FMP Publications

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

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Essential molecular determinants for thyroid hormone transport and first structural implications for monocarboxylate transporter 8
Kinne(*), A., Kleinau, G., Hoefig(*), C. S., Grüters(*), A., Köhrle, J., Krause, G.; Schweizer(*), U.
J Biol Chem, 285:28054-28063

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

Abstract: Monocarboxylate transporter 8 (MCT8, SLC16A2) is a thyroid hormone (TH) transmembrane transport protein mutated in Allan-Herndon-Dudley syndrome, a severe X-linked psychomotor retardation. The neurological and endocrine phenotypes of patients deficient in MCT8 function underscore the physiological significance of carrier-mediated TH transmembrane transport. MCT8 belongs to the major facilitator superfamily of 12 transmembrane-spanning proteins and mediates energy-independent bidirectional transport of iodothyronines across the plasma membrane. Structural information is lacking for all TH transmembrane transporters. To gain insight into structure-function relations in TH transport, we chose human MCT8 as a paradigm. We systematically performed conventional and liquid chromatography-tandem mass spectrometry-based uptake measurements into MCT8-transfected cells using a large number of compounds structurally related to iodothyronines. We found that human MCT8 is specific for L-iodothyronines and requires at least one iodine atom per aromatic ring. Neither thyronamines, decarboxylated metabolites of iodothyronines, nor triiodothyroacetic acid and tetraiodothyroacetic acid, TH derivatives lacking both chiral center and amino group, are substrates for MCT8. The polyphenolic flavonoids naringenin and F21388, potent competitors for TH binding at transthyretin, did not inhibit T(3) transport, suggesting that MCT8 can discriminate its ligand better than transthyretin. Bioinformatic studies and a first molecular homology model of MCT8 suggested amino acids potentially involved in substrate interaction. Indeed, alanine mutation of either Arg(445) (helix 8) or Asp(498) (helix 10) abrogated T(3) transport activity of MCT8, supporting their predicted role in substrate recognition. The MCT8 model allows us to rationalize potential interactions of amino acids including those mutated in patients with Allan-Herndon-Dudley syndrome.

Signaling-sensitive amino acids surround the allosteric ligand binding site of the thyrotropin receptor
Kleinau, G., Haas, A. K., Neumann(*), S., Worth, C. L., Hoyer, I., Furkert, J., Rutz, C., Gershengorn(*), M. C., Schülein, R.; Krause, G.
Faseb j, 24:2347-2354

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

Abstract: The thyrotropin receptor [thyroid-stimulating hormone receptor (TSHR)], a G-protein-coupled receptor (GPCR), is endogenously activated by thyrotropin, which binds to the extracellular region of the receptor. We previously identified a low-molecular-weight (LMW) agonist of the TSHR and predicted its allosteric binding pocket within the receptor's transmembrane domain. Because binding of the LMW agonist probably disrupts interactions or leads to formation of new interactions among amino acid residues surrounding the pocket, we tested whether mutation of residues at these positions would lead to constitutive signaling activity. Guided by molecular modeling, we performed site-directed mutagenesis of 24 amino acids in this spatial region, followed by functional characterization of the mutant receptors in terms of expression and signaling, measured as cAMP accumulation. We found that mutations V421I, Y466A, T501A, L587V, M637C, M637W, S641A, Y643F, L645V, and Y667A located in several helices exhibit constitutive activity. Of note is mutation M637W at position 6.48 in transmembrane helix 6, which has a significant effect on the interaction of the receptor with the LMW agonist. In summary, we found that a high proportion of residues in several helices surrounding the allosteric binding site of LMW ligands in the TSHR when mutated lead to constitutively active receptors. Our findings of signaling-sensitive residues in this region of the transmembrane bundle may be of general importance as this domain appears to be evolutionarily retained among GPCRs.

Principles and Determinants of G-Protein Coupling by the Rhodopsin-Like Thyrotropin Receptor
Kleinau, G., Jaeschke(*), H., Worth, C. L., Mueller(*), S., Gonzalez(*), J., Paschke(*), R.; Krause, G.
Plos One, 5

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

Abstract: In this study we wanted to gain insights into selectivity mechanisms between G-protein-coupled receptors (GPCR) and different subtypes of G-proteins. The thyrotropin receptor (TSHR) binds G-proteins promiscuously and activates both Gs (cAMP) and Gq (IP). Our goal was to dissect selectivity patterns for both pathways in the intracellular region of this receptor. We were particularly interested in the participation of poorly investigated receptor parts. We systematically investigated the amino acids of intracellular loop (ICL) 1 and helix 8 using site-directed mutagenesis alongside characterization of cAMP and IP accumulation. This approach was guided by a homology model of activated TSHR in complex with heterotrimeric Gq, using the X-ray structure of opsin with a bound G-protein peptide as a structural template. We provide evidence that ICL1 is significantly involved in G-protein activation and our model suggests potential interactions with subunits G alpha as well as G beta gamma. Several amino acid substitutions impaired both IP and cAMP accumulation. Moreover, we found a few residues in ICL1 (L440, T441, H443) and helix 8 (R687) that are sensitive for Gq but not for Gs activation. Conversely, not even one residue was found that selectively affects cAMP accumulation only. Together with our previous mutagenesis data on ICL2 and ICL3 we provide here the first systematically completed map of potential interfaces between TSHR and heterotrimeric G-protein. The TSHR/Gq-heterotrimer complex is characterized by more selective interactions than the TSHR/Gs complex. In fact the receptor interface for binding Gs is a subset of that for Gq and we postulate that this may be true for other GPCRs coupling these G-proteins. Our findings support that G-protein coupling and preference is dominated by specific structural features at the intracellular region of the activated GPCR but is completed by additional complementary recognition patterns between receptor and G-protein subtypes.

An interactive web-tool for molecular analyses links naturally occurring mutation data with three-dimensional structures of the rhodopsin-like glycoprotein hormone receptors
Kleinau, G., Kreuchwig, A., Worth, C. L.; Krause, G.
Hum Mutat, 31:E1519-1525

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

Abstract: The collection, description and molecular analysis of naturally occurring (pathogenic) mutations are important for understanding the functional mechanisms and malfunctions of biological units such as proteins. Numerous databases collate a huge amount of functional data or descriptions of mutations, but tools to analyse the molecular effects of genetic variations are as yet poorly provided. The goal of this work was therefore to develop a translational web-application that facilitates the interactive linkage of functional and structural data and which helps improve our understanding of the molecular basis of naturally occurring gain- or loss- of function mutations. Here we focus on the human glycoprotein hormone receptors (GPHRs), for which a huge number of mutations are known to cause diseases. We describe new options for interactive data analyses within three-dimensional structures, which enable the assignment of molecular relationships between structure and function. Strikingly, as the functional data are converted into relational percentage values, the system allows the comparison and classification of data from different GPHR subtypes and different experimental approaches. Our new application has been incorporated into a freely available database and website for the GPHRs (, but the principle development would also be applicable to other macromolecules.

Adaptin' endosomes for synaptic vesicle recycling, learning and memory
Krauss, M.; Haucke, V.
Embo Journal, 29:1313-1315

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: The pathways by which neurotransmitter-filled presynaptic vesicles (SVs) are generated and recycled have been debated for a long time. Glyvuk et al (2010) in this issue of The EMBO Journal describe an unanticipated role for the clathrin adaptor AP-1 and in particular its sigma 1B subunit in SV recycling. SV reformation is defective in sigma 1B-deficient mice, which instead accumulate large endosome-like vacuoles. These defects are paired with reduced motor coordination and long-term spatial memory. This work thus not only provides novel insights into the role of clathrin/AP-1 coats in SV recycling from endosomes, but also unravels a molecular mechanism that may contribute to some forms of X-linked mental retardation.

Microsecond time scale mobility in a solid protein as studied by the 15N R(1rho) site-specific NMR relaxation rates
Krushelnitsky(*), A., Zinkevich(*), T., Reichert(*), D., Chevelkov, V.; Reif, B.
J Am Chem Soc, 132:11850-11853

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: For the first time, we have demonstrated the site-resolved measurement of reliable (i.e., free of interfering effects) (15)N R(1rho) relaxation rates from a solid protein to extract dynamic information on the microsecond time scale. (15)N R(1rho) NMR relaxation rates were measured as a function of the residue number in a (15)N,(2)H-enriched (with 10-20% back-exchanged protons at labile sites) microcrystalline SH3 domain of chicken alpha-spectrin. The experiments were performed at different temperatures and at different spin-lock frequencies, which were realized by on- and off-resonance spin-lock irradiation. The results obtained indicate that the interfering spin-spin contribution to the R(1rho) rate in a perdeuterated protein is negligible even at low spin-lock fields, in contrast to the case for normal protonated samples. Through correlation plots, the R(1rho) rates were compared with previous data for the same protein characterizing different kinds of internal mobility.

Membrane solubilisation and reconstitution by octylglucoside: comparison of synthetic lipid and natural lipid extract by isothermal titration calorimetry
Krylova, O. O., Jahnke, N.; Keller, S.
Biophys Chem, 150:105-111

Tags: Biophysics of Membrane Proteins (Keller)

Abstract: We have studied the solubilisation and reconstitution of lipid membranes composed of either synthetic phosphatidylcholine or Escherichia. coli polar lipid extract by the non-ionic detergent octylglucoside. For both lipid systems, composition-dependent transformations of unilamellar vesicles into micelles or vice versa were followed by high-sensitivity isothermal titration calorimetry. Data obtained over a range of detergent and lipid concentrations could be rationalised in terms of a three-stage phase separation model involving bilayer, bilayer/micelle coexistence, and micellar ranges, yielding the detergent/lipid phase diagrams and the bilayer-to-micelle partition coefficients of both detergent and lipid. The most notable difference between the lipids investigated was a substantial widening of the bilayer/micelle coexistence range for E. coli lipid, which was due to an increased preference of the detergent and a decreased affinity of the lipid for the micellar phase as compared with the bilayer phase. These effects on the bilayer-to-micelle partition coefficients could be explained by the high proportion in E. coli membranes of lipids possessing negative spontaneous curvature, which hampers both their transfer into strongly curved micellar structures as well as the insertion of detergent into condensed bilayers.

An unbalanced translocation unmasks a recessive mutation in the follicle-stimulating hormone receptor (FSHR) gene and causes FSH resistance
Kuechler(*), A., Hauffa(*), B. P., Köninger(*), A., Kleinau, G., Albrecht(*), B., Horsthemke(*), B.; Gromoll(*), J.
European journal of human genetics : EJHG, 18:656-661

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

Abstract: Follicle-stimulating hormone (FSH) mediated by its receptor (FSHR) is pivotal for normal gametogenesis. Inactivating FSHR mutations are known to cause hypergonadotropic hypogonadism with disturbed follicular maturation in females. So far, only very few recessive point mutations have been described. We report on a 17-year-old female with primary amenorrhea, hypergonadotropic hypogonadism and disturbed folliculogenesis. Chromosome analysis detected a seemingly balanced translocation 46,XX,t(2;8)(p16.3or21;p23.1)mat. FSHR sequence analysis revealed a novel non-synonymous point mutation in exon 10 (c.1760C>A, p.Pro587His), but no wild-type allele. The mutation was also found in the father, but not in the mother. Furthermore, molecular-cytogenetic analyses of the breakpoint region on chromosome 2 showed the translocation to be unbalanced, containing a deletion with one breakpoint within the FSHR gene. The deletion size was narrowed down by array analysis to approximately 163 kb, involving exons 9 and 10 of the FSHR gene. Functional studies of the mutation revealed the complete lack of signal transduction presumably caused by a changed conformational structure of transmembrane helix 6. To our knowledge, this is the first description of a compound heterozygosity of an inactivating FSHR point mutation unmasked by a partial deletion. This coincidence of two rare changes caused clinical signs consistent with FSH resistance.

A MAS NMR Study of the Bacterial ABC Transporter ArtMP
Lange, V., Becker-Baldus, J., Kunert, B., van Rossum, B. J., Casagrande(*), F., Engel(*), A., Roske(*), Y., Scheffel(*), F. M., Schneider(*), E.; Oschkinat, H.
Chembiochem, 11:547-555

Tags: Protein Structure (Oschkinat)

Abstract: ATP-binding cassette (ABC) transport systems facilitate the translocation of substances, like amino acids, across cell membranes energised by ATP hydrolysis. This work describes first structural studies on the ABC transporter ArtMP from Geobacillus stearothermophilus in native lipid environment by magic-angle spinning NMR spectroscopy. The 2D crystals of ArtMP and 3D crystals of isolated ArtP were prepared in different nucleotide-bound or -unbound states. From selectively C-13,N-15-labelled ArtP, several sequence-specific assignments were obtained, most of which could be transferred to spectra of ArtMP. Residues Tyr133 and Pro134 protrude directly into the ATP-binding pocket at the interface of the ArtP subunits, and hence, are sensitive monitors for structural changes during nucleotide binding and hydrolysis. Distinct sets of NMR shifts were obtained for ArtP with different phosphorylation states of the ligand. Indications were found for an asymmetric or inhomogeneous state of the ArtP dimer bound with triphosphorylated nucleotides. With this investigation, a model system was established for screening all functional states occurring in one ABC transporter in native lipid environment.

Assignment of dynamic regions in biological solids enabled by spin-state selective NMR experiments
Linser, R., Fink, U.; Reif, B.
J Am Chem Soc, 132:8891-8893

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: Structural investigations are a prerequisite to understand protein function. Intermediate time scale motional processes (ns-micros) are deleterious for NMR of biological solids and obscure the detection of amide moieties in traditional CP based solid-state NMR approaches as well as in regular scalar coupling based experiments. We show that this obstacle can be overcome by using TROSY type techniques in triple resonance experiments, which enable the assignment of resonances in loop regions of a microcrystalline protein. The presented approach provides an exemplified solution for the analysis of secondary structure elements undergoing slow dynamics that might be particularly crucial for understanding protein function.

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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 
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