FMP Publications

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

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Direct assessment of substrate binding to the Neurotransmitter:Sodium Symporter LeuT by solid state NMR
Erlendsson(*), S., Gotfryd(*), K., Larsen, F. H., Mortensen(*), J. S., Geiger, M. A., van Rossum, B. J., Oschkinat, H., Gether(*), U., Teilum(*), K.; Loland(*), C. J.
Elife, 6

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: The Neurotransmitter:Sodium Symporters (NSSs) represent an important class of proteins mediating sodium-dependent uptake of neurotransmitters from the extracellular space. The substrate binding stoichiometry of the bacterial NSS protein, LeuT, and thus the principal transport mechanism, has been heavily debated. Here we used solid state NMR to specifically characterize the bound leucine ligand and probe the number of binding sites in LeuT. We were able to produce high-quality NMR spectra of substrate bound to microcrystalline LeuT samples and identify one set of sodium-dependent substrate-specific chemical shifts. Furthermore, our data show that the binding site mutants F253A and L400S, which probe the major S1 binding site and the proposed S2 binding site, respectively, retain sodium-dependent substrate binding in the S1 site similar to the wild-type protein. We conclude that under our experimental conditions there is only one detectable leucine molecule bound to LeuT.

Design of S-Allylcysteine in Situ Production and Incorporation Based on a Novel Pyrrolysyl-tRNA Synthetase Variant
Exner(*), M. P., Kuenzl(*), T., To(*), T. M., Ouyang(*), Z., Schwagerus, S., Hoesl(*), M. G., Hackenberger, C. P., Lensen(*), M. C., Panke(*), S.; Budisa(*), N.
Chembiochem, 18:85-90

Tags: Chemical Biology II (Hackenberger)

Abstract: The noncanonical amino acid S-allyl cysteine (Sac) is one of the major compounds of garlic extract and exhibits a range of biological activities. It is also a small bioorthogonal alkene tag capable of undergoing controlled chemical modifications, such as photoinduced thiol-ene coupling or Pd-mediated deprotection. Its small size guarantees minimal interference with protein structure and function. Here, we report a simple protocol efficiently to couple in-situ semisynthetic biosynthesis of Sac and its incorporation into proteins in response to amber (UAG) stop codons. We exploited the exceptional malleability of pyrrolysyl-tRNA synthetase (PylRS) and evolved an S-allylcysteinyl-tRNA synthetase (SacRS) capable of specifically accepting the small, polar amino acid instead of its long and bulky aliphatic natural substrate. We succeeded in generating a novel and inexpensive strategy for the incorporation of a functionally versatile amino acid. This will help in the conversion of orthogonal translation from a standard technique in academic research to industrial biotechnology.

A Chemical Disruptor of the ClpX Chaperone Complex Attenuates Multiresistant Staphylococcus aureus Virulence
Fetzer(*), C., Korotkov(*), V. S., Thanert(*), R., Lee(*), K. M., Neuenschwander, M., von Kries, J. P., Medina(*), E.; Sieber(*), S. A.
Angew Chem Int Ed Engl,

Tags: Screening Unit (von Kries)

Abstract: The Staphylococcus aureus ClpXP protease is an important regulator of cell homeostasis and virulence. Here we utilize a high-throughput screen against the ClpXP complex and identify a specific inhibitor of the ClpX chaperone that disrupts its oligomeric state. Synthesis of 34 derivatives revealed that the molecular scaffold is restrictive for diversification with only minor changes tolerated. Subsequent analysis of the most active compound revealed strong attenuation of S. aureus toxin production which was quantified via a customized MS-based assay platform. Transcriptome and whole proteome studies further confirmed the global reduction of virulence and unraveled characteristic signatures of protein expression in compound treated cells. Although these partially matched the pattern of ClpX knockout cells, further depletion of toxins was observed leading to the intriguing perspective that additional virulence pathways may be directly or indirectly addressed by the small molecule.

Complete NMR assignment and conformational analysis of 17-alpha-ethinylestradiol by using RDCs obtained in grafted graphene oxide
Franca(*), J. A., Navarro-Vazquez(*), A., Lei(*), X., Sun, H., Griesinger(*), C.; Hallwass(*), F.
Magn Reson Chem, 55:297-303

Tags: Computational Chemistry and Protein Design (Kühne)

Abstract: The 1 H and 13 C NMR spectra of 17-alpha-ethinylestradiol (EE2), a well-known contraceptive, including diastereotopic methylene groups, were fully assigned with the help of residual dipolar couplings (RDC) measured in the recently developed grafted graphene oxide orienting medium. RDC analysis, which included all 1 DCH couplings and the long-range 2 DCH1 H-C identical with13 C coupling, also pointed to the presence of a minor conformation arising from pseudo-rotation of the steroid B ring. Saturation-transfer difference (STD) measurements revealed that the most likely interaction between EE2 and orienting medium occurred on the C and D ring. Copyright (c) 2016 John Wiley & Sons, Ltd.

Backbone assignment of perdeuterated proteins by solid-state NMR using proton detection and ultrafast magic-angle spinning
Fricke, P., Chevelkov, V., Zinke, M., Giller(*), K., Becker(*), S.; Lange, A.
Nat Protoc, 12:764-782

Tags: Molecular Biophysics (Lange, A.)

Abstract: Solid-state NMR (ssNMR) is a technique that allows the study of protein structure and dynamics at atomic detail. In contrast to X-ray crystallography and cryo-electron microscopy, proteins can be studied under physiological conditions-for example, in a lipid bilayer and at room temperature (0-35 degrees C). However, ssNMR requires considerable amounts (milligram quantities) of isotopically labeled samples. In recent years, 1H-detection of perdeuterated protein samples has been proposed as a method of alleviating the sensitivity issue. Such methods are, however, substantially more demanding to the spectroscopist, as compared with traditional 13C-detected approaches. As a guide, this protocol describes a procedure for the chemical shift assignment of the backbone atoms of proteins in the solid state by 1H-detected ssNMR. It requires a perdeuterated, uniformly 13C- and 15N-labeled protein sample with subsequent proton back-exchange to the labile sites. The sample needs to be spun at a minimum of 40 kHz in the NMR spectrometer. With a minimal set of five 3D NMR spectra, the protein backbone and some of the side-chain atoms can be completely assigned. These spectra correlate resonances within one amino acid residue and between neighboring residues; taken together, these correlations allow for complete chemical shift assignment via a 'backbone walk'. This results in a backbone chemical shift table, which is the basis for further analysis of the protein structure and/or dynamics by ssNMR. Depending on the spectral quality and complexity of the protein, data acquisition and analysis are possible within 2 months.

Cross-over endocytosis of claudins is mediated by interactions via their extracellular loops
Gehne, N., Lamik, A., Lehmann, M., Haseloff, R. F., Andjelkovic(*), A. V.; Blasig, I. E.
Plos One, 12:e0182106

Tags: Molecular Cell Physiology (Blasig, I.E.), Cellular Imaging (Wiesner, Puchkov)

Abstract: Claudins (Cldns) are transmembrane tight junction (TJ) proteins that paracellularly seal endo- and epithelial barriers by their interactions within the TJs. However, the mechanisms allowing TJ remodeling while maintaining barrier integrity are largely unknown. Cldns and occludin are heterophilically and homophilically cross-over endocytosed into neighboring cells in large, double membrane vesicles. Super-resolution microscopy confirmed the presence of Cldns in these vesicles and revealed a distinct separation of Cldns derived from opposing cells within cross-over endocytosed vesicles. Colocalization of cross-over endocytosed Cldn with the autophagosome markers as well as inhibition of autophagosome biogenesis verified involvement of the autophagosomal pathway. Accordingly, cross-over endocytosed Cldns underwent lysosomal degradation as indicated by lysosome markers. Cross-over endocytosis of Cldn5 depended on clathrin and caveolin pathways but not on dynamin. Cross-over endocytosis also depended on Cldn-Cldn-interactions. Amino acid substitutions in the second extracellular loop of Cldn5 (F147A, Q156E) caused impaired cis- and trans-interaction, as well as diminished cross-over endocytosis. Moreover, F147A exhibited an increased mobility in the membrane, while Q156E was not as mobile but enhanced the paracellular permeability. In conclusion, the endocytosis of TJ proteins depends on their ability to interact strongly with each other in cis and trans, and the mobility of Cldns in the membrane is not necessarily an indicator of barrier permeability. TJ-remodeling via cross-over endocytosis represents a general mechanism for the degradation of transmembrane proteins in cell-cell contacts and directly links junctional membrane turnover to autophagy.

Structural determinants of a conserved enantiomer-selective carvone binding pocket in the human odorant receptor OR1A1
Geithe(*), C., Protze, J., Kreuchwig, F., Krause, G.; Krautwurst(*), D.
Cellular and molecular life sciences : CMLS,

Tags: Structural Bioinformatics and Protein Design (Krause, G.)

Abstract: Chirality is a common phenomenon within odorants. Most pairs of enantiomers show only moderate differences in odor quality. One example for enantiomers that are easily discriminated by their odor quality is the carvones: humans significantly distinguish between the spearmint-like (R)-(-)-carvone and caraway-like (S)-(+)-carvone enantiomers. Moreover, for the (R)-(-)-carvone, an anosmia is observed in about 8% of the population, suggesting enantioselective odorant receptors (ORs). With only about 15% de-orphaned human ORs, the lack of OR crystal structures, and few comprehensive studies combining in silico and experimental approaches to elucidate structure-function relations of ORs, knowledge on cognate odorant/OR interactions is still sparse. An adjusted homology modeling approach considering OR-specific proline-caused conformations, odorant docking studies, single-nucleotide polymorphism (SNP) analysis, site-directed mutagenesis, and subsequent functional studies with recombinant ORs in a cell-based, real-time luminescence assay revealed 11 amino acid positions to constitute an enantioselective binding pocket necessary for a carvone function in human OR1A1 and murine Olfr43, respectively. Here, we identified enantioselective molecular determinants in both ORs that discriminate between minty and caraway odor. Comparison with orthologs from 36 mammalian species demonstrated a hominid-specific carvone binding pocket with about 100% conservation. Moreover, we identified loss-of-function SNPs associated with the carvone binding pocket of OR1A1. Given carvone enantiomer-specific receptor activation patterns including OR1A1, our data suggest OR1A1 as a candidate receptor for constituting a carvone enantioselective phenotype, which may help to explain mechanisms underlying a (R)-(-)-carvone-specific anosmia in humans.

Intersectin associates with synapsin and regulates its nanoscale localization and function
Gerth(*), F., Jäpel, M., Pechstein, A., Kochlamazashvili, G., Lehmann, M., Puchkov, D., Onofri(*), F., Benfenati(*), F., Nikonenko(*), A. G., Maritzen, T., Freund(*), C.; Haucke, V.
Proc Natl Acad Sci U S A, 114:12057-12062

Tags: Molecular Pharmacology and Cell Biology (Haucke); Membrane Traffic and Cell Motility (Maritzen)

Abstract: Neurotransmission is mediated by the exocytic release of neurotransmitters from readily releasable synaptic vesicles (SVs) at the active zone. To sustain neurotransmission during periods of elevated activity, release-ready vesicles need to be replenished from the reserve pool of SVs. The SV-associated synapsins are crucial for maintaining this reserve pool and regulate the mobilization of reserve pool SVs. How replenishment of release-ready SVs from the reserve pool is regulated and which other factors cooperate with synapsins in this process is unknown. Here we identify the endocytic multidomain scaffold protein intersectin as an important regulator of SV replenishment at hippocampal synapses. We found that intersectin directly associates with synapsin I through its Src-homology 3 A domain, and this association is regulated by an intramolecular switch within intersectin 1. Deletion of intersectin 1/2 in mice alters the presynaptic nanoscale distribution of synapsin I and causes defects in sustained neurotransmission due to defective SV replenishment. These phenotypes were rescued by wild-type intersectin 1 but not by a locked mutant of intersectin 1. Our data reveal intersectin as an autoinhibited scaffold that serves as a molecular linker between the synapsin-dependent reserve pool and the presynaptic endocytosis machinery.

Structural Basis of the Oncogenic Interaction of Phosphatase PRL-1 with the Magnesium Transporter CNNM2
Gimenez-Mascarell(*), P., Oyenarte(*), I., Hardy(*), S., Breiderhoff(*), T., Stuiver, M., Kostantin(*), E., Diercks(*), T., Pey(*), A. L., Ereno-Orbea(*), J., Martinez-Chantar(*), M. L., Khalaf-Nazzal(*), R., Claverie-Martin(*), F., Müller(*), D., Tremblay(*), M. L.; Martinez-Cruz(*), L. A.
J Biol Chem, 292:786-801

Tags: In-Cell NMR (Selenko)

Abstract: Phosphatases of regenerating liver (PRLs), the most oncogenic of all protein-tyrosine phosphatases (PTPs), play a critical role in metastatic progression of cancers. Recent findings established a new paradigm by uncovering that their association with magnesium transporters of the cyclin M (CNNM) family causes a rise in intracellular magnesium levels that promote oncogenic transformation. Recently, however, essential roles for regulation of the circadian rhythm and reproduction of the CNNM family have been highlighted. Here, we describe the crystal structure of PRL-1 in complex with the Bateman module of CNNM2 (CNNM2BAT), which consists of two cystathionine beta-synthase (CBS) domains (IPR000664) and represents an intracellular regulatory module of the transporter. The structure reveals a heterotetrameric association, consisting of a disc-like homodimer of CNNM2BAT bound to two independent PRL-1 molecules, each one located at opposite tips of the disc. The structure highlights the key role played by Asp-558 at the extended loop of the CBS2 motif of CNNM2 in maintaining the association between the two proteins and proves that the interaction between CNNM2 and PRL-1 occurs via the catalytic domain of the phosphatase. Our data shed new light on the structural basis underlying the interaction between PRL phosphatases and CNNM transporters and provides a hypothesis about the molecular mechanism by which PRL-1, upon binding to CNNM2, might increase the intracellular concentration of Mg2+ thereby contributing to tumor progression and metastasis. The availability of this structure sets the basis for the rational design of compounds modulating PRL-1 and CNNM2 activities.

Loss of Ptpn11 (Shp2) drives satellite cells into quiescence
Griger(*), J., Schneider(*), R., Lahmann(*), I., Schöwel(*9, V., Keller(*), C., Spuler(*), S., Nazare, M.; Birchmeier(*), C.
Elife, 6

Tags: Medicinal Chemistry (Nazare)

Abstract: The equilibrium between proliferation and quiescence of myogenic progenitor and stem cells is tightly regulated to ensure appropriate skeletal muscle growth and repair. The non-receptor tyrosine phosphatase Ptpn11 (Shp2) is an important transducer of growth factor and cytokine signals. Here we combined complex genetic analyses, biochemical studies and pharmacological interference to demonstrate a central role of Ptpn11 in postnatal myogenesis of mice. Loss of Ptpn11 drove muscle stem cells out of the proliferative and into a resting state during muscle growth. This Ptpn11 function was observed in postnatal but not fetal myogenic stem cells. Furthermore, muscle repair was severely perturbed when Ptpn11 was ablated in stem cells due to a deficit in stem cell proliferation and survival. Our data demonstrate a molecular difference in the control of cell cycle withdrawal in fetal and postnatal myogenic stem cells, and assign to Ptpn11 signaling a key function in satellite cell activity.

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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)

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