FMP Publications

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

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A modular toolkit to inhibit proline-rich motif-mediated protein-protein interactions
Opitz, R., Müller, M., Reuter, C., Barone, M., Soicke(*), A., Roske(*), Y., Piotukh, K., Huy(*), P., Beerbaum, M., Wiesner, B., Beyermann, M., Schmieder, P., Freund(*), C., Volkmer, R., Oschkinat, H., Schmalz(*), H. G.; Kühne, R.
Proc Natl Acad Sci U S A, 112:5011-5016

Tags: Computational Chemistry and Protein Design (Kühne), NMR-Supported Structural Biology (Oschkinat), Peptide Chemistry (Hackenberger/ Volkmer), Solution NMR (Schmieder), Peptide Chemistry (Beyermann), Cellular Imaging (Wiesner)

Abstract: Small-molecule competitors of protein-protein interactions are urgently needed for functional analysis of large-scale genomics and proteomics data. Particularly abundant, yet so far undruggable, targets include domains specialized in recognizing proline-rich segments, including Src-homology 3 (SH3), WW, GYF, and Drosophila enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Here, we present a modular strategy to obtain an extendable toolkit of chemical fragments (ProMs) designed to replace pairs of conserved prolines in recognition motifs. As proof-of-principle, we developed a small, selective, peptidomimetic inhibitor of Ena/VASP EVH1 domain interactions. Highly invasive MDA MB 231 breast-cancer cells treated with this ligand showed displacement of VASP from focal adhesions, as well as from the front of lamellipodia, and strongly reduced cell invasion. General applicability of our strategy is illustrated by the design of an ErbB4-derived ligand containing two ProM-1 fragments, targeting the yes-associated protein 1 (YAP1)-WW domain with a fivefold higher affinity.

Design and Stereoselective Synthesis of ProM-2: A Spirocyclic Diproline Mimetic with Polyproline Type II (PPII) Helix Conformation
Reuter(*), C., Opitz, R., Soicke(*), A., Dohmen(*), S., Barone, M., Chiha(*), S., Klein(*), M. T., Neudörfl(*), J. M., Kühne, R.; Schmalz(*), H. G.
Chemistry, 21:8464-8470

Tags: Computational Chemistry and Protein Design (Kühne)

Abstract: With the aim of developing polyproline type II helix (PPII) secondary-structure mimetics for the modulation of prolin-rich-mediated protein-protein interactions, the novel diproline mimetic ProM-2 was designed by bridging the two pyrrolidine rings of a diproline (Pro-Pro) unit through a Z-vinylidene moiety. This scaffold, which closely resembles a section of a PPII helix, was then stereoselectively synthesized by exploiting a ruthenium-catalyzed ring-closing metathesis (RCM) as a late key step. The required vinylproline building blocks, that is, (R)-N-Boc-2-vinylproline (Boc=tert-butyloxycarbonyl) and (S,S)-5-vinylproline-tert-butyl ester, were prepared on a gram scale as pure stereoisomers. The difficult peptide coupling of the sterically demanding building blocks was achieved in good yield and without epimerization by using 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU)/N,N-diisopropylethylamine (DIPEA). The RCM proceeded smoothly in the presence of the Grubbs II catalyst. Stereostructural assignments for several intermediates were secured by X-ray crystallography. As a proof of concept, it was shown that certain peptides containing ProM-2 exhibited improved (canonical) binding towards the Ena/VASP homology 1 (EVH1) domain as a relevant protein interaction target.

Differences in Signal Activation by LH and hCG are Mediated by the LH/CG Receptor's Extracellular Hinge Region
Grzesik, P., Kreuchwig, A., Rutz, C., Furkert, J., Wiesner, B., Schülein, R., Kleinau(*), G., Gromoll(*), J.; Krause, G.
Front Endocrinol (Lausanne), 6:140

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

Abstract: The human lutropin (hLH)/choriogonadotropin (hCG) receptor (LHCGR) can be activated by binding two slightly different gonadotropic glycoprotein hormones, choriogonadotropin (CG) - secreted by the placenta, and lutropin (LH) - produced by the pituitary. They induce different signaling profiles at the LHCGR. This cannot be explained by binding to the receptor's leucine-rich-repeat domain (LRRD), as this binding is similar for the two hormones. We therefore speculate that there are previously unknown differences in the hormone/receptor interaction at the extracellular hinge region, which might help to understand functional differences between the two hormones. We have therefore performed a detailed study of the binding and action of LH and CG at the LHCGR hinge region. We focused on a primate-specific additional exon in the hinge region, which is located between LRRD and the serpentine domain. The segment of the hinge region encoded by exon10 was previously reported to be only relevant to hLH signaling, as the exon10-deletion receptor exhibits decreased hLH signaling, but unchanged hCG signaling. We designed an advanced homology model of the hormone/LHCGR complex, followed by experimental characterization of relevant fragments in the hinge region. In addition, we examined predictions of a helical exon10-encoded conformation by block-wise polyalanine (helix supporting) mutations. These helix preserving modifications showed no effect on hormone-induced signaling. However, introduction of a structure-disturbing double-proline mutant LHCGR-Q303P/E305P within the exon10-helix has, in contrast to exon10-deletion, no impact on hLH, but only on hCG signaling. This opposite effect on signaling by hLH and hCG can be explained by distinct sites of hormone interaction in the hinge region. In conclusion, our analysis provides details of the differences between hLH- and hCG-induced signaling that are mainly determined in the L2-beta loop of the hormones and in the hinge region of the receptor.

Structural insights into thyroid hormone transport mechanisms of the L-type amino acid transporter 2
Hinz, K. M., Meyer, K., Kinne, A., Schülein, R., Köhrle(*), J.; Krause, G.
Mol Endocrinol, 29:933-942

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

Abstract: Thyroid hormones (THs) are transported across cell membranes by different transmembrane transporter proteins. In previous studies, we showed marked 3,3'-diiodothyronine (3,3'-T2) but moderate T3 uptake by the L-type amino acid transporter 2 (Lat2). We have now studied the structure-function relationships of this transporter and TH-like molecules. Our Lat2 homology model is based on 2 crystal structures of the homologous 12-transmembrane helix transporters arginine/agmatine antiporter and amino acid/polyamine/organocation transporter. Model-driven mutagenesis of residues lining an extracellular recognition site and a TH-traversing channel identified 9 sensitive residues. Using Xenopus laevis oocytes as expression system, we found that side chain shortening (N51S, N133S, N248S, and Y130A) expanded the channel and increased 3,3'-T2 transport. Side chain enlargements (T140F, Y130R, and I137M) decreased 3,3'-T2 uptake, indicating channel obstructions. The opposite results with mutations maintaining (F242W) or impairing (F242V) uptake suggest that F242 may have a gating function. Competitive inhibition studies of 14 TH-like compounds revealed that recognition by Lat2 requires amino and carboxylic acid groups. The size of the adjacent hydrophobic group is restricted. Bulky substituents in positions 3 and 5 of the tyrosine ring are allowed. The phenolic ring may be enlarged, provided that the whole molecule is flexible enough to fit into the distinctly shaped TH-traversing channel of Lat2. Taken together, the next Lat2 features were identified 1) TH recognition site; 2) TH-traversing channel in the center of Lat2; and 3) switch site that potentially facilitates intracellular substrate release. Together with identified substrate features, these data help to elucidate the molecular mechanisms and role of Lat2 in T2 transport.

Involvement of the L-Type Amino Acid Transporter Lat2 in the Transport of 3,3'-Diiodothyronine across the Plasma Membrane
Kinne, A., Wittner, M., Wirth(*), E. K., Hinz, K. M., Schülein, R., Köhrle(*), J.; Krause, G.
Eur Thyroid J, 4:42-50

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

Abstract: Thyroid hormones are transported across cell membranes by transmembrane transporter proteins, for example by members of the monocarboxylate transporter (MCT) and the L-type amino acid transporter (LAT) families. LATs consist of a light chain (e.g. LAT2) and a heavy chain (CD98), which is essential for their cell surface expression and functionality. The specificity of Lat2 for thyroid hormones and their metabolites and its role in their transport was not fully clear. This fact motivated us to establish a cell system to elucidate the uptake of thyroid hormones and their metabolites by mouse Lat2. The coinjection of cRNA coding for Lat2 and CD98 into Xenopus laevis oocytes resulted in a markedly increased level of 3,3'-diiodo-L-thyronine (3,3'-T2) and to some extent also enhanced T3 transport. To gain insight into properties of thyroid hormones and their metabolites transported by Lat2, we inhibited 3,3'-T2 uptake by various iodothyronine derivatives. T1 and T2 derivatives as well as 2-aminobicyclo-[2, 2,1]-heptane-2-carboxylic acid strongly competed with 3,3'-T2 uptake. In addition, we performed T2 uptake measurements with the thyroid hormone-specific transporter MCT8. For both Lat2 and MCT8, Km values in a low micromolar range were calculated. We demonstrated that oocytes are a suitable system for thyroid hormone transport studies mediated by Lat2. Our data indicates that Lat2 compared to other thyroid hormone transporters prefers 3,3'-T2 as the substrate. Thus, Lat2 might contribute to the availability of thyroid hormone by importing and/or exporting 3,3'-T2, which is generated either by T3 inactivation or by rapid deiodinase 1-mediated rT3 degradation.

Defining a conformational consensus motif in cotransin-sensitive signal sequences: a proteomic and site-directed mutagenesis study
Klein, W., Westendorf, C., Schmidt, A., Conill-Cortes, M., Rutz, C., Blohs, M., Beyermann, M., Protze, J., Krause, G., Krause, E.; Schülein, R.
Plos One, 10:e0120886

Tags: Protein Trafficking (Schülein), Mass Spectrometry (Krause, E.), Structural Bioinformatics and Protein Design (Krause, G.), Peptide Chemistry (Beyermann)

Abstract: The cyclodepsipeptide cotransin was described to inhibit the biosynthesis of a small subset of proteins by a signal sequence-discriminatory mechanism at the Sec61 protein-conducting channel. However, it was not clear how selective cotransin is, i.e. how many proteins are sensitive. Moreover, a consensus motif in signal sequences mediating cotransin sensitivity has yet not been described. To address these questions, we performed a proteomic study using cotransin-treated human hepatocellular carcinoma cells and the stable isotope labelling by amino acids in cell culture technique in combination with quantitative mass spectrometry. We used a saturating concentration of cotransin (30 micromolar) to identify also less-sensitive proteins and to discriminate the latter from completely resistant proteins. We found that the biosynthesis of almost all secreted proteins was cotransin-sensitive under these conditions. In contrast, biosynthesis of the majority of the integral membrane proteins was cotransin-resistant. Cotransin sensitivity of signal sequences was neither related to their length nor to their hydrophobicity. Instead, in the case of signal anchor sequences, we identified for the first time a conformational consensus motif mediating cotransin sensitivity.

Assembly and function of claudins: Structure-function relationships based on homology models and crystal structures
Krause, G., Protze, J.; Piontek(*), J.
Semin Cell Dev Biol, 42:3-12

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

Abstract: The tetra-span transmembrane proteins of the claudin family are critical components of formation and function of tight junctions (TJ). Homo- and heterophilic side-by-side (cis) and intercellular head-to-head (trans) interactions of 27 claudin-subtypes regulate tissue-specifically the paracellular permeability and/or tightness between epithelial or endothelial cells. This review highlights the functional impact that has been identified for particular claudin residues by relating them to structural features and architectural characteristics in the light of structural advances, which have been contributed by homology models, cryo-electron microscopy and crystal structures. The differing contributions to the TJ functionalities by claudins are dissected for the transmembrane region, the first and the second extracellular loop of claudins separately. Their particular impact to oligomerisation and TJ strand- and pore-formation is surveyed. Detailed knowledge about structure-function relationships about claudins helps to reveal the molecular mechanisms of TJ assembly and regulation of paracellular permeability, which is yet not fully understood.

Directed structural modification of Clostridium perfringens enterotoxin to enhance binding to claudin-5
Protze, J., Eichner, M., Piontek, A., Dinter, S., Rossa, J., Blecharz(*), K. G., Vajkoczy(*), P., Piontek(*), J.; Krause, G.
Cellular and molecular life sciences : CMLS, 72:1417-1432

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

Abstract: Clostridium perfringens enterotoxin (CPE) binds to distinct claudins (Clds), which regulate paracellular barrier functions in endo- and epithelia. The C-terminal domain (cCPE) has the potential for selective claudin modulation, since it only binds to a subset of claudins, e.g., Cld3 and Cld4 (cCPE receptors). Cld5 (non-CPE receptor) is a main constituent in tight junctions (TJ) of the blood-brain barrier. We aimed to reveal claudin recognition mechanisms of cCPE and to create a basis for a Cld5-binder. By utilizing structure-based interaction models, mutagenesis and assays of cCPE-binding to the TJ-free cell line HEK293, transfected with human Cld1 and murine Cld5, we showed how cCPE-binding to Cld1 and Cld5 is prevented by two residues in extracellular loop 2 of Cld1 (Asn(150) and Thr(153)) and Cld5 (Asp(149) and Thr(151)). Binding to Cld5 is especially attenuated by the lack of a bulky hydrophobic residue like leucine at position 151. By downsizing the binding pocket and compensating for the lack of this leucine residue, we created a novel cCPE-variant; cCPEY306W/S313H binds Cld5 with nanomolar affinity (K d 33 +/- 10 nM). Finally, the effective binding to endogenously Cld5-expressing blood-brain barrier model cells (murine microvascular endothelial cEND cell line) suggests cCPEY306W/S313H as basis for Cld5-specific modulation to improve paracellular drug delivery, or to target claudin overexpressing tumors.

Transport of Iodothyronines by Human L-Type Amino Acid Transporters
Zevenbergen(*), C., Meima(*), M. E., Lima de Souza(*), E. C., Peeters(*), R. P., Kinne, A., Krause, G., Visser(*), W. E.; Visser(*), T. J.
Endocrinology, 156:4345-4355

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

Abstract: Thyroid hormone (TH) transporters facilitate cellular TH influx and efflux, which is paramount for normal physiology. The L-type amino acid transporters LAT1 and LAT2 are known to facilitate TH transport. However, the role of LAT3, LAT4, and LAT5 is still unclear. Therefore, the aim of this study was to further characterize TH transport by LAT1 and LAT2 and to explore possible TH transport by LAT3, LAT4, and LAT5. FLAG-LAT1-5 constructs were transiently expressed in COS1 cells. LAT1 and LAT2 were cotransfected with the CD98 heavy chain. Cellular transport was measured using 10 nM (125)I-labeled T4, T3, rT3, 3,3'-T2, and 10 muM [(125)I]3'-iodotyrosine (MIT) as substrates. Intracellular metabolism of these substrates was determined in cells cotransfected with either of the LATs with type 1 or type 3 deiodinase. LAT1 facilitated cellular uptake of all substrates and LAT2 showed a net uptake of T3, 3,3'-T2, and MIT. Expression of LAT3 or LAT4 did not affect transport of T4 and T3 but resulted in the decreased cellular accumulation of 3,3'-T2 and MIT. LAT5 did not facilitate the transport of any substrate. Cotransfection with LAT3 or LAT4 strongly diminished the cellular accumulation of 3,3'-T2 and MIT by LAT1 and LAT2. These data were confirmed by metabolism studies. LAT1 and LAT2 show distinct preferences for the uptake of the different iodocompounds, whereas LAT3 and LAT4 specifically facilitate the 3,3'-T2 and MIT efflux. Together our findings suggest that different sets of transporters with specific influx or efflux capacities may cooperate to regulate the cellular thyroid state.

SEPT9 negatively regulates ubiquitin-dependent downregulation of EGFR
Diesenberg, K., Beerbaum, M., Fink, U., Schmieder, P.; Krauss, M.
J Cell Sci, 128:397-407

Tags: Molecular Pharmacology and Cell Biology (Haucke), Solution NMR (Schmieder)

Abstract: Septins constitute a family of GTP-binding proteins that are involved in a variety of biological processes. Several isoforms have been implicated in disease, but the molecular mechanisms underlying pathogenesis are poorly understood. Here, we show that depletion of SEPT9 decreases surface levels of epidermal growth factor receptors (EGFRs) by enhancing receptor degradation. We identify a consensus motif within the SEPT9 N-terminal domain that supports its association with the adaptor protein CIN85 (also known as SH3KBP1). We further show CIN85-SEPT9 to be localized exclusively to the plasma membrane, where SEPT9 is recruited to EGF-engaged receptors in a CIN85-dependent manner. Finally, we demonstrate that SEPT9 negatively regulates EGFR degradation by preventing the association of the ubiquitin ligase Cbl with CIN85, resulting in reduced EGFR ubiquitylation. Taken together, these data provide a mechanistic explanation of how SEPT9, though acting exclusively at the plasma membrane, impairs the sorting of EGFRs into the degradative pathway.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
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