FMP Publications

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

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References
Temperature dependence of cross-effect dynamic nuclear polarization in rotating solids: advantages of elevated temperatures
Geiger, M. A., Orwick-Rydmark, M., Marker, K., Franks, W. T., Akhmetzyanov(*), D., Stöppler, D., Zinke, M., Specker, E., Nazare, M., Diehl, A., van Rossum, B. J., Aussenac(*), F., Prisner(*), T., Akbey, Ü.; Oschkinat, H.
Phys Chem Chem Phys, 18:30696-30704
(2016)

Tags: NMR-Supported Structural Biology (Oschkinat), Medicinal Chemistry (Nazare), Molecular Biophysics (Lange, A.)

Abstract: Dynamic nuclear polarization exploits electron spin polarization to boost signal-to-noise in magic-angle-spinning (MAS) NMR, creating new opportunities in materials science, structural biology, and metabolomics studies. Since protein NMR spectra recorded under DNP conditions can show improved spectral resolution at 180-200 K compared to 110 K, we investigate the effects of AMUPol and various deuterated TOTAPOL isotopologues on sensitivity and spectral resolution at these temperatures, using proline and reproducibly prepared SH3 domain samples. The TOTAPOL deuteration pattern is optimized for protein DNP MAS NMR, and signal-to-noise per unit time measurements demonstrate the high value of TOTAPOL isotopologues for Protein DNP MAS NMR at 180-200 K. The combined effects of enhancement, depolarization, and proton longitudinal relaxation are surprisingly sample-specific. At 200 K, DNP on SH3 domain standard samples yields a 15-fold increase in signal-to-noise over a sample without radicals. 2D and 3D NCACX/NCOCX spectra were recorded at 200 K within 1 and 13 hours, respectively. Decreasing enhancements with increasing 2H-content at the CH2 sites of the TEMPO rings in CD3-TOTAPOL highlight the importance of protons in a sphere of 4-6 A around the nitroxyl group, presumably for polarization pickup from electron spins.

Structural analysis of a signal peptide inside the ribosome tunnel by DNP MAS NMR
Lange, S., Franks, W. T., Rajagopalan(*), N., Döring(*), K., Geiger, M. A., Linden, A., van Rossum, B. J., Kramer(*), G., Bukau(*), B.; Oschkinat, H.
Sci Adv, 2:e1600379
(2016)

Tags: NMR-Supported Structural Biology (Oschkinat), Molecular Biophysics (Lange, A.)

Abstract: Proteins are synthesized in cells by ribosomes and, in parallel, prepared for folding or targeting. While ribosomal protein synthesis is progressing, the nascent chain exposes amino-terminal signal sequences or transmembrane domains that mediate interactions with specific interaction partners, such as the signal recognition particle (SRP), the SecA-adenosine triphosphatase, or the trigger factor. These binding events can set the course for folding in the cytoplasm and translocation across or insertion into membranes. A distinction of the respective pathways depends largely on the hydrophobicity of the recognition sequence. Hydrophobic transmembrane domains stabilize SRP binding, whereas less hydrophobic signal sequences, typical for periplasmic and outer membrane proteins, stimulate SecA binding and disfavor SRP interactions. In this context, the formation of helical structures of signal peptides within the ribosome was considered to be an important factor. We applied dynamic nuclear polarization magic-angle spinning nuclear magnetic resonance to investigate the conformational states of the disulfide oxidoreductase A (DsbA) signal peptide stalled within the exit tunnel of the ribosome. Our results suggest that the nascent chain comprising the DsbA signal sequence adopts an extended structure in the ribosome with only minor populations of helical structure.

Surface Binding of TOTAPOL Assists Structural Investigations of Amyloid Fibrils by Dynamic Nuclear Polarization NMR Spectroscopy
Nagaraj, M., Franks, T. W., Saeidpour(*), S., Schubeis(*), T., Oschkinat, H., Ritter(*), C.; van Rossum, B. J.
Chembiochem, 17:1308-1311
(2016)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Dynamic nuclear polarization (DNP) NMR can enhance sensitivity but often comes at the price of a substantial loss of resolution. Two major factors affect spectral quality: low-temperature heterogeneous line broadening and paramagnetic relaxation enhancement (PRE) effects. Investigations by NMR spectroscopy, isothermal titration calorimetry (ITC), and EPR revealed a new substantial affinity of TOTAPOL to amyloid surfaces, very similar to that shown by the fluorescent dye thioflavin-T (ThT). As a consequence, DNP spectra with remarkably good resolution and still reasonable enhancement could be obtained at very low TOTAPOL concentrations, typically 400 times lower than commonly employed. These spectra yielded several long-range constraints that were difficult to obtain without DNP. Our findings open up new strategies for structural studies with DNP NMR spectroscopy on amyloids that can bind the biradical with affinity similar to that shown towards ThT.

Dynamic Nuclear Polarization Provides New Insights into Chromophore Structure in Phytochrome Photoreceptors
Stöppler, D., Song(*), C., van Rossum, B. J., Geiger, M. A., Lang(*), C., Mroginski(*), M. A., Jagtap(*), A. P., Sigurdsson(*), S. T., Matysik(*), J., Hughes(*), J.; Oschkinat, H.
Angew Chem Int Ed Engl, 55:16017-16020
(2016)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Phytochromes are red/far-red photochromic photoreceptors acting as master regulators of development in higher plants, thereby controlling transcription of about 20 % of their genes. Light-induced isomerization of the bilin chromophore leads to large rearrangements in protein structure, whereby the role of protonation dynamics and charge distribution is of particular interest. To help unravel the inherent mechanisms, we present two-dimensional dynamic nuclear polarization (DNP) enhanced solid-state magic-angle spinning (MAS) NMR spectra of the functional sensory module of the cyanobacterial phytochrome Cph1. To this end, the pyrrole ring nitrogen signals were assigned unequivocally, enabling us to locate the positive charge of the phycocyanobilin (PCB) chromophore. To help analyze proton exchange pathways, the proximity of PCB ring nitrogen atoms and functionally relevant H2 O molecules was also determined. Our study demonstrates the value of DNP in biological solid-state MAS NMR spectroscopy.

A Small-Molecule Antagonist of the beta-Catenin/TCF4 Interaction Blocks the Self-Renewal of Cancer Stem Cells and Suppresses Tumorigenesis
Fang(*), L., Zhu(*), Q., Neuenschwander, M., Specker, E., Wulf-Goldenberg(*), A., Weis(*), W. I., von Kries, J. P.; Birchmeier(*), W.
Cancer research, 76:891-901
(2016)

Tags: Screening Unit (von Kries)

Abstract: Wnt/beta-catenin signaling is a highly conserved pathway essential for embryogenesis and tissue homeostasis. However, deregulation of this pathway can initiate and promote human malignancies, especially of the colon and head and neck. Therefore, Wnt/beta-catenin signaling represents an attractive target for cancer therapy. We performed high-throughput screening using AlphaScreen and ELISA techniques to identify small molecules that disrupt the critical interaction between beta-catenin and the transcription factor TCF4 required for signal transduction. We found that compound LF3, a 4-thioureido-benzenesulfonamide derivative, robustly inhibited this interaction. Biochemical assays revealed clues that the core structure of LF3 was essential for inhibition. LF3 inhibited Wnt/beta-catenin signals in cells with exogenous reporters and in colon cancer cells with endogenously high Wnt activity. LF3 also suppressed features of cancer cells related to Wnt signaling, including high cell motility, cell-cycle progression, and the overexpression of Wnt target genes. However, LF3 did not cause cell death or interfere with cadherin-mediated cell-cell adhesion. Remarkably, the self-renewal capacity of cancer stem cells was blocked by LF3 in concentration-dependent manners, as examined by sphere formation of colon and head and neck cancer stem cells under nonadherent conditions. Finally, LF3 reduced tumor growth and induced differentiation in a mouse xenograft model of colon cancer. Collectively, our results strongly suggest that LF3 is a specific inhibitor of canonical Wnt signaling with anticancer activity that warrants further development for preclinical and clinical studies as a novel cancer therapy.

Substrate Hunting for the Myxobacterial CYP260A1 Revealed New 1alpha-Hydroxylated Products from C-19 Steroids
Khatri(*), Y., Ringle(*), M., Lisurek, M., von Kries, J. P., Zapp(*), J.; Bernhardt(*), R.
Chembiochem, 17:90-101
(2016)

Tags: Screening Unit (von Kries), Structural Bioinformatics and Protein Design (Krause, G.)

Abstract: Cytochromes P450 catalyze a variety of synthetically useful reactions. However, it is difficult to determine their physiological or artificial functions when a plethora of orphan P450 systems are present in a genome. CYP260A1 from Sorangium cellulosum So ce56 is a new member among the 21 available P450s in the strain. To identify putative substrates for CYP260A1 we used high-throughput screening of a compound library (ca. 17,000 ligands). Structural analogues of the type I hits were searched for biotechnologically relevant compounds, and this led us to select C-19 steroids as potential substrates. We identified efficient surrogate redox partners for CYP260A1, and an Escherichia coli-based whole-cell biocatalyst system was developed to convert testosterone, androstenedione, and their derivatives methyltestosterone and 11-oxoandrostenedione. A detailed (1) H and (13) C NMR characterization of the product(s) from C-19 steroids revealed that CYP260A1 is the very first 1alpha-steroid hydroxylase.

Bimodal antagonism of PKA signalling by ARHGAP36
Eccles(*), R. L., Czajkowski(*), M. T., Barth(*), C., Müller(*), P. M., McShane(*), E., Grunwald(*), S., Beaudette(*), P., Mecklenburg(*), N., Volkmer, R., Zühlke(*), K., Dittmar(*), G., Selbach(*), M., Hammes(*), A., Daumke(*), O., Klussmann(*), E., Urbe(*), S.; Rocks(*), O.
Nat Commun, 7:12963
(2016)

Tags: Peptide Synthesis (Hackenberger/Volkmer)

Abstract: Protein kinase A is a key mediator of cAMP signalling downstream of G-protein-coupled receptors, a signalling pathway conserved in all eukaryotes. cAMP binding to the regulatory subunits (PKAR) relieves their inhibition of the catalytic subunits (PKAC). Here we report that ARHGAP36 combines two distinct inhibitory mechanisms to antagonise PKA signalling. First, it blocks PKAC activity via a pseudosubstrate motif, akin to the mechanism employed by the protein kinase inhibitor proteins. Second, it targets PKAC for rapid ubiquitin-mediated lysosomal degradation, a pathway usually reserved for transmembrane receptors. ARHGAP36 thus dampens the sensitivity of cells to cAMP. We show that PKA inhibition by ARHGAP36 promotes derepression of the Hedgehog signalling pathway, thereby providing a simple rationale for the upregulation of ARHGAP36 in medulloblastoma. Our work reveals a new layer of PKA regulation that may play an important role in development and disease.

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.

Structural disorder of monomeric alpha-synuclein persists in mammalian cells
Theillet, F. X., Binolfi, A., Bekei, B., Martorana(*), A., Rose, H. M., Stuiver, M., Verzini, S., Lorenz, D., van Rossum, M., Goldfarb(*), D.; Selenko, P.
Nature, 530:45-50
(2016)

Tags: In-Cell NMR (Selenko), Cellular Imaging (Wiesner)

Abstract: Intracellular aggregation of the human amyloid protein alpha-synuclein is causally linked to Parkinson's disease. While the isolated protein is intrinsically disordered, its native structure in mammalian cells is not known. Here we use nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy to derive atomic-resolution insights into the structure and dynamics of alpha-synuclein in different mammalian cell types. We show that the disordered nature of monomeric alpha-synuclein is stably preserved in non-neuronal and neuronal cells. Under physiological cell conditions, alpha-synuclein is amino-terminally acetylated and adopts conformations that are more compact than when in buffer, with residues of the aggregation-prone non-amyloid-beta component (NAC) region shielded from exposure to the cytoplasm, which presumably counteracts spontaneous aggregation. These results establish that different types of crowded intracellular environments do not inherently promote alpha-synuclein oligomerization and, more generally, that intrinsic structural disorder is sustainable in mammalian cells.

5-Aryl-2-(naphtha-1-yl)sulfonamido-thiazol-4(5H)-ones as clathrin inhibitors
Robertson(*), M. J., Horatscheck, A., Sauer, S., von Kleist(*), L., Baker, J. R., Stahlschmidt, W., Nazare, M., Whiting(*), A., Chau(*), N., Robinson(*), P. J., Haucke, V.; McCluskey(*), A.
Org Biomol Chem, 14:11266-11278
(2016)

Tags: Molecular Pharmacology and Cell Biology (Haucke), Medicinal Chemistry (Nazare)

Abstract: The development of a (Z)-5-((6,8-dichloro-4-oxo-4H-chromen-3-yl)methylene)-2-thioxothiazolidin-4-one (2), rhodanine-based lead that led to the Pitstop(R) 2 family of clathrin inhibitors is described herein. Head group substitution and bioisosteric replacement of the rhodanine core with a 2-aminothiazol-4(5H)-one scaffold eliminated off target dynamin activity. A series of N-substituents gave first phenylglycine (20, IC50 approximately 20 muM) then phenyl (25, IC50 approximately 7.1 muM) and 1-napthyl sulfonamide (26, Pitstop(R) 2 compound, IC50 approximately 1.9 muM) analogues with good activity, validating this approach. A final library exploring the head group resulted in three analogues displaying either slight improvements or comparable activity (33, 38, and 29 with IC50 approximately 1.4, 1.6 and 1.8 muM respectively) and nine others with IC50 < 10 muM. These results were rationalized using in silico docking studies. Docking studies predicted enhanced Pitstop(R) 2 family binding, not a loss of binding, within the Pistop(R) groove of the reported clathrin mutant invalidating recent assumptions of poor selectivity for this family of clathrin inhibitors.

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