular NMR
Maintaining a stable fold for recombinant proteins is challenging, especially when working with highly purified and concentrated samples at temperatures >20 °C. Therefore, it is worthwhile to screen for different buffer components that can stabilize protein samples. Thermal shift assays or ThermoFluor(®) provide a high-throughput screening method to assess the thermal stability of a sample under several conditions simultaneously. Here, we describe a thermal shift assay that is designed to optimize conditions for nuclear magnetic resonance studies, which typically require stable samples at high concentration and ambient (or higher) temperature. We demonstrate that for two challenging proteins, the multicomponent screen helped to identify ingredients that increased protein stability, leading to clear improvements in the quality of the spectra. Thermal shift assays provide an economic and time-efficient method to find optimal conditions for NMR structural studies.
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Fluorometry
Magnetic Resonance Spectroscopy
Nuclear Magnetic Resonance, Biomolecular
Protein Stability
Proteins
Temperature
Optimization of protein samples for NMR using thermal shift assays.
Article2018-06-12T23:17:09ZMaintaining a stable fold for recombinant proteins is challenging, especially when working with highly purified and concentrated samples at temperatures >20 °C. Therefore, it is worthwhile to screen for different buffer components that can stabilize protein samples. Thermal shift assays or ThermoFluor(®) provide a high-throughput screening method to assess the thermal stability of a sample under several conditions simultaneously. Here, we describe a thermal shift assay that is designed to optimize conditions for nuclear magnetic resonance studies, which typically require stable samples at high concentration and ambient (or higher) temperature. We demonstrate that for two challenging proteins, the multicomponent screen helped to identify ingredients that increased protein stability, leading to clear improvements in the quality of the spectra. Thermal shift assays provide an economic and time-efficient method to find optimal conditions for NMR structural studies.oai:repository.helmholtz-hzi.de:10033/6212962019-08-30T11:37:00Zcom_10033_311308col_10033_621053
Graziadei, Andrea
Masiewicz, Pawel
Lapinaite, Audrone
Carlomagno, Teresa
Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
2018-02-26T09:36:56Z
2018-02-26T09:36:56Z
2016-05
Archaea box C/D enzymes methylate two distinct substrate rRNA sequences with different efficiency. 2016, 22 (5):764-72 RNA
1469-9001
26925607
10.1261/rna.054320.115
http://hdl.handle.net/10033/621296
RNA (New York, N.Y.)
RNA modifications confer complexity to the 4-nucleotide polymer; nevertheless, their exact function is mostly unknown. rRNA 2'-O-ribose methylation concentrates to ribosome functional sites and is important for ribosome biogenesis. The methyl group is transferred to rRNA by the box C/D RNPs: The rRNA sequence to be methylated is recognized by a complementary sequence on the guide RNA, which is part of the enzyme. In contrast to their eukaryotic homologs, archaeal box C/D enzymes can be assembled in vitro and are used to study the mechanism of 2'-O-ribose methylation. In Archaea, each guide RNA directs methylation to two distinct rRNA sequences, posing the question whether this dual architecture of the enzyme has a regulatory role. Here we use methylation assays and low-resolution structural analysis with small-angle X-ray scattering to study the methylation reaction guided by the sR26 guide RNA fromPyrococcus furiosus We find that the methylation efficacy at sites D and D' differ substantially, with substrate D' turning over more efficiently than substrate D. This observation correlates well with structural data: The scattering profile of the box C/D RNP half-loaded with substrate D' is similar to that of the holo complex, which has the highest activity. Unexpectedly, the guide RNA secondary structure is not responsible for the functional difference at the D and D' sites. Instead, this difference is recapitulated by the nature of the first base pair of the guide-substrate duplex. We suggest that substrate turnover may occur through a zip mechanism that initiates at the 5'-end of the product.
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Archaea
Enzymes
Methylation
Mutation
Nucleic Acid Conformation
RNA, Ribosomal
Archaea box C/D enzymes methylate two distinct substrate rRNA sequences with different efficiency.
Article2018-06-13T21:37:14ZRNA modifications confer complexity to the 4-nucleotide polymer; nevertheless, their exact function is mostly unknown. rRNA 2'-O-ribose methylation concentrates to ribosome functional sites and is important for ribosome biogenesis. The methyl group is transferred to rRNA by the box C/D RNPs: The rRNA sequence to be methylated is recognized by a complementary sequence on the guide RNA, which is part of the enzyme. In contrast to their eukaryotic homologs, archaeal box C/D enzymes can be assembled in vitro and are used to study the mechanism of 2'-O-ribose methylation. In Archaea, each guide RNA directs methylation to two distinct rRNA sequences, posing the question whether this dual architecture of the enzyme has a regulatory role. Here we use methylation assays and low-resolution structural analysis with small-angle X-ray scattering to study the methylation reaction guided by the sR26 guide RNA fromPyrococcus furiosus We find that the methylation efficacy at sites D and D' differ substantially, with substrate D' turning over more efficiently than substrate D. This observation correlates well with structural data: The scattering profile of the box C/D RNP half-loaded with substrate D' is similar to that of the holo complex, which has the highest activity. Unexpectedly, the guide RNA secondary structure is not responsible for the functional difference at the D and D' sites. Instead, this difference is recapitulated by the nature of the first base pair of the guide-substrate duplex. We suggest that substrate turnover may occur through a zip mechanism that initiates at the 5'-end of the product.oai:repository.helmholtz-hzi.de:10033/6215102019-08-30T11:29:45Zcom_10033_311308col_10033_621053
Marchanka, Alexander
Stanek, Jan
Pintacuda, Guido
Carlomagno, Teresa
Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
2018-10-09T14:22:41Z
2018-10-09T14:22:41Z
2018-08-21
1364-548X
29974085
10.1039/c8cc04437f
http://hdl.handle.net/10033/621510
Fast (4100 kHz) magic angle spinning solid-state NMR allows
combining high-sensitive proton detection with the absence of an
intrinsic molecular weight limit. Using this technique we observe for
the first time narrow 1H RNA resonances and assign nucleotide spin
systems with only 200 lg of uniformly 13C,15N-labelled RNA.
Attribution-NonCommercial-ShareAlike 3.0 United States
http://creativecommons.org/licenses/by-nc-sa/3.0/us/
Rapid access to RNA resonances by proton-detected solid-state NMR at >100 kHz MAS.
Article
Chemical communications (Cambridge, England)2018-10-09T14:22:41Zoai:repository.helmholtz-hzi.de:10033/6219402019-09-17T02:30:05Zcom_10033_311308col_10033_621053
Danilenko, Nataliya
Lercher, Lukas
Kirkpatrick, John
Gabel, Frank
Codutti, Luca
Carlomagno, Teresa
HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany
2019-09-16T12:53:14Z
2019-09-16T12:53:14Z
2019-08-06
2041-1723
10.1038/s41467-019-11410-7
http://hdl.handle.net/10033/621940
Nature Communications
Histones, the principal protein components of chromatin, contain long disordered sequences, which are extensively post-translationally modified. Although histone chaperones are known to control both the activity and specificity of histone-modifying enzymes, the mechanisms promoting modification of highly disordered substrates, such as lysine-acetylation within the N-terminal tail of histone H3, are not understood. Here, to understand how histone chaperones Asf1 and Vps75 together promote H3 K9-acetylation, we establish the solution structural model of the acetyltransferase Rtt109 in complex with Asf1 and Vps75 and the histone dimer H3:H4. We show that Vps75 promotes K9-acetylation by engaging the H3 N-terminal tail in fuzzy electrostatic interactions with its disordered C-terminal domain, thereby confining the H3 tail to a wide central cavity faced by the Rtt109 active site. These fuzzy interactions between disordered domains achieve localization of lysine residues in the H3 tail to the catalytic site with minimal loss of entropy, and may represent a common mechanism of enzymatic reactions involving highly disordered substrates.
en
Springer Science and Business Media LLC
Attribution-NonCommercial-ShareAlike 4.0 International
http://creativecommons.org/licenses/by-nc-sa/4.0/
General Biochemistry, Genetics and Molecular Biology
General Physics and Astronomy
General Chemistry
Histone chaperone exploits intrinsic disorder to switch acetylation specificity
Article
10
12019-09-16T12:53:14Zoai:repository.helmholtz-hzi.de:10033/6221262020-02-12T12:12:43Zcom_10033_311308col_10033_621053
Lapinaite, Audrone
Carlomagno, Teresa
Gabel, Frank
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
2020-02-11T14:17:33Z
2020-02-11T14:17:33Z
2020-01-01
Methods Mol Biol. 2020;2113:165-188. doi: 10.1007/978-1-0716-0278-2_13.
1940-6029
32006315
10.1007/978-1-0716-0278-2_13
http://hdl.handle.net/10033/622126
Methods in molecular biology
Small-angle neutron scattering (SANS) provides structural information on biomacromolecules and their complexes in dilute solutions at the nanometer length scale. The overall dimensions, shapes, and interactions can be probed and compared to information obtained by complementary structural biology techniques such as crystallography, NMR, and EM. SANS, in combination with solvent H2O/D2O exchange and/or deuteration, is particularly well suited to probe the internal structure of RNA-protein (RNP) complexes since neutrons are more sensitive than X-rays to the difference in scattering length densities of proteins and RNA, with respect to an aqueous solvent. In this book chapter we provide a practical guide on how to carry out SANS experiments on RNP complexes, as well as possibilities of data analysis and interpretation.
en
Springer
Attribution-NonCommercial-ShareAlike 4.0 International
http://creativecommons.org/licenses/by-nc-sa/4.0/
Contrast variation
Deuterium labeling
Macromolecular complex
Protein
RNA
RNP
Small-angle neutron scattering (SANS)
Structural biology
Small-Angle Neutron Scattering of RNA-Protein Complexes.
Article
Methods in molecular biology (Clifton, N.J.)
oai:repository.helmholtz-hzi.de:10033/6221732020-03-12T03:28:45Zcom_10033_311308col_10033_621053
Marasco, M
Berteotti, A
Weyershaeuser, J
Thorausch, N
Sikorska, J
Krausze, J
Brandt, H J
Kirkpatrick, J
Rios, P
Schamel, W W
Köhn, M
Carlomagno, T
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
2020-02-26T09:37:24Z
2020-02-26T09:37:24Z
2020-01-01
Sci Adv. 2020 Jan 31;6(5):eaay4458. doi: 10.1126/sciadv.aay4458. eCollection 2020 Jan.
2375-2548
32064351
10.1126/sciadv.aay4458
http://hdl.handle.net/10033/622173
Science Advances
In cancer, the programmed death-1 (PD-1) pathway suppresses T cell stimulation and mediates immune escape. Upon stimulation, PD-1 becomes phosphorylated at its immune receptor tyrosine-based inhibitory motif (ITIM) and immune receptor tyrosine-based switch motif (ITSM), which then bind the Src homology 2 (SH2) domains of SH2-containing phosphatase 2 (SHP2), initiating T cell inactivation. The SHP2-PD-1 complex structure and the exact functions of the two SH2 domains and phosphorylated motifs remain unknown. Here, we explain the structural basis and provide functional evidence for the mechanism of PD-1-mediated SHP2 activation. We demonstrate that full activation is obtained only upon phosphorylation of both ITIM and ITSM: ITSM binds C-SH2 with strong affinity, recruiting SHP2 to PD-1, while ITIM binds N-SH2, displacing it from the catalytic pocket and activating SHP2. This binding event requires the formation of a new inter-domain interface, offering opportunities for the development of novel immunotherapeutic approaches.
en
American Association for the Advancement of Science
Attribution-NonCommercial-ShareAlike 4.0 International
http://creativecommons.org/licenses/by-nc-sa/4.0/
Molecular mechanism of SHP2 activation by PD-1 stimulation.
Article
Science advances2020-02-26T09:37:25Zoai:repository.helmholtz-hzi.de:10033/6222102020-03-18T02:01:28Zcom_10033_311308col_10033_621053
Ahmed, Mumdooh
Marchanka, Alexander
Carlomagno, Teresa
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
2020-03-17T14:29:17Z
2020-03-17T14:29:17Z
2020-02-05
Angew Chem Int Ed Engl. 2020 Feb 5. doi: 10.1002/anie.201915465.
32023357
10.1002/anie.201915465
http://hdl.handle.net/10033/622210
1521-3773
Angewandte Chemie (International ed. in English)
Solid-state NMR (ssNMR) is applicable to high molecular-weight (MW) protein assemblies in a non-amorphous precipitate. The technique yields atomic resolution structural information on both soluble and insoluble particles without limitations of MW or requirement of crystals. Herein, we propose and demonstrate an approach that yields the structure of protein-RNA complexes (RNP) solely from ssNMR data. Instead of using low-sensitivity magnetization transfer steps between heteronuclei of the protein and the RNA, we measure paramagnetic relaxation enhancement effects elicited on the RNA by a paramagnetic tag coupled to the protein. We demonstrate that this data, together with chemical-shift-perturbation data, yields an accurate structure of an RNP complex, starting from the bound structures of its components. The possibility of characterizing protein-RNA interactions by ssNMR may enable applications to large RNP complexes, whose structures are not accessible by other methods.
en
Wiley
Attribution-NonCommercial-ShareAlike 4.0 International
http://creativecommons.org/licenses/by-nc-sa/4.0/
RNA recognition
paramagnetic relaxation enhancement
protein-RNA complex
solid-state NMR
structure determination
Structure of a Protein-RNA Complex by Solid-State NMR Spectroscopy.
Article
Angewandte Chemie (International ed. in English)
Germany2020-03-17T14:29:18Zoai:repository.helmholtz-hzi.de:10033/6222232020-04-08T02:45:06Zcom_10033_311308col_10033_621053
Graziadei, Andrea
Gabel, Frank
Kirkpatrick, John
Carlomagno, Teresa
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstraße 7, 38124 Braunschweig, Germany.
2020-04-07T09:49:31Z
2020-04-07T09:49:31Z
2020-03-23
Elife. 2020 Mar 23;9. pii: 50027. doi: 10.7554/eLife.50027.
32202498
10.7554/eLife.50027
http://hdl.handle.net/10033/622223
2050-084X
eLife
2'-O-rRNA methylation, which is essential in eukaryotes and archaea, is catalysed by the Box C/D RNP complex in an RNA-guided manner. Despite the conservation of the methylation sites, the abundance of site-specific modifications shows variability across species and tissues, suggesting that rRNA methylation may provide a means of controlling gene expression. As all Box C/D RNPs are thought to adopt a similar structure, it remains unclear how the methylation efficiency is regulated. Here, we provide the first structural evidence that, in the context of the Box C/D RNP, the affinity of the catalytic module fibrillarin for the substrate-guide helix is dependent on the RNA sequence outside the methylation site, thus providing a mechanism by which both the substrate and guide RNA sequences determine the degree of methylation. To reach this result, we develop an iterative structure-calculation protocol that exploits the power of integrative structural biology to characterize conformational ensembles.
en
eLife Sciences Publications
Attribution-NonCommercial-ShareAlike 4.0 International
http://creativecommons.org/licenses/by-nc-sa/4.0/
Box C/D RNP
NMR
SAS
biochemistry
chemical biology
conformational equilibrium
integrative structural biology
molecular biophysics
rRNA methylation
structural biology
The guide sRNA sequence determines the activity level of box C/D RNPs.
Article
9
eLife
England2020-04-07T09:49:31Zoai:repository.helmholtz-hzi.de:10033/6222692020-05-27T08:03:46Zcom_10033_311308col_10033_621053
Marasco, Michelangelo
Kirkpatrick, John P
Carlomagno, Teresa
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
2020-05-25T11:26:19Z
2020-05-25T11:26:19Z
2020-04-01
Biomol NMR Assign. 2020;10.1007/s12104-020-09941-y. doi:10.1007/s12104-020-09941-y
32236803
10.1007/s12104-020-09941-y
http://hdl.handle.net/10033/622269
1874-270X
Biomolecular NMR assignments
Inhibition of immune checkpoint receptor Programmed Death-1 (PD-1) via monoclonal antibodies is an established anticancer immunotherapeutic approach. This treatment has been largely successful; however, its high cost demands equally effective, more affordable alternatives. To date, the development of drugs targeting downstream players in the PD-1-dependent signaling pathway has been hampered by our poor understanding of the molecular details of the intermolecular interactions involved in the pathway. Activation of PD-1 leads to phosphorylation of two signaling motifs located in its cytoplasmic domain, the immune tyrosine inhibitory motif (ITIM) and immune tyrosine switch motif (ITSM), which recruit and activate protein tyrosine phosphatase SHP2. This interaction is mediated by the two Src homology 2 (SH2) domains of SHP2, termed N-SH2 and C-SH2, which recognize phosphotyrosines pY223 and pY248 of ITIM and ITSM, respectively. SHP2 then propagates the inhibitory signal, ultimately leading to suppression of T cell functionality. In order to facilitate mechanistic structural studies of this signaling pathway, we report the resonance assignments of the complexes formed by the signaling motifs of PD-1 and the SH2 domains of SHP2.
en
Springer
Attribution-NonCommercial-ShareAlike 4.0 International
http://creativecommons.org/licenses/by-nc-sa/4.0/
Immunotherapy
PD-1
SH2 domains
SHP2
1H, 13C, 15N chemical shift assignments of SHP2 SH2 domains in complex with PD-1 immune-tyrosine motifs.
Article
Biomolecular NMR assignments
Netherlands2020-05-25T11:26:21Zoai:repository.helmholtz-hzi.de:10033/6224282020-09-05T01:55:07Zcom_10033_311308col_10033_621053
Mahieu, Emilie
Covès, Jacques
Krüger, Georg
Martel, Anne
Moulin, Martine
Carl, Nico
Härtlein, Michael
Carlomagno, Teresa
Franzetti, Bruno
Gabel, Frank
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
2020-09-04T10:50:05Z
2020-09-04T10:50:05Z
2020-06-24
Biophys J. 2020;119(2):375-388. doi:10.1016/j.bpj.2020.06.015.
32640186
10.1016/j.bpj.2020.06.015
http://hdl.handle.net/10033/622428
1542-0086
Biophysical journal
The proteasome is a key player of regulated protein degradation in all kingdoms of life. Although recent atomic structures have provided snapshots on a number of conformations, data on substrate states and populations during the active degradation process in solution remain scarce. Here, we use time-resolved small-angle neutron scattering of a deuterium-labeled GFPssrA substrate and an unlabeled archaeal PAN-20S system to obtain direct structural information on substrate states during ATP-driven unfolding and subsequent proteolysis in solution. We find that native GFPssrA structures are degraded in a biexponential process, which correlates strongly with ATP hydrolysis, the loss of fluorescence, and the buildup of small oligopeptide products. Our solution structural data support a model in which the substrate is directly translocated from PAN into the 20S proteolytic chamber, after a first, to our knowledge, successful unfolding process that represents a point of no return and thus prevents dissociation of the complex and the release of harmful, aggregation-prone products.
en
Elsevier
Attribution-NonCommercial-ShareAlike 4.0 International
http://creativecommons.org/licenses/by-nc-sa/4.0/
Observing Protein Degradation by the PAN-20S Proteasome by Time-Resolved Neutron Scattering.
Article
119
2
375
388
Biophysical journal
United States2020-09-04T10:50:06Zoai:repository.helmholtz-hzi.de:10033/6227082021-01-29T01:51:21Zcom_10033_311308col_10033_621053
Danilenko, Nataliya
Lercher, Lukas
Kirkpatrick, John
Gabel, Frank
Codutti, Luca
Carlomagno, Teresa
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
2021-01-28T13:16:42Z
2021-01-28T13:16:42Z
2019-08-06
Nat Commun. 2019 Aug 6;10(1):3435. doi: 10.1038/s41467-019-11410-7.
31387991
10.1038/s41467-019-11410-7
http://hdl.handle.net/10033/622708
2041-1723
Nature communications
Histones, the principal protein components of chromatin, contain long disordered sequences, which are extensively post-translationally modified. Although histone chaperones are known to control both the activity and specificity of histone-modifying enzymes, the mechanisms promoting modification of highly disordered substrates, such as lysine-acetylation within the N-terminal tail of histone H3, are not understood. Here, to understand how histone chaperones Asf1 and Vps75 together promote H3 K9-acetylation, we establish the solution structural model of the acetyltransferase Rtt109 in complex with Asf1 and Vps75 and the histone dimer H3:H4. We show that Vps75 promotes K9-acetylation by engaging the H3 N-terminal tail in fuzzy electrostatic interactions with its disordered C-terminal domain, thereby confining the H3 tail to a wide central cavity faced by the Rtt109 active site. These fuzzy interactions between disordered domains achieve localization of lysine residues in the H3 tail to the catalytic site with minimal loss of entropy, and may represent a common mechanism of enzymatic reactions involving highly disordered substrates.
en
Nature Pulishing Group
Attribution 4.0 International
http://creativecommons.org/licenses/by/4.0/
Histone chaperone exploits intrinsic disorder to switch acetylation specificity.
Article
10
1
3435
Nature communications
England2021-01-28T13:16:43Zoai:repository.helmholtz-hzi.de:10033/6229232021-07-15T13:38:37Zcom_10033_311308col_10033_621053
Wang, Ying
Kirkpatrick, John
Zur Lage, Susanne
Korn, Sophie M
Neißner, Konstantin
Schwalbe, Harald
Schlundt, Andreas
Carlomagno, Teresa
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
2021-07-05T14:02:36Z
2021-07-05T14:02:36Z
2021-03-26
Biomol NMR Assign. 2021 Mar 26:1–9. doi: 10.1007/s12104-021-10019-6. Epub ahead of print.
33770349
10.1007/s12104-021-10019-6
http://hdl.handle.net/10033/622923
1874-270X
Biomolecular NMR assignments
The current COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has become a worldwide health crisis, necessitating coordinated scientific research and urgent identification of new drug targets for treatment of COVID-19 lung disease. The covid19-nmr consortium seeks to support drug development by providing publicly accessible NMR data on the viral RNA elements and proteins. The SARS-CoV-2 genome comprises a single RNA of about 30 kb in length, in which 14 open reading frames (ORFs) have been annotated, and encodes approximately 30 proteins. The first two-thirds of the SARS-CoV-2 genome is made up of two large overlapping open-reading-frames (ORF1a and ORF1b) encoding a replicase polyprotein, which is subsequently cleaved to yield 16 so-called non-structural proteins. The non-structural protein 1 (Nsp1), which is considered to be a major virulence factor, suppresses host immune functions by associating with host ribosomal complexes at the very end of its C-terminus. Furthermore, Nsp1 facilitates initiation of viral RNA translation via an interaction of its N-terminal domain with the 5' untranslated region (UTR) of the viral RNA. Here, we report the near-complete backbone chemical-shift assignments of full-length SARS-CoV-2 Nsp1 (19.8 kDa), which reveal the domain organization, secondary structure and backbone dynamics of Nsp1, and which will be of value to further NMR-based investigations of both the biochemical and physiological functions of Nsp1.
en
SPringer
Attribution 4.0 International
http://creativecommons.org/licenses/by/4.0/
5′ untranslated region
NMR spectroscopy
New drug targets
Non-structural proteins
Nsp1
SARS-CoV-2
1H, 13C, and 15N backbone chemical-shift assignments of SARS-CoV-2 non-structural protein 1 (leader protein)
Article
Biomolecular NMR assignments
Netherlands2021-07-05T14:02:37Zoai:repository.helmholtz-hzi.de:10033/6229302021-07-09T01:46:28Zcom_10033_311308col_10033_621053
Marasco, Michelangelo
Carlomagno, Teresa
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
2021-07-08T12:55:02Z
2021-07-08T12:55:02Z
2020-05-27
J Struct Biol X. 2020 May 27;4:100026. doi: 10.1016/j.yjsbx.2020.100026.
32647828
10.1016/j.yjsbx.2020.100026
http://hdl.handle.net/10033/622930
2590-1524
Journal of structural biology: X
Phosphotyrosine (pY) signaling is instrumental to numerous cellular processes. pY recognition occurs through specialized protein modules, among which the Src-homology 2 (SH2) domain is the most common. SH2 domains are small protein modules with an invariant fold, and are present in more than a hundred proteins with different function. Here we ask the question of how such a structurally conserved, small protein domain can recognize distinct phosphopeptides with the breath of binding affinity, specificity and kinetic parameters necessary for proper control of pY-dependent signaling and rapid cellular response. We review the current knowledge on structure, thermodynamics and kinetics of SH2-phosphopeptide complexes and conclude that selective phosphopeptide recognition is governed by both structure and dynamics of the SH2 domain, as well as by the kinetics of the binding events. Further studies on the thermodynamic and kinetic properties of SH2-phosphopeptide complexes, beyond their structure, are required to understand signaling regulation.
en
Elsevier
Attribution 4.0 International
http://creativecommons.org/licenses/by/4.0/
Binding specificity
Phosphotyrosine
SH2 domain
pY signalling
Specificity and regulation of phosphotyrosine signaling through SH2 domains.
Article
4
100026
Journal of structural biology: X
United States2021-07-08T12:55:03Zoai:repository.helmholtz-hzi.de:10033/6229342021-07-13T01:42:01Zcom_10033_311308col_10033_621053
Höfler, Simone
Lukat, Peer
Blankenfeldt, Wulf
Carlomagno, Teresa
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
2021-07-12T12:49:33Z
2021-07-12T12:49:33Z
2021-01-22
RNA. 2021 Apr;27(4):496-512. doi: 10.1261/rna.077396.120. Epub 2021 Jan 22.
33483369
10.1261/rna.077396.120
http://hdl.handle.net/10033/622934
1469-9001
RNA (New York, N.Y.)
Ribosomal RNA (rRNA) carries extensive 2'-O-methyl marks at functionally important sites. This simple chemical modification is thought to confer stability, promote RNA folding, and contribute to generate a heterogenous ribosome population with a yet-uncharacterized function. 2'-O-methylation occurs both in archaea and eukaryotes and is accomplished by the Box C/D RNP enzyme in an RNA-guided manner. Extensive and partially conflicting structural information exists for the archaeal enzyme, while no structural data is available for the eukaryotic enzyme. The yeast Box C/D RNP consists of a guide RNA, the RNA-primary binding protein Snu13, the two scaffold proteins Nop56 and Nop58, and the enzymatic module Nop1. Here we present the high-resolution structure of the eukaryotic Box C/D methyltransferase Nop1 from Saccharomyces cerevisiae bound to the amino-terminal domain of Nop56. We discuss similarities and differences between the interaction modes of the two proteins in archaea and eukaryotes and demonstrate that eukaryotic Nop56 recruits the methyltransferase to the Box C/D RNP through a protein-protein interface that differs substantially from the archaeal orthologs. This study represents a first achievement in understanding the evolution of the structure and function of these proteins from archaea to eukaryotes.
en
Cold Spring Harbour Laboratory Press
Attribution 4.0 International
http://creativecommons.org/licenses/by/4.0/
2′-O-methylation
Fibrillarin
Nop56
eukaryotic Box C/D RNP
protein–protein complex structure
High-resolution structure of eukaryotic Fibrillarin interacting with Nop56 amino-terminal domain.
Article
27
4
496
512
RNA (New York, N.Y.)
United States2021-01-14T00:00:00Zoai:repository.helmholtz-hzi.de:10033/6229362021-07-14T01:42:30Zcom_10033_311308col_10033_621053
Altincekic, Nadide
Korn, Sophie Marianne
Qureshi, Nusrat Shahin
Dujardin, Marie
Ninot-Pedrosa, Martí
Abele, Rupert
Abi Saad, Marie Jose
Alfano, Caterina
Almeida, Fabio C L
Alshamleh, Islam
de Amorim, Gisele Cardoso
Anderson, Thomas K
Anobom, Cristiane D
Anorma, Chelsea
Bains, Jasleen Kaur
Bax, Adriaan
Blackledge, Martin
Blechar, Julius
Böckmann, Anja
Brigandat, Louis
Bula, Anna
Bütikofer, Matthias
Camacho-Zarco, Aldo R
Carlomagno, Teresa
Caruso, Icaro Putinhon
Ceylan, Betül
Chaikuad, Apirat
Chu, Feixia
Cole, Laura
Crosby, Marquise G
de Jesus, Vanessa
Dhamotharan, Karthikeyan
Felli, Isabella C
Ferner, Jan
Fleischmann, Yanick
Fogeron, Marie-Laure
Fourkiotis, Nikolaos K
Fuks, Christin
Fürtig, Boris
Gallo, Angelo
Gande, Santosh L
Gerez, Juan Atilio
Ghosh, Dhiman
Gomes-Neto, Francisco
Gorbatyuk, Oksana
Guseva, Serafima
Hacker, Carolin
Häfner, Sabine
Hao, Bing
Hargittay, Bruno
Henzler-Wildman, K
Hoch, Jeffrey C
Hohmann, Katharina F
Hutchison, Marie T
Jaudzems, Kristaps
Jović, Katarina
Kaderli, Janina
Kalniņš, Gints
Kaņepe, Iveta
Kirchdoerfer, Robert N
Kirkpatrick, John
Knapp, Stefan
Krishnathas, Robin
Kutz, Felicitas
Zur Lage, Susanne
Lambertz, Roderick
Lang, Andras
Laurents, Douglas
Lecoq, Lauriane
Linhard, Verena
Löhr, Frank
Malki, Anas
Bessa, Luiza Mamigonian
Martin, Rachel W
Matzel, Tobias
Maurin, Damien
McNutt, Seth W
Mebus-Antunes, Nathane Cunha
Meier, Beat H
Meiser, Nathalie
Mompeán, Miguel
Monaca, Elisa
Montserret, Roland
Mariño Perez, Laura
Moser, Celine
Muhle-Goll, Claudia
Neves-Martins, Thais Cristtina
Ni, Xiamonin
Norton-Baker, Brenna
Pierattelli, Roberta
Pontoriero, Letizia
Pustovalova, Yulia
Ohlenschläger, Oliver
Orts, Julien
Da Poian, Andrea T
Pyper, Dennis J
Richter, Christian
Riek, Roland
Rienstra, Chad M
Robertson, Angus
Pinheiro, Anderson S
Sabbatella, Raffaele
Salvi, Nicola
Saxena, Krishna
Schulte, Linda
Schiavina, Marco
Schwalbe, Harald
Silber, Mara
Almeida, Marcius da Silva
Sprague-Piercy, Marc A
Spyroulias, Georgios A
Sreeramulu, Sridhar
Tants, Jan-Niklas
Tārs, Kaspars
Torres, Felix
Töws, Sabrina
Treviño, Miguel Á
Trucks, Sven
Tsika, Aikaterini C
Varga, Krisztina
Wang, Ying
Weber, Marco E
Weigand, Julia E
Wiedemann, Christoph
Wirmer-Bartoschek, Julia
Wirtz Martin, Maria Alexandra
Zehnder, Johannes
Hengesbach, Martin
Schlundt, Andreas
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
2021-07-13T14:17:13Z
2021-07-13T14:17:13Z
2021-05-10
ront Mol Biosci. 2021 May 10;8:653148. doi: 10.3389/fmolb.2021.653148.
2296-889X
34041264
10.3389/fmolb.2021.653148
http://hdl.handle.net/10033/622936
Frontiers in molecular biosciences
The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium's collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.
en
Frontiers
Attribution 4.0 International
http://creativecommons.org/licenses/by/4.0/
COVID-19
NMR spectroscopy
SARS-CoV-2
accessory proteins
cell-free protein synthesis
intrinsically disordered region
nonstructural proteins
structural proteins
Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications.
Article
8
653148
Frontiers in molecular biosciences
Switzerland2021-07-13T14:17:14Zoai:repository.helmholtz-hzi.de:10033/6230252021-09-14T01:52:45Zcom_10033_311308col_10033_621053
Marasco, Michelangelo
Kirkpatrick, John
Nanna, Vittoria
Sikorska, Justyna
Carlomagno, Teresa
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
2021-09-13T12:16:40Z
2021-09-13T12:16:40Z
2021-04-20
Comput Struct Biotechnol J. 2021 Apr 20;19:2398-2415. doi: 10.1016/j.csbj.2021.04.040.
2001-0370
34025932
10.1016/j.csbj.2021.04.040
http://hdl.handle.net/10033/623025
Computational and structural biotechnology journal
SHP2 is a ubiquitous protein tyrosine phosphatase, whose activity is regulated by phosphotyrosine (pY)-containing peptides generated in response to extracellular stimuli. Its crystal structure reveals a closed, auto-inhibited conformation in which the N-terminal Src homology 2 (N-SH2) domain occludes the catalytic site of the phosphatase (PTP) domain. High-affinity mono-phosphorylated peptides promote catalytic activity by binding to N-SH2 and disrupting the interaction with the PTP. The mechanism behind this process is not entirely clear, especially because N-SH2 is incapable of accommodating complete peptide binding when SHP2 is in the auto-inhibited state. Here, we show that pY performs an essential role in this process; in addition to its contribution to overall peptide-binding energy, pY-recognition leads to enhanced dynamics of the N-SH2 EF and BG loops via an allosteric communication network, which destabilizes the N-SH2–PTP interaction surface and simultaneously generates a fully accessible binding pocket for the C-terminal half of the phosphopeptide. Subsequently, full binding of the phosphopeptide is associated with the stabilization of activated SHP2. We demonstrate that this allosteric network exists only in N-SH2, which is directly involved in the regulation of SHP2 activity, while the C-terminal SH2 domain (C-SH2) functions primarily to recruit high-affinity bidentate phosphopeptides.
en
Elsevier
Attribution 4.0 International
http://creativecommons.org/licenses/by/4.0/
Allosteric coupling
Molecular dynamics
NMR spectroscopy
PD-1
SHP2
Phosphotyrosine couples peptide binding and SHP2 activation via a dynamic allosteric network.
Article
19
2398
2415
Computational and structural biotechnology journal
Netherlands2021-09-13T12:16:41Z