FMP Publications

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

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References
A Self-Assembled Oligopeptide as a Versatile NMR Alignment Medium for the Measurement of Residual Dipolar Couplings in Methanol
Lei(*), X. X., Qiu(*), F., Sun, H., Bai(*), L. W., Wang(*), W. X., Xiang(*), W. S.; Xiao(*), H. P.
Angew Chem Int Edit, 56:12857-12861
(2017)

Tags: Computational Chemistry and Protein Design (Kühne)

Abstract: Residual dipolar coupling (RDC) is a powerful structural parameter for the determination of the constitution, conformation, and configuration of organic molecules. Herein, we report the first liquid crystal-based orienting medium that is compatible with MeOH, thus enabling RDC acquisitions of a wide range of intermediate to polar organic molecules. The liquid crystals were produced from self-assembled oligopeptide nanotubes (AAKLVFF), which are stable at very low concentrations. The presented alignment medium is highly homogeneous, and the size of RDCs can be scaled with the concentration of the peptide. To assess the accuracy of the RDC measurement by employing this new medium, seven bioactive natural products from different classes were chosen and analyzed. The straightforward preparation of the anisotropic alignment sample will offer a versatile and robust protocol for the routine RDC measurement of natural products.

Subtype-specific block of voltage-gated K+ channels by mu-conopeptides
Leipold(*), E., Ullrich, F., Thiele(*), M., Tietze(*), A. A., Terlau(*), H., Imhof(*), D.; Heinemann(*), S. H.
Biochem. Biophys. Res. Commun., 482:1135-1140
(2017)

Tags: Physiology and Pathophysiology of Ion Transport (Jentsch)

Abstract: The neurotoxic cone snail peptide mu-GIIIA specifically blocks skeletal muscle voltage-gated sodium (Na(V)1.4) channels. The related conopeptides mu-PIIIA and mu-SIIIA, however, exhibit a wider activity spectrum by also inhibiting the neuronal Na-V channels Na-V 1.2 and Na-V 1.7. Here we demonstrate that those mu-conopeptides with a broader target range also antagonize select subtypes of voltage-gated potassium channels of the K(v)1 family: mu-PIIIA and mu-SIIIA inhibited K(V)1.1 and K(V)1.6 channels in the nanomolar range, while being inactive on subtypes K(V)1.2-1.5 and K(V)2.1. Construction and electro-physiological evaluation of chimeras between K(V)1.5 and K(V)1.6 revealed that these toxins block K-V channels involving their pore regions; the subtype specificity is determined in part by the sequence close to the selectivity filter but predominantly by the so-called turret domain, i.e. the extracellular loop connecting the pore with transmembrane segment S5. Conopeptides mu-SIIIA and mu-PIIIA thus, are not specific for Na-V channels, and the known structure of some K-V channel subtypes may provide access to structural insight into the molecular interaction between-conopeptides and their target channels. (C) 2016 Elsevier Inc. All rights reserved.

Evidence for Heterodimerization and Functional Interaction of the Angiotensin Type 2 Receptor and the Receptor MAS
Leonhardt(*), J., Villela(*), D. C., Teichmann, A., Munter(*), L. M., Mayer(*), M. C., Mardahl(*), M., Kirsch(*), S., Namsolleck(*), P., Lucht(*), K., Benz(*), V., Alenina(*), N., Daniell(*), N., Horiuchi(*), M., Iwai(*), M., Multhaup(*), G., Schülein, R., Bader(*), M., Santos(*), R. A., Unger(*), T.; Steckelings(*), U. M.
Hypertension,
(2017)

Tags: Protein Trafficking (Schülein), Cellular Imaging (Wiesner)

Abstract: The angiotensin type 2 receptor (AT2R) and the receptor MAS are receptors of the protective arm of the renin-angiotensin system. They mediate strikingly similar actions. Moreover, in various studies, AT2R antagonists blocked the effects of MAS agonists and vice versa. Such cross-inhibition may indicate heterodimerization of these receptors. Therefore, this study investigated the molecular and functional interplay between MAS and the AT2R. Molecular interactions were assessed by fluorescence resonance energy transfer and by cross correlation spectroscopy in human embryonic kidney-293 cells transfected with vectors encoding fluorophore-tagged MAS or AT2R. Functional interaction of AT2R and MAS was studied in astrocytes with CX3C chemokine receptor-1 messenger RNA expression as readout. Coexpression of fluorophore-tagged AT2R and MAS resulted in a fluorescence resonance energy transfer efficiency of 10.8 +/- 0.8%, indicating that AT2R and MAS are capable to form heterodimers. Heterodimerization was verified by competition experiments using untagged AT2R and MAS. Specificity of dimerization of AT2R and MAS was supported by lack of dimerization with the transient receptor potential cation channel, subfamily C-member 6. Dimerization of the AT2R was abolished when it was mutated at cysteine residue 35. AT2R and MAS stimulation with the respective agonists, Compound 21 or angiotensin-(1-7), significantly induced CX3C chemokine receptor-1 messenger RNA expression. Effects of each agonist were blocked by an AT2R antagonist (PD123319) and also by a MAS antagonist (A-779). Knockout of a single of these receptors made astrocytes unresponsive for both agonists. Our results suggest that MAS and the AT2R form heterodimers and that-at least in astrocytes-both receptors functionally depend on each other.

Helical Polyisocyanopeptides as Lyotropic Liquid Crystals for Measuring Residual Dipolar Couplings
Li(*), G. W., Cao(*), J. M., Zong(*), W., Hu(*), L., Hu(*), M. L., Lei(*), X., Sun, H.; Tan(*), R. X.
Chemistry, 23:7653-7656
(2017)

Tags: Computational Chemistry and Protein Design (Kühne)

Abstract: Residual dipolar couplings (RDC) emerged to be an important structural parameter for organic and biomolecules. Herein, a new helical polyisocyanopeptide (l,l-PIAF-OBn) that forms lyotropic liquid crystals (LLC) in CDCl3 is proposed as a novel weakly orienting medium for acquiring residual dipolar couplings (RDCs) of organic molecules. We demonstrate its application for the structural elucidation of strychnine and triptolide.

Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones
Liokatis, S.
J Vis Exp,
(2017)

Tags: In-Cell NMR (Selenko)

Abstract: Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications (PTMs). They are newly identified species with unknown means of establishment and functional implications. Current analytical methods are inadequate to detect the copy-specific occurrence of PTMs on the nucleosomal sister histones. This protocol presents a biochemical method for the in vitro reconstitution of nucleosomes containing differentially isotope-labeled sister histones. The generated complex can be also asymmetrically modified, after including a premodified histone pool during refolding of histone subcomplexes. These asymmetric nucleosome preparations can be readily reacted with histone-modifying enzymes to study modification cross-talk mechanisms imposed by the asymmetrically pre-incorporated PTM using nuclear magnetic resonance (NMR) spectroscopy. Particularly, the modification reactions in real-time can be mapped independently on the two sister histones by performing different types of NMR correlation experiments, tailored for the respective isotope type. This methodology provides the means to study crosstalk mechanisms that contribute to the formation and propagation of asymmetric PTM patterns on nucleosomal complexes.

Optimized fragmentation schemes and data analysis strategies for proteome-wide cross-link identification
Liu, F., Lössl(*), P., Scheltema(*), R., Viner(*), R.; Heck(*), A. J. R.
Nat Commun, 8:15473
(2017)

Tags: Mass Spectrometry (Krause, E.)

Abstract: We describe optimized fragmentation schemes and data analysis strategies substantially enhancing the depth and accuracy in identifying protein cross-links using non-restricted whole proteome databases. These include a novel hybrid data acquisition strategy to sequence cross-links at both MS2 and MS3 level and a new algorithmic design XlinkX v2.0 for data analysis. As proof-of-concept we investigated proteome-wide protein interactions in E. coli and HeLa cell lysates, respectively, identifying 1,158 and 3,301 unique cross-links at approximately 1% false discovery rate. These protein interaction repositories provide meaningful structural information on many endogenous macromolecular assemblies, as we showcase on several protein complexes involved in translation, protein folding and carbohydrate metabolism.

Facilitating identification of minimal protein binding domains by cross-linking mass spectrometry
Liu(*), Q., Remmelzwaal(*), S., Heck(*), A. J. R., Akhmanova(*), A.; Liu, F.
Sci Rep, 7:13453
(2017)

Tags: Mass Spectrometry (Krause, E.)

Abstract: Characterization of protein interaction domains is crucial for understanding protein functions. Here we combine cross-linking mass spectrometry (XL-MS) with deletion analysis to accurately locate minimal protein interaction domains. As a proof of concept, we investigated in detail the binding interfaces of two protein assemblies: the complex formed by MICAL3, ELKS and Rab8A, which is involved in exocytosis, and the complex of SLAIN2, CLASP2 and ch-TOG, which controls microtubule dynamics. We found that XL-MS provides valuable information to efficiently guide the design of protein fragments that are essential for protein interaction. However, we also observed a number of cross-links between polypeptide regions that were dispensable for complex formation, especially among intrinsically disordered sequences. Collectively, our results indicate that XL-MS, which renders distance restrains of linked residue pairs, accelerates the characterization of protein binding regions in combination with other biochemical approaches.

Human iPSC-Derived Neural Progenitors Are an Effective Drug Discovery Model for Neurological mtDNA Disorders
Lorenz(*), C., Lesimple(*), P., Bukowiecki(*), R., Zink(*), A., Inak(*), G., Mlody(*), B., Singh(*), M., Semtner(*), M., Mah(*), N., Aure(*), K., Leong(*), M., Zabiegalov(*), O., Lyras(*), E. M., Pfiffer(*), V., Fauler(*), B., Eichhorst, J., Wiesner, B., Huebner(*), N., Priller(*), J., Mielke(*), T., Meierhofer(*), D., Izsvak(*), Z., Meier(*), J. C., Bouillaud(*), F., Adjaye(*), J., Schuelke(*), M., Wanker(*), E. E., Lombes(*), A.; Prigione(*), A.
Cell stem cell,
(2017)

Tags: Cellular Imaging (Wiesner)

Abstract: Mitochondrial DNA (mtDNA) mutations frequently cause neurological diseases. Modeling of these defects has been difficult because of the challenges associated with engineering mtDNA. We show here that neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) retain the parental mtDNA profile and exhibit a metabolic switch toward oxidative phosphorylation. NPCs derived in this way from patients carrying a deleterious homoplasmic mutation in the mitochondrial gene MT-ATP6 (m.9185T>C) showed defective ATP production and abnormally high mitochondrial membrane potential (MMP), plus altered calcium homeostasis, which represents a potential cause of neural impairment. High-content screening of FDA-approved drugs using the MMP phenotype highlighted avanafil, which we found was able to partially rescue the calcium defect in patient NPCs and differentiated neurons. Overall, our results show that iPSC-derived NPCs provide an effective model for drug screening to target mtDNA disorders that affect the nervous system.

Selective transport of neurotransmitters and modulators by distinct volume-regulated LRRC8 anion channels
Lutter, D., Ullrich, F., Lueck, J. C., Kempa(*), S.; Jentsch, T. J.
J Cell Sci, 130:1122-1133
(2017)

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: In response to swelling, mammalian cells release chloride and organic osmolytes through volume-regulated anion channels (VRACs). VRACs are heteromers of LRRC8A and other LRRC8 isoforms (LRRC8B to LRRC8E), which are co-expressed in HEK293 and most other cells. The spectrum of VRAC substrates and its dependence on particular LRRC8 isoforms remains largely unknown. We show that, besides the osmolytes taurine and myo-inositol, LRRC8 channels transport the neurotransmitters glutamate, aspartate and gamma-aminobutyric acid (GABA) and the co-activator D-serine. HEK293 cells engineered to express defined subsets of LRRC8 isoforms were used to elucidate the subunit-dependence of transport. Whereas LRRC8D was crucial for the translocation of overall neutral compounds like myo-inositol, taurine and GABA, and sustained the transport of positively charged lysine, flux of negatively charged aspartate was equally well supported by LRRC8E. Disruption of LRRC8B or LRRC8C failed to decrease the transport rates of all investigated substrates, but their inclusion into LRRC8 heteromers influenced the substrate preference of VRAC. This suggested that individual VRACs can contain three or more different LRRC8 subunits, a conclusion confirmed by sequential co-immunoprecipitations. Our work suggests a composition-dependent role of VRACs in extracellular signal transduction.

Deciphering the Multisite Interactions of a Protein and Its Ligand at Atomic Resolution by Using Sensitive Paramagnetic Effects
Ma(*), F. H., Wang(*), X., Chen(*), J. L., Wen(*), X., Sun, H.; Su(*), X. C.
Chemistry, 23:926-934
(2017)

Tags: Computational Chemistry and Protein Design (Kühne)

Abstract: Quantitative analysis of multisite interactions between a protein and its binding partner at atomic resolution is complicated because locating the binding sites is difficult and differentiating the flexibility of each binding site is even more elusive. Introduction of a paramagnetic metal center close to the binding pocket greatly attenuates the signals in the NMR spectrum upon binding. Herein, the multisite binding of hen egg white lysozyme (HEWL) with lanthanide complexes [Ln(DPA)3 ]3- (DPA=dipicolinic acid) was analyzed with sensitive paramagnetic NMR spectroscopy. Paramagnetic relaxation enhancement (PRE) revealed that HEWL interacts with [Ln(DPA)3 ]3- at four major binding sites in aqueous solution, which is in contrast to a previous X-ray structural analysis. The varied binding affinities for the ligands and different flexibilities at each binding site were in good agreement with atomistic molecular dynamics (MD) simulations. The present work demonstrates that a combination of paramagnetic NMR spectroscopy and MD simulations is a powerful tool to delineate the multisite interactions of a protein with its binding partner at atomic resolution, in terms of both affinity and flexibility.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
Robert-Roessle-Str. 10
13125 Berlin, Germany
+4930 94793 - 100 
+4930 94793 - 109 (Fax)
info(at)fmp-berlin.de

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