FMP Publications

Our publications are recorded in a searchable database since 2010, updates will be added regularly.

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References
Modulations of DNA Contacts by Linker Histones and Post-translational Modifications Determine the Mobility and Modifiability of Nucleosomal H3 Tails
Stützer(*), A., Liokatis, S., Kiesel(*), A., Schwarzer, D., Sprangers(*), R., Söding(*), J., Selenko, P.; Fischle(*), W.
Mol Cell, 61:247-259
(2016)

Tags: In-Cell NMR (Selenko), Protein Chemistry (Schwarzer)

Abstract: Post-translational histone modifications and linker histone incorporation regulate chromatin structure and genome activity. How these systems interface on a molecular level is unclear. Using biochemistry and NMR spectroscopy, we deduced mechanistic insights into the modification behavior of N-terminal histone H3 tails in different nucleosomal contexts. We find that linker histones generally inhibit modifications of different H3 sites and reduce H3 tail dynamics in nucleosomes. These effects are caused by modulations of electrostatic interactions of H3 tails with linker DNA and largely depend on the C-terminal domains of linker histones. In agreement, linker histone occupancy and H3 tail modifications segregate on a genome-wide level. Charge-modulating modifications such as phosphorylation and acetylation weaken transient H3 tail-linker DNA interactions, increase H3 tail dynamics, and, concomitantly, enhance general modifiability. We propose that alterations of H3 tail-linker DNA interactions by linker histones and charge-modulating modifications execute basal control mechanisms of chromatin function.

Chemoselective synthesis and analysis of naturally occurring phosphorylated cysteine peptides
Bertran-Vicente, J., Penkert, M., Nieto-Garcia, O., Jeckelmann(*), J. M., Schmieder, P., Krause, E.; Hackenberger, C. P.
Nat Commun, 7:12703
(2016)

Tags: Chemical Biology II (Hackenberger), Mass Spectrometry (Krause, E), Solution NMR (Schmieder)

Abstract: In contrast to protein O-phosphorylation, studying the function of the less frequent N- and S-phosphorylation events have lagged behind because they have chemical features that prevent their manipulation through standard synthetic and analytical methods. Here we report on the development of a chemoselective synthetic method to phosphorylate Cys side-chains in unprotected peptides. This approach makes use of a reaction between nucleophilic phosphites and electrophilic disulfides accessible by standard methods. We achieve the stereochemically defined phosphorylation of a Cys residue and verify the modification using electron-transfer higher-energy dissociation (EThcD) mass spectrometry. To demonstrate the use of the approach in resolving biological questions, we identify an endogenous Cys phosphorylation site in IICB(Glc), which is known to be involved in the carbohydrate uptake from the bacterial phosphotransferase system (PTS). This new chemical and analytical approach finally allows further investigating the functions and significance of Cys phosphorylation in a wide range of crucial cellular processes.

The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6
Boschert(*), V., Frisch(*), C., Back(*), J. W., van Pee(*), K., Weidauer(*), S. E., Muth(*), E. M., Schmieder, P., Beerbaum, M., Knappik(*), A., Timmerman(*), P.; Mueller(*), T. D.
Open biology, 6
(2016)

Tags: Solution NMR (Schmieder)

Abstract: The glycoprotein sclerostin has been identified as a negative regulator of bone growth. It exerts its function by interacting with the Wnt co-receptor LRP5/6, blocks the binding of Wnt factors and thereby inhibits Wnt signalling. Neutralizing anti-sclerostin antibodies are able to restore Wnt activity and enhance bone growth thereby presenting a new osteoanabolic therapy approach for diseases such as osteoporosis. We have generated various Fab antibodies against human and murine sclerostin using a phage display set-up. Biochemical analyses have identified one Fab developed against murine sclerostin, AbD09097 that efficiently neutralizes sclerostin's Wnt inhibitory activity. In vitro interaction analysis using sclerostin variants revealed that this neutralizing Fab binds to sclerostin's flexible second loop, which has been shown to harbour the LRP5/6 binding motif. Affinity maturation was then applied to AbD09097, providing a set of improved neutralizing Fab antibodies which particularly bind human sclerostin with enhanced affinity. Determining the crystal structure of AbD09097 provides first insights into how this antibody might recognize and neutralize sclerostin. Together with the structure-function relationship derived from affinity maturation these new data will foster the rational design of new and highly efficient anti-sclerostin antibodies for the therapy of bone loss diseases such as osteoporosis.

Chemical shift assignments and secondary structure prediction for Q4DY78, a conserved kinetoplastid-specific protein from Trypanosoma cruzi
D'Andrea(*), E. D., Diehl, A., Schmieder, P., Oschkinat, H.; Pires(*), J. R.
Biomol NMR Assign, 10:325-328
(2016)

Tags: NMR-Supported Structural Biology (Oschkinat), Solution NMR (Schmieder)

Abstract: Trypanosoma cruzi, Trypanosma brucei and Leishmania spp. are kinetoplastid protozoa causative agents of Chagas disease, sleeping sickness and leishmaniasis, respectively, neglected tropical diseases estimated to infect millions of people worldwide. Their genome sequencing has revealed approximately 50 % of genes encoding hypothetical proteins of unknown function, opening possibilities for novel target identification and drug discovery. Q4DY78 is a putative essential protein from T. cruzi conserved in the related kinetoplastids and divergent from mammalian host proteins. Here we report the (1)H, (15)N, and (13)C chemical shift assignments and secondary structure analysis of the Q4DY78 protein as basis for NMR structure determination, functional analysis and drug screening.

Intradomain Allosteric Network Modulates Calcium Affinity of the C-Type Lectin Receptor Langerin
Hanske(*), J., Aleksic(*), S., Ballaschk, M., Jurk(*), M., Shanina(*), E., Beerbaum, M., Schmieder, P., Keller(*), B. G.; Rademacher(*), C.
J Am Chem Soc, 138:12176-12186
(2016)

Tags: Solution NMR (Schmieder)

Abstract: Antigen uptake and processing by innate immune cells is crucial to initiate the immune response. Therein, the endocytic C-type lectin receptors serve as pattern recognition receptors, detecting pathogens by their glycan structures. Herein, we studied the carbohydrate recognition domain of Langerin, a C-type lectin receptor involved in the host defense against viruses such as HIV and influenza as well as bacteria and fungi. Using a combination of nuclear magnetic resonance and molecular dynamics simulations, we unraveled the molecular determinants underlying cargo capture and release encoded in the receptor architecture. Our findings revealed receptor dynamics over several time scales associated with binding and release of the essential cofactor Ca(2+) controlled by the coupled motions of two loops. Applying mutual information theory and site-directed mutagenesis, we identified an allosteric intradomain network that modulates the Ca(2+) affinity depending on the pH, thereby promoting fast ligand release.

Effects of Halide Ions on the Carbamidocyclophane Biosynthesis in Nostoc sp. CAVN2
Preisitsch(*), M., Heiden(*), S. E., Beerbaum, M., Niedermeyer(*), T. H., Schneefeld(*), M., Herrmann(*), J., Kumpfmüller(*), J., Thürmer(*), A., Neidhardt(*), I., Wiesner(*), C., Daniel(*), R., Müller(*), R., Bange(*), F. C., Schmieder, P., Schweder(*), T.; Mundt(*), S.
Mar Drugs, 14:21
(2016)

Tags: Solution NMR (Schmieder)

Abstract: In this study, the influence of halide ions on [7.7]paracyclophane biosynthesis in the cyanobacterium Nostoc sp. CAVN2 was investigated. In contrast to KI and KF, supplementation of the culture medium with KCl or KBr resulted not only in an increase of growth but also in an up-regulation of carbamidocyclophane production. LC-MS analysis indicated the presence of chlorinated, brominated, but also non-halogenated derivatives. In addition to 22 known cylindrocyclophanes and carbamidocyclophanes, 27 putative congeners have been detected. Nine compounds, carbamidocyclophanes M-U, were isolated, and their structural elucidation by 1D and 2D NMR experiments in combination with HRMS and ECD analysis revealed that they are brominated analogues of chlorinated carbamidocyclophanes. Quantification of the carbamidocyclophanes showed that chloride is the preferably utilized halide, but incorporation is reduced in the presence of bromide. Evaluation of the antibacterial activity of 30 [7.7]paracyclophanes and related derivatives against selected pathogenic Gram-positive and Gram-negative bacteria exhibited remarkable effects especially against methicillin- and vancomycin-resistant staphylococci and Mycobacterium tuberculosis. For deeper insights into the mechanisms of biosynthesis, the carbamidocyclophane biosynthetic gene cluster in Nostoc sp. CAVN2 was studied. The gene putatively coding for the carbamoyltransferase has been identified. Based on bioinformatic analyses, a possible biosynthetic assembly is discussed.

Green tea reduces body fat via upregulation of neprilysin
Muenzner, M., Tappenbeck(*), N., Gembardt(*), F., Rülke, R., Furkert, J., Melzig(*), M. F., Siems, W. E., Brockmann(*), G. A.; Walther(*), T.
Int J Obes (Lond), 40:1850-1855
(2016)

Tags: Biochemical Neurobiology (Siems)

Abstract: BACKGROUND/OBJECTIVE: Consumption of green tea has become increasingly popular, particularly because of claimed reduction in body weight. We recently reported that animals with pharmacological inhibition (by candoxatril) or genetic absence of the endopeptidase neprilysin (NEP) develop an obese phenotype. We now investigated the effect of green tea extract (in drinking water) on body weight and body composition and the mediating role of NEP. SUBJECTS/METHODS: To elucidate the role of NEP in mediating the beneficial effects of green tea extract, 'Berlin fat mice' or NEP-deficient mice and their age- and gender-matched wild-type controls received the extract in two different doses (300 or 600 mg kg-1 body weight per day) in the drinking water. RESULTS: In 'Berlin fat mice', 51 days of green tea treatment did not only prevent fat accumulation (control: day 0: 30.5% fat, day 51: 33.1%; NS) but also reduced significant body fat (green tea: day 0: 27.8%, day 51: 20.9%, P<0.01) and body weight below the initial levels. Green tea reduced food intake. This was paralleled by a selective increase in peripheral (in kidney 17%, in intestine 92%), but not central NEP expression and activity, leading to downregulation of orexigens (like galanin and neuropeptide Y (NPY)) known to be physiological substrates of NEP. Consequently, in NEP-knockout mice, green tea extract failed to reduce body fat/weight. CONCLUSIONS: Our data generate experimental proof for the assumed effects of green tea on body weight and the key role for NEP in such process, and thus open a new avenue for the treatment of obesity.

Intracellular repair of oxidation-damaged alpha-synuclein fails to target C-terminal modification sites
Binolfi, A., Limatola, A., Verzini, S., Kosten, J., Theillet, F. X., Rose, H. M., Bekei, B., Stuiver, M., van Rossum, M.; Selenko, P.
Nat Commun, 7:10251
(2016)

Tags: In-Cell NMR (Selenko)

Abstract: Cellular oxidative stress serves as a common denominator in many neurodegenerative disorders, including Parkinson's disease. Here we use in-cell NMR spectroscopy to study the fate of the oxidation-damaged Parkinson's disease protein alpha-synuclein (alpha-Syn) in non-neuronal and neuronal mammalian cells. Specifically, we deliver methionine-oxidized, isotope-enriched alpha-Syn into cultured cells and follow intracellular protein repair by endogenous enzymes at atomic resolution. We show that N-terminal alpha-Syn methionines Met1 and Met5 are processed in a stepwise manner, with Met5 being exclusively repaired before Met1. By contrast, C-terminal methionines Met116 and Met127 remain oxidized and are not targeted by cellular enzymes. In turn, persisting oxidative damage in the C-terminus of alpha-Syn diminishes phosphorylation of Tyr125 by Fyn kinase, which ablates the necessary priming event for Ser129 modification by CK1. These results establish that oxidative stress can lead to the accumulation of chemically and functionally altered alpha-Syn in cells.

In-Cell Protein Structures from 2D NMR Experiments
Müntener(*), T., Haussinger(*), D., Selenko, P.; Theillet, F. X.
J Phys Chem Lett, 7:2821-2825
(2016)

Tags: In-Cell NMR (Selenko)

Abstract: In-cell NMR spectroscopy provides atomic resolution insights into the structural properties of proteins in cells, but it is rarely used to solve entire protein structures de novo. Here, we introduce a paramagnetic lanthanide-tag to simultaneously measure protein pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs) to be used as input for structure calculation routines within the Rosetta program. We employ this approach to determine the structure of the protein G B1 domain (GB1) in intact Xenopus laevis oocytes from a single set of 2D in-cell NMR experiments. Specifically, we derive well-defined GB1 ensembles from low concentration in-cell NMR samples ( approximately 50 muM) measured at moderate magnetic field strengths (600 MHz), thus offering an easily accessible alternative for determining intracellular protein structures.

Opposing effects of Elk-1 multisite phosphorylation shape its response to ERK activation
Mylona(*), A., Theillet, F. X., Foster(*), C., Cheng(*), T. M., Miralles(*), F., Bates(*), P. A., Selenko, P.; Treisman(*), R.
Science, 354:233-237
(2016)

Tags: In-Cell NMR (Selenko)

Abstract: Multisite phosphorylation regulates many transcription factors, including the serum response factor partner Elk-1. Phosphorylation of the transcriptional activation domain (TAD) of Elk-1 by the protein kinase ERK at multiple sites potentiates recruitment of the Mediator transcriptional coactivator complex and transcriptional activation, but the roles of individual phosphorylation events had remained unclear. Using time-resolved nuclear magnetic resonance spectroscopy, we found that ERK2 phosphorylation proceeds at markedly different rates at eight TAD sites in vitro, which we classified as fast, intermediate, and slow. Mutagenesis experiments showed that phosphorylation of fast and intermediate sites promoted Mediator interaction and transcriptional activation, whereas modification of slow sites counteracted both functions, thereby limiting Elk-1 output. Progressive Elk-1 phosphorylation thus ensures a self-limiting response to ERK activation, which occurs independently of antagonizing phosphatase activity.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
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