FMP Publications

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

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References
Untangling a Repetitive Amyloid Sequence: Correlating Biofilm-Derived and Segmentally Labeled Curli Fimbriae by Solid-State NMR Spectroscopy
Schubeis(*), T., Yuan(*), P., Ahmed(*), M., Nagaraj, M., van Rossum, B. J.; Ritter(*), C.
Angew Chem Int Ed Engl, 54:14669-14672
(2015)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Curli are functional bacterial amyloids produced by an intricate biogenesis machinery. Insights into their folding and regulation can advance our understanding of amyloidogenesis. However, gaining detailed structural information of amyloids, and their tendency for structural polymorphisms, remains challenging. Herein we compare high-quality solid-state NMR spectra from biofilm-derived and recombinantly produced curli and provide evidence that they adopt a similar, well-defined beta-solenoid arrangement. Curli subunits consist of five sequence repeats, resulting in severe spectral overlap. Using segmental isotope labeling, we obtained the unambiguous sequence-specific resonance assignments and secondary structure of one repeat, and demonstrate that all repeats are most likely structurally equivalent.

Solid-state NMR Study of the YadA Membrane-Anchor Domain in the Bacterial Outer Membrane
Shahid, S. A., Nagaraj, M., Chauhan(*), N., Franks, T. W., Bardiaux(*), B., Habeck(*), M., Orwick-Rydmark(*), M., Linke(*), D.; van Rossum, B. J.
Angew Chem Int Ed Engl, 54:12602-12606
(2015)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: MAS-NMR was used to study the structure and dynamics at ambient temperatures of the membrane-anchor domain of YadA (YadA-M) in a pellet of the outer membrane of E. coli in which it was expressed. YadA is an adhesin from the pathogen Yersinia enterocolitica that is involved in interactions with the host cell, and it is a model protein for studying the autotransport process. Existing assignments were sucessfully transferred to a large part of the YadA-M protein in the E. coli lipid environment by using (13) C-(13) C DARR and PDSD spectra at different mixing times. The chemical shifts in most regions of YadA-M are unchanged relative to those in microcrystalline YadA-M preparations from which a structure has previously been solved, including the ASSA region that is proposed to be involved in transition-state hairpin formation for transport of the soluble domain. Comparisons of the dynamics between the microcrystalline and membrane-embedded samples indicate greater flexibility of the ASSA region in the outer-membrane preparation at physiological temperatures. This study will pave the way towards MAS-NMR structure determination of membrane proteins, and a better understanding of functionally important dynamic residues in native membrane environments.

Selective inhibitors of the protein tyrosine phosphatase SHP2 block cellular motility and growth of cancer cells in vitro and in vivo
Grosskopf, S., Eckert, C., Arkona(*), C., Radetzki, S., Böhm(*), K., Heinemann(*), U., Wolber(*), G., von Kries, J. P., Birchmeier(*), W.; Rademann(*), J.
Chemmedchem, 10:815-826
(2015)

Tags: Medicinal Chemistry (Rademann), Screening Unit (von Kries)

Abstract: Selective inhibitors of the protein tyrosine phosphatase SHP2 (src homology region 2 domain phosphatase; PTPN11), an enzyme that is deregulated in numerous human tumors, were generated through a combination of chemical synthesis and structure-based rational design. Seventy pyridazolon-4-ylidenehydrazinyl benzenesulfonates were prepared and evaluated in enzyme assays. The binding modes of active inhibitors were simulated in silico using a newly generated crystal structure of SHP2. The most powerful compound, GS-493 (4-(2Z)-2-[1,3-bis(4-nitrophenyl)-5-oxo-1,5-dihydro-4H-pyrazol-4-yliden]hydrazin obenzenesulfonic acid; 25) inhibited SHP2 with an IC50 value of 71+/-15 nM in the enzyme assay and was 29- and 45-fold more active toward SHP2 than against related SHP1 and PTP1B. In cell culture experiments compound 25 was found to block hepatocyte growth factor (HGF)-stimulated epithelial-mesenchymal transition of human pancreatic adenocarcinoma (HPAF) cells, as indicated by a decrease in the minimum neighbor distances of cells. Moreover, 25 inhibited cell colony formation in the non-small-cell lung cancer cell line LXFA 526L in soft agar. Finally, 25 was observed to inhibit tumor growth in a murine xenograft model. Therefore, the novel specific compound 25 strengthens the hypothesis that SHP2 is a relevant protein target for the inhibition of mobility and invasiveness of cancer cells.

Exploring monovalent and multivalent peptides for the inhibition of FBP21-tWW
Henning(*), L. M., Bhatia(*), S., Bertazzon(*), M., Marczynke(*), M., Seitz(*), O., Volkmer, R., Haag(*), R.; Freund(*), C.
Beilstein J Org Chem, 11:701-706
(2015)

Tags: Peptide Synthesis (Hackenberger/Volkmer)

Abstract: The coupling of peptides to polyglycerol carriers represents an important route towards the multivalent display of protein ligands. In particular, the inhibition of low affinity intracellular protein-protein interactions can be addressed by this design. We have applied this strategy to develop binding partners for FBP21, a protein which is important for the splicing of pre-mRNA in the nucleus of eukaryotic cells. Firstly, by using phage display the optimized sequence WPPPPRVPR was derived which binds with K Ds of 80 muM and 150 microM to the individual WW domains and with a K D of 150 muM to the tandem-WW1-WW2 construct. Secondly, this sequence was coupled to a hyperbranched polyglycerol (hPG) that allowed for the multivalent display on the surface of the dendritic polymer. This novel multifunctional hPG-peptide conjugate displayed a K D of 17.6 microM which demonstrates that the new carrier provides a venue for the future inhibition of proline-rich sequence recognition by FBP21 during assembly of the spliceosome.

A modular toolkit to inhibit proline-rich motif-mediated protein-protein interactions
Opitz, R., Müller, M., Reuter, C., Barone, M., Soicke(*), A., Roske(*), Y., Piotukh, K., Huy(*), P., Beerbaum, M., Wiesner, B., Beyermann, M., Schmieder, P., Freund(*), C., Volkmer, R., Oschkinat, H., Schmalz(*), H. G.; Kühne, R.
Proc Natl Acad Sci U S A, 112:5011-5016
(2015)

Tags: Computational Chemistry and Protein Design (Kühne), NMR-Supported Structural Biology (Oschkinat), Peptide Chemistry (Hackenberger/ Volkmer), Solution NMR (Schmieder), Peptide Chemistry (Beyermann), Cellular Imaging (Wiesner)

Abstract: Small-molecule competitors of protein-protein interactions are urgently needed for functional analysis of large-scale genomics and proteomics data. Particularly abundant, yet so far undruggable, targets include domains specialized in recognizing proline-rich segments, including Src-homology 3 (SH3), WW, GYF, and Drosophila enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Here, we present a modular strategy to obtain an extendable toolkit of chemical fragments (ProMs) designed to replace pairs of conserved prolines in recognition motifs. As proof-of-principle, we developed a small, selective, peptidomimetic inhibitor of Ena/VASP EVH1 domain interactions. Highly invasive MDA MB 231 breast-cancer cells treated with this ligand showed displacement of VASP from focal adhesions, as well as from the front of lamellipodia, and strongly reduced cell invasion. General applicability of our strategy is illustrated by the design of an ErbB4-derived ligand containing two ProM-1 fragments, targeting the yes-associated protein 1 (YAP1)-WW domain with a fivefold higher affinity.

The membrane remodeling protein Pex11p activates the GTPase Dnm1p during peroxisomal fission
Williams(*), C., Opalinski(*), L., Landgraf(*), C., Costello(*), J., Schrader(*), M., Krikken(*), A. M., Knoops(*), K., Kram(*), A. M., Volkmer, R.; van der Klei(*), I. J.
Proc Natl Acad Sci U S A, 112:6377-6382
(2015)

Tags: Peptide Synthesis (Hackenberger/Volkmer)

Abstract: The initial phase of peroxisomal fission requires the peroxisomal membrane protein Peroxin 11 (Pex11p), which remodels the membrane, resulting in organelle elongation. Here, we identify an additional function for Pex11p, demonstrating that Pex11p also plays a crucial role in the final step of peroxisomal fission: dynamin-like protein (DLP)-mediated membrane scission. First, we demonstrate that yeast Pex11p is necessary for the function of the GTPase Dynamin-related 1 (Dnm1p) in vivo. In addition, our data indicate that Pex11p physically interacts with Dnm1p and that inhibiting this interaction compromises peroxisomal fission. Finally, we demonstrate that Pex11p functions as a GTPase activating protein (GAP) for Dnm1p in vitro. Similar observations were made for mammalian Pex11beta and the corresponding DLP Drp1, indicating that DLP activation by Pex11p is conserved. Our work identifies a previously unknown requirement for a GAP in DLP function.

Alterations in creatine metabolism observed in experimental autoimmune myocarditis using ex vivo proton magic angle spinning MRS
Muench(*), F., Retel, J., Jeuthe(*), S., (*)D, O. h.-I., van Rossum, B., Wassilew(*), K., Schmerler(*), P., Kuehne(*), T., Berger(*), F., Oschkinat, H.; Messroghli(*), D. R.
Nmr Biomed, 28:1625-1633
(2015)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Experimental autoimmune myocarditis (EAM) in rodents is an accepted model of myocarditis and dilated cardiomyopathy (DCM). Altered metabolism is thought to play an important role in the pathogenesis of DCM and heart failure (HF). Study of the metabolism may provide new diagnostic information and insights into the mechanisms of myocarditis and HF. Proton MRS ((1)H-MRS) has not yet been used to study the changes occurring in myocarditis and subsequent HF. We aimed to explore the changes in creatine metabolism using this model and compare them with the findings in healthy animals. Myocardial function of male young Lewis rats with EAM was quantified by performing left ventricular ejection fraction (LVEF) analysis in short-axis cine images throughout the whole heart. Inflammatory cellular infiltrate was assessed by immunohistochemistry. Myocardial tissue was analyzed using ex vivo proton magic angle spinning MRS ((1)H-MAS-MRS). Myocarditis was confirmed histologically by the presence of an inflammatory cellular infiltrate and CD68 positive staining. A significant increase in the metabolic ratio of Tau/tCr (taurine/total creatine) obtained by (1)H-MAS-MRS was observed in myocarditis compared with healthy controls (21 d acute EAM, 4.38 (+/-0.23); 21 d control, 2.84 (+/-0.08); 35 d chronic EAM, 4.47 (+/-0.83); 35 d control, 2.59 (+/-0.38); P < 0.001). LVEF was reduced in diseased animals (EAM, 55.2% (+/-11.3%); control, 72.6% (+/-3.8%); P < 0.01) and correlated with Tau/tCr ratio (R = 0.937, P < 0.001). Metabolic alterations occur acutely with the development of myocarditis. Myocardial Tau/tCr ratio as detected by (1)H-MRS correlates with LVEF and is able to differentiate between healthy myocardium and myocardium from rats with EAM.

Theoretical aspects of Magic Angle Spinning - Dynamic Nuclear Polarization
Mentink-Vigier(*), F., Akbey, Ü., Oschkinat, H., Vega(*), S.; Feintuch(*), A.
J Magn Reson, 258:102-120
(2015)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Magic Angle Spinning (MAS) combined with Dynamic Nuclear Polarization (DNP) has been proven in recent years to be a very powerful method for increasing solid-state NMR signals. Since the advent of biradicals such as TOTAPOL to increase the nuclear polarization new classes of radicals, with larger molecular weight and/or different spin properties have been developed. These have led to unprecedented signal gain, with varying results for different experimental parameters, in particular the microwave irradiation strength, the static field, and the spinning frequency. Recently it has been demonstrated that sample spinning imposes DNP enhancement processes that differ from the active DNP mechanism in static samples as upon sample spinning the DNP enhancements are the results of energy level anticrossings occurring periodically during each rotor cycle. In this work we present experimental results with regards to the MAS frequency dependence of the DNP enhancement profiles of four nitroxide-based radicals at two different sets of temperature, 110 and 160K. In fact, different magnitudes of reduction in enhancement are observed with increasing spinning frequency. Our simulation code for calculating MAS-DNP powder enhancements of small model spin systems has been improved to extend our studies of the influence of the interaction and relaxation parameters on powder enhancements. To achieve a better understanding we simulated the spin dynamics of a single three-spin system ea-eb-n during its steady state rotor periods and used the Landau-Zener formula to characterize the influence of the different anti-crossings on the polarizations of the system and their necessary action for reaching steady state conditions together with spin relaxation processes. Based on these model calculations we demonstrate that the maximum steady state nuclear polarization cannot become larger than the maximum polarization difference between the two electrons during the steady state rotor cycle. This study also shows the complexity of the MAS-DNP process and therefore the necessity to rely on numerical simulations for understanding parametric dependencies of the enhancements. Finally an extension of the spin system up to five spins allowed us to probe the first steps of the transfer of polarization from the nuclei coupled to the electrons to further away nuclei, demonstrating a decrease in the spin-diffusion barrier under MAS conditions.

Copper binding to the N-terminally acetylated, naturally occurring form of alpha-synuclein induces local helical folding
Miotto(*), M. C., Valiente-Gabioud(*), A. A., Rossetti(*), G., Zweckstetter(*), M., Carloni(*), P., Selenko, P., Griesinger(*), C., Binolfi, A.; Fernandez(*), C. O.
J Am Chem Soc, 137:6444-6447
(2015)

Tags: In-Cell NMR (Selenko)

Abstract: Growing evidence supports a link between brain copper homeostasis, the formation of alpha-synuclein (AS)-copper complexes, and the development of Parkinson disease (PD). Recently it was demonstrated that the physiological form of AS is N-terminally acetylated (AcAS). Here we used NMR spectroscopy to structurally characterize the interaction between Cu(I) and AcAS. We found that the formation of an AcAS-Cu(I) complex at the N-terminal region stabilizes local conformations with alpha-helical secondary structure and restricted motility. Our work provides new evidence into the metallo-biology of PD and opens new lines of research as the formation of AcAS-Cu(I) complex might impact on AcAS membrane binding and aggregation.

Phenothiazine-derived antipsychotic drugs inhibit dynamin and clathrin-mediated endocytosis
Daniel(*), J. A., Chau(*), N., Abdel-Hamid(*), M. K., Hu(*), L., von Kleist, L., Whiting(*), A., Krishnan(*), S., Maamary(*), P., Joseph(*), S. R., Simpson(*), F., Haucke, V., McCluskey(*), A.; Robinson(*), P. J.
Traffic, 16:635-654
(2015)

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Chlorpromazine is a phenothiazine-derived antipsychotic drug (APD) that inhibits clathrin-mediated endocytosis (CME) in cells by an unknown mechanism. We examined whether its action and that of other APDs might be mediated by the GTPase activity of dynamin. Eight of eight phenothiazine-derived APDs inhibited dynamin I (dynI) in the 2-12 microm range, the most potent being trifluoperazine (IC50 2.6 +/- 0.7 microm). They also inhibited dynamin II (dynII) at similar concentrations. Typical and atypical APDs not based on the phenothiazine scaffold were 8- to 10-fold less potent (haloperidol and clozapine) or were inactive (droperidol, olanzapine and risperidone). Kinetic analysis showed that phenothiazine-derived APDs were lipid competitive, while haloperidol was uncompetitive with lipid. Accordingly, phenothiazine-derived APDs inhibited dynI GTPase activity stimulated by lipids but not by various SH3 domains. All dynamin-active APDs also inhibited transferrin (Tfn) CME in cells at related potencies. Structure-activity relationships (SAR) revealed dynamin inhibition to be conferred by a substituent group containing a terminal tertiary amino group at the N2 position. Chlorpromazine was previously proposed to target AP-2 recruitment in the formation of clathrin-coated vesicles (CCV). However, neither chlorpromazine nor thioridazine affected AP-2 interaction with amphiphysin or clathrin. Super-resolution microscopy revealed that chlorpromazine blocks neither clathrin recruitment by AP-2, nor AP-2 recruitment, showing that CME inhibition occurs downstream of CCV formation. Overall, potent dynamin inhibition is a shared characteristic of phenothiazine-derived APDs, but not other typical or atypical APDs, and the data indicate that dynamin is their likely in-cell target in endocytosis.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
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13125 Berlin, Germany
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info(at)fmp-berlin.de

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