FMP Publications

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

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Bidirectional binding of invariant chain peptides to an MHC class II molecule
Günther, S., Schlundt, A., Sticht, J., Roske(*), Y., Heinemann(*), U., Wiesmüller(*), K. H., Jung(*), G., Falk(*), K., Rötzschke(*), O.; Freund, C.
Proc Natl Acad Sci U S A, 107:22219-22224

Tags: Protein Engineering (Freund)

Abstract: T-cell recognition of peptides bound to MHC class II (MHCII) molecules is a central event in cell-mediated adaptive immunity. The current paradigm holds that prebound class II-associated invariant chain peptides (CLIP) and all subsequent antigens maintain a canonical orientation in the MHCII binding groove. Here we provide evidence for MHCII-bound CLIP inversion. NMR spectroscopy demonstrates that the interconversion from the canonical to the inverse alignment is a dynamic process, and X-ray crystallography shows that conserved MHC residues form a hydrogen bond network with the peptide backbone in both orientations. The natural catalyst HLA-DM accelerates peptide reorientation and the exchange of either canonically or inversely bound CLIP against antigenic peptide. Thus, noncanonical MHC-CLIP displays the hallmarks of a structurally and functionally intact antigen-presenting complex.

Glycogen synthase kinase 3beta interaction protein functions as an A-kinase anchoring protein
Hundsrucker, C., Skroblin, P., Christian, F., Zenn(*), H. M., Popara, V., Joshi, M., Eichhorst, J., Wiesner, B., Herberg(*), F. W., Reif, B., Rosenthal(*), W.; Klussmann, E.
J Biol Chem, 285:5507-5521

Tags: Anchored Signalling (Klussmann), Solid-State NMR Spectroscopy (Reif), Cellular Imaging (Wiesner)

Abstract: A-kinase anchoring proteins (AKAPs) include a family of scaffolding proteins that target protein kinase A (PKA) and other signaling proteins to cellular compartments and thereby confine the activities of the associated proteins to distinct regions within cells. AKAPs bind PKA directly. The interaction is mediated by the dimerization and docking domain of regulatory subunits of PKA and the PKA-binding domain of AKAPs. Analysis of the interactions between the dimerization and docking domain and various PKA-binding domains yielded a generalized motif allowing the identification of AKAPs. Our bioinformatics and peptide array screening approaches based on this signature motif identified GSKIP (glycogen synthase kinase 3beta interaction protein) as an AKAP. GSKIP directly interacts with PKA and GSK3beta (glycogen synthase kinase 3beta). It is widely expressed and facilitates phosphorylation and thus inactivation of GSK3beta by PKA. GSKIP contains the evolutionarily conserved domain of unknown function 727. We show here that this domain of GSKIP and its vertebrate orthologues binds both PKA and GSK3beta and thereby provides a mechanism for the integration of PKA and GSK3beta signaling pathways.

Solid-state NMR and SAXS studies provide a structural basis for the activation of alphaB-crystallin oligomers
Jehle, S., Rajagopal(*), P., Bardiaux, B., Markovic, S., Kühne, R., Stout(*), J. R., Higman, V. A., Klevit(*), R. E., van Rossum, B. J.; Oschkinat, H.
Nat Struct Mol Biol, 17:1037-1042

Tags: Protein Structure (Oschkinat), Computational Chemistry/ Drug Design (Kühne)

Abstract: The small heat shock protein alphaB-crystallin (alphaB) contributes to cellular protection against stress. For decades, high-resolution structural studies on oligomeric alphaB have been confounded by its polydisperse nature. Here, we present a structural basis of oligomer assembly and activation of the chaperone using solid-state NMR and small-angle X-ray scattering (SAXS). The basic building block is a curved dimer, with an angle of approximately 121 degrees between the planes of the beta-sandwich formed by alpha-crystallin domains. The highly conserved IXI motif covers a substrate binding site at pH 7.5. We observe a pH-dependent modulation of the interaction of the IXI motif with beta4 and beta8, consistent with a pH-dependent regulation of the chaperone function. N-terminal region residues Ser59-Trp60-Phe61 are involved in intermolecular interaction with beta3. Intermolecular restraints from NMR and volumetric restraints from SAXS were combined to calculate a model of a 24-subunit alphaB oligomer with tetrahedral symmetry.

Intermolecular protein-RNA interactions revealed by 2D 31P-15N magic angle spinning solid-state NMR spectroscopy
Jehle(*), S., Falb(*), M., Kirkpatrick(*), J. P., Oschkinat, H., van Rossum, B. J., Althoff(*), G.; Carlomagno(*), T.
J Am Chem Soc, 132:3842-3846

Tags: Protein Structure (Oschkinat)

Abstract: The structural investigation of large RNP complexes by X-ray crystallography can be a difficult task due to the flexibility of the RNA and of the protein-RNA interfaces, which may hinder crystallization. In these cases, NMR spectroscopy is an attractive alternative to crystallography, although the large size of typical RNP complexes may limit the applicability of solution NMR. Solid-state NMR spectroscopy, however, is not subject to any intrinsic limitations with respect to the size of the object under investigation, with restrictions imposed solely by the sensitivity of the instrumentation. In addition, it does not require large, well-ordered crystals and can therefore be applied to flexible, partially disordered complexes. Here we show for the first time that solid-state NMR spectroscopy can be used to probe intermolecular interactions at the protein-RNA interface in RNP complexes. Distances between the (15)N nuclei of the protein backbone and the (31)P nuclei of the RNA backbone can be measured in TEDOR experiments and used as restraints in structure calculations. The distance measurement is accurate, as proven for the test case of the L7Ae-box C/D RNA complex, for which a crystal structure is available. The results presented here reveal the as yet unexplored potential of solid-state NMR spectroscopy in the investigation of large RNP complexes.

Modulation of G-protein coupled receptor sample quality by modified cell-free expression protocols: A case study of the human endothelin A receptor
Junge(*), F., Luh(*), L. M., Proverbio(*), D., Schäfer(*), B., Abele(*), R., Beyermann, M., Dötsch(*), V.; Bernhard(*), F.
J Struct Biol, 172:94-106

Tags: Peptide Synthesis (Beyermann)

Abstract: G-protein coupled receptors still represent one of the most challenging targets in membrane protein research. Here we present a strategic approach for the cell-free synthesis of these complex membrane proteins exemplified by the preparative scale production of the human endothelin A receptor. The versatility of the cell-free expression system was used to modulate sample quality by alteration of detergents hence presenting different solubilization environments to the synthesized protein at different stages of the production process. Sample properties after co-translational and post-translational solubilization have been analysed by evaluation of homogeneity, protein stability and receptor ligand binding competence. This is a first quality evaluation of a membrane protein obtained in two different cell-free expression modes and we demonstrate that both can be used for the production of ligand-binding competent endothelin A receptor in quantities sufficient for structural approaches. The presented strategy of cell-free expression protocol development could serve as basic guideline for the production of related receptors in similar systems. (C) 2010 Elsevier Inc. All rights reserved.

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.

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.

Lysosomal pathology and osteopetrosis upon loss of H+-driven lysosomal Cl- accumulation
Weinert, S., Jabs, S., Supanchart(*), C., Schweizer(*), M., Gimber, N., Richter, M., Rademann, J., Stauber, T., Kornak(*), U.; Jentsch, T. J.
Science, 328:1401-1403

Tags: Physiology and Pathology of Ion Transport (Jentsch), Medicinal Chemistry (Rademann)

Abstract: During lysosomal acidification, proton-pump currents are thought to be shunted by a chloride ion (Cl-) channel, tentatively identified as ClC-7. Surprisingly, recent data suggest that ClC-7 instead mediates Cl-/proton (H+) exchange. We generated mice carrying a point mutation converting ClC-7 into an uncoupled (unc) Cl- conductor. Despite maintaining lysosomal conductance and normal lysosomal pH, these Clcn7(unc/unc) mice showed lysosomal storage disease like mice lacking ClC-7. However, their osteopetrosis was milder, and they lacked a coat color phenotype. Thus, only some roles of ClC-7 Cl-/H+ exchange can be taken over by a Cl- conductance. This conductance was even deleterious in Clcn7(+/unc) mice. Clcn7(-/-) and Clcn7(unc/unc) mice accumulated less Cl- in lysosomes than did wild-type mice. Thus, lowered lysosomal chloride may underlie their common phenotypes.

The KCNQ5 potassium channel mediates a component of the afterhyperpolarization current in mouse hippocampus
Tzingounis(*), A. V., Heidenreich, M., Kharkovets(*), T., Spitzmaul, G., Jensen(*), H. S., Nicoll(*), R. A.; Jentsch, T. J.
P Natl Acad Sci USA, 107:10232-10237

Tags: Physiology and Pathology of Ion Transport (Jentsch

Abstract: Mutations in KCNQ2 and KCNQ3 voltage-gated potassium channels lead to neonatal epilepsy as a consequence of their key role in regulating neuronal excitability. Previous studies in the brain have focused primarily on these KCNQ family members, which contribute to M-currents and afterhyperpolarization conductances in multiple brain areas. In contrast, the function of KCNQ5 (Kv7.5), which also displays widespread expression in the brain, is entirely unknown. Here, we developed mice that carry a dominant negative mutation in the KCNQ5 pore to probe whether it has a similar function as other KCNQ channels. This mutation renders KCNQ5(dn)-containing homomeric and heteromeric channels nonfunctional. We find that Kcnq5(dn/dn) mice are viable and have normal brain morphology. Furthermore, expression and neuronal localization of KCNQ2 and KCNQ3 subunits are unchanged. However, in the CA3 area of hippocampus, a region that highly expresses KCNQ5 channels, the medium and slow afterhyperpolarization currents are significantly reduced. In contrast, neither current is affected in the CA1 area of the hippocampus, a region with low KCNQ5 expression. Our results demonstrate that KCNQ5 channels contribute to the afterhyperpolarization currents in hippocampus in a cell type-specific manner.

A cation counterflux supports lysosomal acidification
Steinberg(*), B. E., Huynh(*), K. K., Brodovitch(*), A., Jabs, S., Stauber, T., Jentsch, T. J.; Grinstein(*), S.
J Cell Biol, 189:1171-1186

Tags: Physiology and Pathology of Ion Transport (Jentsch

Abstract: The profound luminal acidification essential for the degradative function of lysosomes requires a counter-ion flux to dissipate an opposing voltage that would prohibit proton accumulation. It has generally been assumed that a parallel anion influx is the main or only counter-ion transport that enables acidification. Indeed, defective anion conductance has been suggested as the mechanism underlying attenuated lysosome acidification in cells deficient in CFTR or ClC-7. To assess the individual contribution of counter-ions to acidification, we devised means of reversibly and separately permeabilizing the plasma and lysosomal membranes to dialyze the cytosol and lysosome lumen in intact cells, while ratiometrically monitoring lysosomal pH. Replacement of cytosolic Cl(-) with impermeant anions did not significantly alter proton pumping, while the presence of permeant cations in the lysosomal lumen supported acidification. Accordingly, the lysosomes were found to acidify to the same pH in both CFTR- and ClC-7-deficient cells. We conclude that cations, in addition to chloride, can support lysosomal acidification and defects in lysosomal anion conductance cannot explain the impaired microbicidal capacity of CF phagocytes.

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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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