FMP Publications

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

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Research Resource: Novel Structural Insights Bridge Gaps in Glycoprotein Hormone Receptor Analyses
Kreuchwig, A., Kleinau(*), G.; Krause, G.
Mol Endocrinol, 27:1357-1363

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

Abstract: The first version of a glycoprotein hormone receptor (GPHR) information resource was designed to link functional with structural GPHR information, in order to support sequence-structure-function analysis of the LH, FSH, and TSH receptors ( However, structural information on a binding- and signaling-sensitive extracellular fragment (similar to 100 residues), the hinge region, had been lacking. A new FSHR crystal structure of the hormone-bound extracellular domain has recently been solved. The structure comprises the leucine-rich repeat domain and most parts of the hinge region. We have not only integrated the new FSHR/FSH structure and the derived homology models of TSHR/TSH, LHCGR/CG, and LHCGR/LH into our web-based information resource, but have additionally provided novel tools to analyze the advanced structural features, with the common characteristics and distinctions between GPHRs, in a more precise manner. The hinge region with its second hormone-binding site allows us to assign functional data to the new structural features between hormone and receptor, such as binding details of a sulfated tyrosine (conserved throughout the GPHRs) extending into a pocket of the hormone. We have also implemented a protein interface analysis tool that enables the identification and visualization of extracellular contact points between interaction partners. This provides a starting point for comparing the binding patterns of GPHRs. Together with the mutagenesis data stored in the database, this will help to decipher the essential residues for ligand recognition and the molecular mechanisms of signal transduction, extending from the extracellular hormone-binding site toward the intracellular G protein-binding sites.

Lipophilic prodrugs of a triazole-containing colchicine analogue in liposomes: Biological effects on human tumor cells
Kuznetsova(*), N. R., Svirshchevskaya(*), E. V., Sitnikov(*), N. S., Abodo(*), L., Sutorius(*), H., Zapke, J., Velder(*), J., Thomopoulou(*), P., Oschkinat, H., Prokop(*), A., Schmalz(*), H. G., Fedorov(*), A. Y.; Vodovozova(*), E. L.
Russ J Bioorg Chem+, 39:543-552

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Colchicine site binders-blockers of tubulin polymerization-are potential antimitotic agents for anticancer therapy. To reduce their systemic toxicity and improve biodistribution, encapsulation in nanosized liposomes may be employed. Liposomes present a convenient means for preparation of injectable for-mulations of hydrophobic compounds, however colchicine as such is known to leak through the lipid bilayer. In this study, newly synthesized triazole-containing analogues of colchicine and allocolchicine, and their palmitic and oleic esters (lipophilic prodrugs) were tested for anti-proliferative activity and apoptosis-inducing potential. In contrast to colchicine conjugates, whose activities ranged with those of colchicine, allocolchicine derivatives exhibited drastically lower effects and were discarded. Liposomes of about 100 nm in diameter composed of egg phosphatidylcholine-yeast phosphatidylinositol-palmitic or oleic prodrug, 8: 1: 1, by mol, were prepared by standard extrusion technique and tested in a panel of four human tumor cell lines. Liposome formulations preserved the biological activities of the parent colchicinoid the most towards human epithelial tumor cells. Moreover, liposomal form of the oleoyl bearing colchicinoid inhibited cell proliferation more efficiently than free lipophilic prodrug. Due to substantial loading capacity of the liposomes, the dispersions contain sufficient concentration of the active agent to test wide dose range in experiments on systemic administration to animals.

A conformational intermediate in glutamate receptor activation
Lau(*), A. Y., Salazar, H., Blachowicz(*), L., Ghisi, V., Plested, A. J.; Roux(*), B.
Neuron, 79:492-503

Tags: Molecular Neuroscience and Biophysics (Plested)

Abstract: Ionotropic glutamate receptors (iGluRs) transduce the chemical signal of neurotransmitter release into membrane depolarization at excitatory synapses in the brain. The opening of the transmembrane ion channel of these ligand-gated receptors is driven by conformational transitions that are induced by the association of glutamate molecules to the ligand-binding domains (LBDs). Here, we describe the crystal structure of a GluA2 LBD tetramer in a configuration that involves an approximately 30 degrees rotation of the LBD dimers relative to the crystal structure of the full-length receptor. The configuration is stabilized by an engineered disulfide crosslink. Biochemical and electrophysiological studies on full-length receptors incorporating either this crosslink or an engineered metal bridge show that this LBD configuration corresponds to an intermediate state of receptor activation. GluA2 activation therefore involves a combination of both intra-LBD (cleft closure) and inter-LBD dimer conformational transitions. Overall, these results provide a comprehensive structural characterization of an iGluR intermediate state.

Molecular biology of maize Ac/Ds elements: an overview
Lazarow, K., Doll(*), M. L.; Kunze(*), R.
Methods Mol Biol, 1057:59-82

Tags: Screening Unit (von Kries)

Abstract: Maize Activator (Ac) is one of the prototype transposable elements of the hAT transposon superfamily, members of which were identified in plants, fungi, and animals. The autonomous Ac and nonautonomous Dissociation (Ds) elements are mobilized by the single transposase protein encoded by Ac. To date Ac/Ds transposons were shown to be functional in approximately 20 plant species and have become the most widely used transposable elements for gene tagging and functional genomics approaches in plants. In this chapter we review the biology, regulation, and transposition mechanism of Ac/Ds elements in maize and heterologous plants. We discuss the parameters that are known to influence the functionality and transposition efficiency of Ac/Ds transposons and need to be considered when designing Ac transposase expression constructs and Ds elements for application in heterologous plant species.

Common gating of both CLC transporter subunits underlies voltage-dependent activation of the 2Cl-/1H+ exchanger ClC-7/Ostm1
Ludwig, C. F., Ullrich, F., Leisle, L., Stauber, T.; Jentsch, T. J.
J Biol Chem, 288:28611-28619

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: CLC anion transporters form dimers that function either as Cl(-) channels or as electrogenic Cl(-)/H(+) exchangers. CLC channels display two different types of "gates," "protopore" gates that open and close the two pores of a CLC dimer independently of each other and common gates that act on both pores simultaneously. ClC-7/Ostm1 is a lysosomal 2Cl(-)/1H(+) exchanger that is slowly activated by depolarization. This gating process is drastically accelerated by many CLCN7 mutations underlying human osteopetrosis. Making use of some of these mutants, we now investigate whether slow voltage activation of plasma membrane-targeted ClC-7/Ostm1 involves protopore or common gates. Voltage activation of wild-type ClC-7 subunits was accelerated by co-expressing an excess of ClC-7 subunits carrying an accelerating mutation together with a point mutation rendering these subunits transport-deficient. Conversely, voltage activation of a fast ClC-7 mutant could be slowed by co-expressing an excess of a transport-deficient mutant. These effects did not depend on whether the accelerating mutation localized to the transmembrane part or to cytoplasmic cystathionine-beta-synthase (CBS) domains of ClC-7. Combining accelerating mutations in the same subunit did not speed up gating further. No currents were observed when ClC-7 was truncated after the last intramembrane helix. Currents and slow gating were restored when the C terminus was co-expressed by itself or fused to the C terminus of the beta-subunit Ostm1. We conclude that common gating underlies the slow voltage activation of ClC-7. It depends on the CBS domain-containing C terminus that does not require covalent binding to the membrane domain of ClC-7.

NMR Spectroscopy of Soluble Protein Complexes at One Mega-Dalton and Beyond
Mainz, A., Religa, T. L., Sprangers, R., Linser, R., Kay, L. E.; Reif, B.
Angew Chem Int Edit, 52:8746-8751

Tags: Solid-State NMR Spectroscopy (Reif)

Completion of proteomic data sets by Kd measurement using cell-free synthesis of site-specifically labeled proteins
Majkut, P., Claussnitzer(*), I., Merk(*), H., Freund(*), C., Hackenberger, C. P.; Gerrits(*), M.
Plos One, 8:e82352

Tags: Chemical Biology II (Hackenberger)

Abstract: The characterization of phosphotyrosine mediated protein-protein interactions is vital for the interpretation of downstream pathways of transmembrane signaling processes. Currently however, there is a gap between the initial identification and characterization of cellular binding events by proteomic methods and the in vitro generation of quantitative binding information in the form of equilibrium rate constants (Kd values). In this work we present a systematic, accelerated and simplified approach to fill this gap: using cell-free protein synthesis with site-specific labeling for pull-down and microscale thermophoresis (MST) we were able to validate interactions and to establish a binding hierarchy based on Kd values as a completion of existing proteomic data sets. As a model system we analyzed SH2-mediated interactions of the human T-cell phosphoprotein ADAP. Putative SH2 domain-containing binding partners were synthesized from a cDNA library using Expression-PCR with site-specific biotinylation in order to analyze their interaction with fluorescently labeled and in vitro phosphorylated ADAP by pull-down. On the basis of the pull-down results, selected SH2's were subjected to MST to determine Kd values. In particular, we could identify an unexpectedly strong binding of ADAP to the previously found binding partner Rasa1 of about 100 nM, while no evidence of interaction was found for the also predicted SH2D1A. Moreover, Kd values between ADAP and its known binding partners SLP-76 and Fyn were determined. Next to expanding data on ADAP suggesting promising candidates for further analysis in vivo, this work marks the first Kd values for phosphotyrosine/SH2 interactions on a phosphoprotein level.

Reduced activity of alkaline phosphatase due to host-guest interactions with humic superstructures
Mazzei(*), P., Oschkinat, H.; Piccolo(*), A.
Chemosphere, 93:1972-1979

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Nuclear Magnetic Resonance (NMR) spectroscopy was applied to directly study the interactions between the alkaline phosphatase enzyme (AP) and two different humic acids from a volcanic soil (HA-V) and a Lignite deposit (HA-L). Addition of humic matter to enzyme solutions caused signals broadening in H-1-NMR spectra, and progressive decrease and increase of enzyme relaxation (T-1 and T-2) and correlation (tau(c)) times, respectively. Spectroscopic changes were explained with formation of ever larger weaklybound humic-enzyme complexes, whose translational and rotational motion was increasingly restricted. NMR diffusion experiments also showed that the AP diffusive properties were progressively reduced with formation of large humic-enzyme complexes. The more hydrophobic HA-L affected spectral changes more than the more hydrophilic HA-V. H-1-NMR spectra also showed the effect of progressively greater humic-enzyme complexes on the hydrolysis of an enzyme substrate, the 4-nitrophenyl phosphate disodium salt hexahydrate (p-NPP). While AP catalysis concomitantly decreased NMR signals of p-NPP and increased those of nitrophenol, addition of humic matter progressively and significantly slowed down the rate of change for these signals. In agreement with the observed spectral changes, the AP catalytic activity was more largely inhibited by HA-L than by HA-V. Contrary to previous studies, in which humic-enzyme interactions were only indirectly assumed from changes in spectrophotometric behavior of enzyme substrates, the direct measurements of AP behavior by NMR spectroscopy indicated that humic materials formed weakly-bound host-guest complexes with alkaline phosphatase, and the enzyme catalytic activity was thereby significantly inhibited. These results suggest that the role of extracellular enzymes in soils may be considerably reduced when they come in contact with organic matter dissolved in the soil solution. (C) 2013 Elsevier Ltd. All rights reserved.

State-dependent FRET reports calcium- and voltage-dependent gating-ring motions in BK channels
Miranda(*), P., Contreras(*), J. E., Plested, A. J., Sigworth(*), F. J., Holmgren(*), M.; Giraldez(*), T.
Proc Natl Acad Sci U S A, 110:5217-5222

Tags: Molecular Neuroscience and Biophysics (Plested)

Abstract: Large-conductance voltage- and calcium-dependent potassium channels (BK, "Big K+") are important controllers of cell excitability. In the BK channel, a large C-terminal intracellular region containing a "gating-ring" structure has been proposed to transduce Ca(2+) binding into channel opening. Using patch-clamp fluorometry, we have investigated the calcium and voltage dependence of conformational changes of the gating-ring region of BK channels, while simultaneously monitoring channel conductance. Fluorescence resonance energy transfer (FRET) between fluorescent protein inserts indicates that Ca(2+) binding produces structural changes of the gating ring that are much larger than those predicted by current X-ray crystal structures of isolated gating rings.

Stabilization of peptides for intracellular applications by phosphoramidate-linked polyethylene glycol chains
Nischan, N., Chakrabarti(*), A., Serwa, R. A., Bovee-Geurts(*), P. H., Brock(*), R.; Hackenberger, C. P.
Angew Chem Int Ed Engl, 52:11920-11924

Tags: Chemical Biology II (Hackenberger)

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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
+4930 94793 - 100 
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