FMP Publications

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

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Peptide-polymer ligands for a tandem WW-domain, an adaptive multivalent protein-protein interaction: lessons on the thermodynamic fitness of flexible ligands
Koschek, K., Durmaz(*), V., Krylova, O., Wieczorek, M., Gupta(*), S., Richter, M., Bujotzek(*), A., Fischer(*), C., Haag(*), R., Freund, C., Weber(*), M.; Rademann, J.
Beilstein J Org Chem, 11:837-847

Tags: Medicinal Chemistry (Rademann), Protein Engineering (Freund), Peptide-Lipid-Interaction/ Peptide Transport (Dathe)

Abstract: Three polymers, poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA), hyperbranched polyglycerol (hPG), and dextran were investigated as carriers for multivalent ligands targeting the adaptive tandem WW-domain of formin-binding protein (FBP21). Polymer carriers were conjugated with 3-9 copies of the proline-rich decapeptide GPPPRGPPPR-NH2 (P1). Binding of the obtained peptide-polymer conjugates to the tandem WW-domain was investigated employing isothermal titration calorimetry (ITC) to determine the binding affinity, the enthalpic and entropic contributions to free binding energy, and the stoichiometry of binding for all peptide-polymer conjugates. Binding affinities of all multivalent ligands were in the microM range, strongly amplified compared to the monovalent ligand P1 with a K D > 1 mM. In addition, concise differences were observed, pHPMA and hPG carriers showed moderate affinity and bound 2.3-2.8 peptides per protein binding site resulting in the formation of aggregates. Dextran-based conjugates displayed affinities down to 1.2 microM, forming complexes with low stoichiometry, and no precipitation. Experimental results were compared with parameters obtained from molecular dynamics simulations in order to understand the observed differences between the three carrier materials. In summary, the more rigid and condensed peptide-polymer conjugates based on the dextran scaffold seem to be superior to induce multivalent binding and to increase affinity, while the more flexible and dendritic polymers, pHPMA and hPG are suitable to induce crosslinking upon binding.

Sensitivity and resolution of proton detected spectra of a deuterated protein at 40 and 60 kHz magic-angle-spinning
Nieuwkoop, A. J., Franks, W. T., Rehbein, K., Diehl, A., Akbey, Ü., Engelke(*), F., Emsley(*), L., Pintacuda(*), G.; Oschkinat, H.
J Biomol NMR, 61:161-171

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: The use of small rotors capable of very fast magic-angle spinning (MAS) in conjunction with proton dilution by perdeuteration and partial reprotonation at exchangeable sites has enabled the acquisition of resolved, proton detected, solid-state NMR spectra on samples of biological macromolecules. The ability to detect the high-gamma protons, instead of carbons or nitrogens, increases sensitivity. In order to achieve sufficient resolution of the amide proton signals, rotors must be spun at the maximum rate possible given their size and the proton back-exchange percentage tuned. Here we investigate the optimal proton back-exchange ratio for triply labeled SH3 at 40 kHz MAS. We find that spectra acquired on 60 % back-exchanged samples in 1.9 mm rotors have similar resolution at 40 kHz MAS as spectra of 100 % back-exchanged samples in 1.3 mm rotors spinning at 60 kHz MAS, and for (H)NH 2D and (H)CNH 3D spectra, show 10-20 % higher sensitivity. For 100 % back-exchanged samples, the sensitivity in 1.9 mm rotors is superior by a factor of 1.9 in (H)NH and 1.8 in (H)CNH spectra but at lower resolution. For (H)C(C)NH experiments with a carbon-carbon mixing period, this sensitivity gain is lost due to shorter relaxation times and less efficient transfer steps. We present a detailed study on the sensitivity of these types of experiments for both types of rotors, which should enable experimentalists to make an informed decision about which type of rotor is best for specific applications.

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.

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.

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.

N-Terminal Signal Peptides of G Protein-Coupled Receptors: Significance for Receptor Biosynthesis, Trafficking, and Signal Transduction
Rutz, C., Klein, W.; Schülein, R.
Prog Mol Biol Transl Sci, 132:267-287

Tags: Protein Trafficking (Schülein)

Abstract: Signal sequences play a key role during the first steps of the intracellular transport of G protein-coupled receptors (GPCRs). They are involved in targeting of the nascent chains to the membrane of the endoplasmic reticulum (ER) and initiate integration of the newly synthesized receptors into this compartment. Two classes of signal sequences are known: N-terminal signal peptides, which are usually cleaved-off following ER insertion and internal signal sequences, the so-called signal anchor sequences, which form part of the mature proteins. About 5-10% of the GPCRs contain N-terminal signal peptides; the vast majority possesses signal anchor sequences. The reason why only a subset of GPCRs require signal peptides for ER targeting/insertion was addressed in the past decade by a limited number of studies indicating that the presence of signal peptides facilitates N-tail translocation at the ER membrane. Interestingly, recent work showed that signal peptides of GPCRs do not only serve "classical" functions in the early secretory pathway. Uncleaved pseudo signal peptides may regulate receptor densities in the plasma membrane, receptor dimerization, and G protein coupling selectivity. On the other hand, even cleaved and released peptides may have post-ER functions. In this review, we summarize the current knowledge about cleavable signal peptides of GPCRs and address also the question whether these sequences may serve as future drug targets in pharmacology.

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.

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.

Alterations in creatine metabolism observed in experimental autoimmune myocarditis using ex vivo proton magic angle spinning MRS
Muench(*), F., Retel, J., Jeuthe(*), S., (*)D, O. h.-I., van Rossum, B., Wassilew(*), K., Schmerler(*), P., Kuehne(*), T., Berger(*), F., Oschkinat, H.; Messroghli(*), D. R.
Nmr Biomed, 28:1625-1633

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Experimental autoimmune myocarditis (EAM) in rodents is an accepted model of myocarditis and dilated cardiomyopathy (DCM). Altered metabolism is thought to play an important role in the pathogenesis of DCM and heart failure (HF). Study of the metabolism may provide new diagnostic information and insights into the mechanisms of myocarditis and HF. Proton MRS ((1)H-MRS) has not yet been used to study the changes occurring in myocarditis and subsequent HF. We aimed to explore the changes in creatine metabolism using this model and compare them with the findings in healthy animals. Myocardial function of male young Lewis rats with EAM was quantified by performing left ventricular ejection fraction (LVEF) analysis in short-axis cine images throughout the whole heart. Inflammatory cellular infiltrate was assessed by immunohistochemistry. Myocardial tissue was analyzed using ex vivo proton magic angle spinning MRS ((1)H-MAS-MRS). Myocarditis was confirmed histologically by the presence of an inflammatory cellular infiltrate and CD68 positive staining. A significant increase in the metabolic ratio of Tau/tCr (taurine/total creatine) obtained by (1)H-MAS-MRS was observed in myocarditis compared with healthy controls (21 d acute EAM, 4.38 (+/-0.23); 21 d control, 2.84 (+/-0.08); 35 d chronic EAM, 4.47 (+/-0.83); 35 d control, 2.59 (+/-0.38); P < 0.001). LVEF was reduced in diseased animals (EAM, 55.2% (+/-11.3%); control, 72.6% (+/-3.8%); P < 0.01) and correlated with Tau/tCr ratio (R = 0.937, P < 0.001). Metabolic alterations occur acutely with the development of myocarditis. Myocardial Tau/tCr ratio as detected by (1)H-MRS correlates with LVEF and is able to differentiate between healthy myocardium and myocardium from rats with EAM.

Selective inhibitors of the protein tyrosine phosphatase SHP2 block cellular motility and growth of cancer cells in vitro and in vivo
Grosskopf, S., Eckert, C., Arkona(*), C., Radetzki, S., Böhm(*), K., Heinemann(*), U., Wolber(*), G., von Kries, J. P., Birchmeier(*), W.; Rademann(*), J.
Chemmedchem, 10:815-826

Tags: Medicinal Chemistry (Rademann), Screening Unit (von Kries)

Abstract: Selective inhibitors of the protein tyrosine phosphatase SHP2 (src homology region 2 domain phosphatase; PTPN11), an enzyme that is deregulated in numerous human tumors, were generated through a combination of chemical synthesis and structure-based rational design. Seventy pyridazolon-4-ylidenehydrazinyl benzenesulfonates were prepared and evaluated in enzyme assays. The binding modes of active inhibitors were simulated in silico using a newly generated crystal structure of SHP2. The most powerful compound, GS-493 (4-(2Z)-2-[1,3-bis(4-nitrophenyl)-5-oxo-1,5-dihydro-4H-pyrazol-4-yliden]hydrazin obenzenesulfonic acid; 25) inhibited SHP2 with an IC50 value of 71+/-15 nM in the enzyme assay and was 29- and 45-fold more active toward SHP2 than against related SHP1 and PTP1B. In cell culture experiments compound 25 was found to block hepatocyte growth factor (HGF)-stimulated epithelial-mesenchymal transition of human pancreatic adenocarcinoma (HPAF) cells, as indicated by a decrease in the minimum neighbor distances of cells. Moreover, 25 inhibited cell colony formation in the non-small-cell lung cancer cell line LXFA 526L in soft agar. Finally, 25 was observed to inhibit tumor growth in a murine xenograft model. Therefore, the novel specific compound 25 strengthens the hypothesis that SHP2 is a relevant protein target for the inhibition of mobility and invasiveness of cancer cells.

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