• 1H, 13C, 15N chemical shift assignments of SHP2 SH2 domains in complex with PD-1 immune-tyrosine motifs.

      Marasco, Michelangelo; Kirkpatrick, John P; Carlomagno, Teresa; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer, 2020-04-01)
      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.
    • Archaea box C/D enzymes methylate two distinct substrate rRNA sequences with different efficiency.

      Graziadei, Andrea; Masiewicz, Pawel; Lapinaite, Audrone; Carlomagno, Teresa; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-05)
      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.
    • A bromodomain-DNA interaction facilitates acetylation-dependent bivalent nucleosome recognition by the BET protein BRDT.

      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. (2016-12-19)
      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.
    • CD4 T Cell Dependent Colitis Exacerbation Following Re-Exposure of Mycobacterium avium ssp. paratuberculosis.

      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; et al. (2017)
      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.
    • The guide sRNA sequence determines the activity level of box C/D RNPs.

      Graziadei, Andrea; Gabel, Frank; Kirkpatrick, John; Carlomagno, Teresa; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstraße 7, 38124 Braunschweig, Germany. (eLife Sciences Publications, 2020-03-23)
      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.
    • Histone chaperone exploits intrinsic disorder to switch acetylation specificity

      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 (Springer Science and Business Media LLC, 2019-08-06)
      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.
    • The histone chaperone sNASP binds a conserved peptide motif within the globular core of histone H3 through its TPR repeats.

      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.. (2016-04-20)
      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.
    • Molecular mechanism of SHP2 activation by PD-1 stimulation.

      Marasco, M; Berteotti, A; Weyershaeuser, J; Thorausch, N; Sikorska, J; Krausze, J; Brandt, H J; Kirkpatrick, J; Rios, P; Schamel, W W; et al. (American Association for the Advancement of Science, 2020-01-01)
      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.
    • Observing Protein Degradation by the PAN-20S Proteasome by Time-Resolved Neutron Scattering.

      Mahieu, Emilie; Covès, Jacques; Krüger, Georg; Martel, Anne; Moulin, Martine; Carl, Nico; Härtlein, Michael; Carlomagno, Teresa; Franzetti, Bruno; Gabel, Frank; et al. (Elsevier, 2020-06-24)
      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.
    • Optimization of protein samples for NMR using thermal shift assays.

      Kozak, Sandra; Lercher, Lukas; Karanth, Megha N; Meijers, Rob; Carlomagno, Teresa; Boivin, Stephane; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016)
      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.
    • Rapid access to RNA resonances by proton-detected solid-state NMR at >100 kHz MAS.

      Marchanka, Alexander; Stanek, Jan; Pintacuda, Guido; Carlomagno, Teresa; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-08-21)
      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.
    • RNA structure determination by solid-state NMR spectroscopy.

      Marchanka, Alexander; Simon, Bernd; Althoff-Ospelt, Gerhard; Carlomagno, Teresa; Helmholtzzentrum für Infektionsforschung, 38124 Braunschweig (2015)
      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.
    • Small-Angle Neutron Scattering of RNA-Protein Complexes.

      Lapinaite, Audrone; Carlomagno, Teresa; Gabel, Frank; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer, 2020-01-01)
      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.
    • Structural characterization of the Asf1-Rtt109 interaction and its role in histone acetylation.

      Lercher, Lukas; Danilenko, Nataliya; Kirkpatrick, John; Carlomagno, Teresa; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017-12-29)
      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.
    • Structure of a Protein-RNA Complex by Solid-State NMR Spectroscopy.

      Ahmed, Mumdooh; Marchanka, Alexander; Carlomagno, Teresa; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley, 2020-02-05)
      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.
    • The structure of the SOLE element of oskar mRNA.

      Simon, Bernd; Masiewicz, Pawel; Ephrussi, Anne; Carlomagno, Teresa; Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany. (2015-06-18)
      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.