FMP Publications

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

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References
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
(2010)

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.

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
(2010)

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.

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
(2010)

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.

Optimum levels of exchangeable protons in perdeuterated proteins for proton detection in MAS solid-state NMR spectroscopy
Akbey, Ü., Lange, S., Trent Franks, W., Linser, R., Rehbein, K., Diehl, A., van Rossum, B. J., Reif, B.; Oschkinat, H.
J Biomol NMR, 46:67-73
(2010)

Tags: Protein Structure (Oschkinat), Solid-State NMR Spectroscopy (Reif)

Abstract: We present a systematic study of the effect of the level of exchangeable protons on the observed amide proton linewidth obtained in perdeuterated proteins. Decreasing the amount of D(2)O employed in the crystallization buffer from 90 to 0%, we observe a fourfold increase in linewidth for both (1)H and (15)N resonances. At the same time, we find a gradual increase in the signal-to-noise ratio (SNR) for (1)H-(15)N correlations in dipolar coupling based experiments for H(2)O concentrations of up to 40%. Beyond 40%, a significant reduction in SNR is observed. Scalar-coupling based (1)H-(15)N correlation experiments yield a nearly constant SNR for samples prepared with < or =30% H(2)O. Samples in which more H(2)O is employed for crystallization show a significantly reduced NMR intensity. Calculation of the SNR by taking into account the reduction in (1)H T (1) in samples containing more protons (SNR per unit time), yields a maximum SNR for samples crystallized using 30 and 40% H(2)O for scalar and dipolar coupling based experiments, respectively. A sensitivity gain of 3.8 is obtained by increasing the H(2)O concentration from 10 to 40% in the CP based experiment, whereas the linewidth only becomes 1.5 times broader. In general, we find that CP is more favorable compared to INEPT based transfer when the number of possible (1)H,(1)H interactions increases. At low levels of deuteration (> or =60% H(2)O in the crystallization buffer), resonances from rigid residues are broadened beyond detection. All experiments are carried out at MAS frequency of 24 kHz employing perdeuterated samples of the chicken alpha-spectrin SH3 domain.

Dynamic nuclear polarization of deuterated proteins
Akbey, Ü., Franks, W. T., Linden, A., Lange, S., Griffin(*), R. G., van Rossum, B. J.; Oschkinat, H.
Angew Chem Int Ed Engl, 49:7803-7806
(2010)

Tags: Protein Structure (Oschkinat)

Identification of small-molecule scaffolds for p450 inhibitors
von Kries, J. P., Warrier(*), T.; Podust(*), L. M.
Curr Protoc Microbiol, Chapter 17:Unit17 14
(2010)

Tags: Screening Unit (von Kries)

Abstract: Mycobacterium tuberculosis cytochrome P450 enzymes (CYP) attract ongoing interest for their pharmacological development potential, driving direct screening efforts against potential CYP targets with the ultimate goal of developing potent CYP-specific inhibitors and/or molecular probes to address M. tuberculosis biology. The property of CYP enzymes to shift the ferric heme Fe Soret band in response to ligand binding provides the basis for an experimental platform for high-throughput screening (HTS) of compound libraries to select chemotypes with high binding affinities to the target. Promising compounds can be evaluated in in vitro assays or in vivo disease models and further characterized by x-ray crystallography, leading to optimization strategies to assist drug design. Protocols are provided for compound library screening, analysis of inhibitory potential, and co-crystallization with the target CYP, as well as expression and purification of soluble CYP enzymes.

Design of chemical libraries with potentially bioactive molecules applying a maximum common substructure concept
Lisurek, M., Rupp, B., Wichard, J., Neuenschwander, M., von Kries, J. P., Frank(*), R., Rademann, J.; Kühne, R.
Mol Divers, 14:401-408
(2010)

Tags: Screening Unit (von Kries), Medicinal Chemistry (Rademann), Computational Chemistry/Drug Design (Kühne)

Abstract: Success in small molecule screening relies heavily on the preselection of compounds. Here, we present a strategy for the enrichment of chemical libraries with potentially bioactive compounds integrating the collected knowledge of medicinal chemistry. Employing a genetic algorithm, substructures typically occurring in bioactive compounds were identified using the World Drug Index. Availability of compounds containing the selected substructures was analysed in vendor libraries, and the substructure-specific sublibraries were assembled. Compounds containing reactive, undesired functional groups were omitted. Using a diversity filter for both physico-chemical properties and the substructure composition, the compounds of all the sublibraries were ranked. Accordingly, a screening collection of 16,671 compounds was selected. Diversity and chemical space coverage of the collection indicate that it is highly diverse and well-placed in the chemical space spanned by bioactive compounds. Furthermore, secondary assay-validated hits presented in this study show the practical relevance of our library design strategy.

The use of small molecule high-throughput screening to identify inhibitors of the proteinase 3-NB1 interaction
Choi(*), M., Eulenberg(*), C., Rolle(*), S., von Kries, J. P., Luft(*), F. C.; Kettritz(*), R.
Clin Exp Immunol, 161:389-396
(2010)

Tags: Screening Unit (von Kries)

Abstract: P>Anti-neutrophil cytoplasmic antibodies (ANCA) to proteinase 3 (PR3) are found in patients with small-vessel vasculitis. PR3-ANCA bind strongly to membrane PR3 (mPR3) that is presented by the NB1 receptor. We performed high-throughput screening using a small molecule library to identify compounds that inhibit PR3-NB1 binding. We established a human embryonic kidney (HEK293) cell-based system, where approximately 95 +/- 2% of the NB1-transfected cells expressed the NB1 receptor on the cell surface. Addition of 0 center dot 1 mu g/ml human PR3 to 104 NB1-expressing HEK293 cells resulted in PR3 binding that was detected by immunofluorescence using a fluorescence plate reader assay. We identified 13 of 20 000 molecules that inhibited PR3 binding by > 70%. Seven of 13 substances showed reproducible inhibition in four additional validation experiments. Two selected compounds (27519 and 27549) demonstrated a dose-dependent inhibition over a range from 6 center dot 25 to 100 mu M as measured by the plate reader assay. We used flow cytometry as a second assay, and found that both compounds reproducibly inhibited PR3 binding to NB1-transfected HEK293 cells at 50 mu M (inhibition to 42 +/- 4% with compound 27519 and to 47 +/- 6% with compound 27549 compared to the dimethylsulphoxide control). Furthermore, compounds 27519 and 27549 also inhibited binding of exogenous PR3 to human neutrophils. In contrast, the compounds did not decrease mPR3 expression on resting neutrophils, but reduced the tumour necrosis factor-alpha-mediated mPR3 increase on NB1pos neutrophils when present continuously during the assay. The findings suggest that small inhibitory compounds provide a potential therapeutic tool to reduce mPR3 by preventing its binding to NB1.

Renal ACE2 expression and activity is unaltered during established hypertension in adult SHRSP and TGR(mREN2)27
Kamilic(*), J., Hamming(*), I., Kreutz(*), R., Bolbrinker(*), J., Siems, W. E., Nassar(*), I., Sluimer(*), J. C., Walther(*), T., Navis(*), G. J.; van Goor(*), H.
Hypertens Res, 33:123-128
(2010)

Tags: Biochemical Neurobiology (Siems)

Abstract: Differential renal expression of a homolog of the angiotensin-converting enzyme (ACE), that is, ACE2, has been implicated as a genetic basis of polygenetic hypertension in the stroke-prone spontaneously hypertensive rat model. However, data on the role of ACE2 in hypertension are still inconclusive. Therefore, we analyzed kidney ACE2 mRNA, ACE2 protein and ACE2 enzyme activities in the adult polygenetic stroke-prone spontaneously hypertensive rat (SHRSP) and the monogenetic TGR(mREN2)27 rat models, in comparison with their normotensive reference strains, Wistar-Kyoto (WKY) and Spraque-Dawley (SD) rats, respectively. Kidney ACE2 mRNA was studied using quantitative real-time reverse transcriptase-PCR (RT-PCR) in cortex and medulla, whereas protein expression was scored semiquantitatively in detail in different renal structures using immunohistochemistry. Furthermore, total renal tissue ACE2 activity was measured using a fluorimetric assay that was specified by the ACE2 inhibitor DX600. In SHRSP and homozygous TGR(mREN2)27 rats with established hypertension, kidney ACE2 mRNA, protein and tissue ACE2 activities were not different from their respective WKY and SD reference strain, respectively. In addition, when we looked at renal localization, we found ACE2 protein to be predominantly present in glomeruli and endothelium with weak staining in distal and negative staining in proximal tubuli. Thus, our data challenge previous work that implicates ACE2 as a candidate gene for hypertension in SHRSP by reporting a significant reduction of ACE2 in the kidneys of SHRSP. Taken together, renal ACE2 is not altered in the SHRSP and TGR(mREN2)27 genetic rat models with established hypertension. Hypertension Research (2010) 33, 123-128; doi: 10.1038/hr.2009.191; published online 20 November 2009

Effects of ACE2 inhibition in the post-myocardial infarction heart
Kim(*), M. A., Yang(*), D., Kida(*), K., Molotkova(*), N., Yeo(*), S. J., Varki(*), N., Iwata(*), M., Dalton(*), N. D., Peterson(*), K. L., Siems, W. E., Walther(*), T., Cowling(*), R. T., Kjekshus(*), J.; Greenberg(*), B.
Journal of cardiac failure, 16:777-785
(2010)

Tags: Biochemical Neurobiology (Siems)

Abstract: BACKGROUND: There is evidence that angiotensin-converting enzyme 2 (ACE2) is cardioprotective. To assess this in the post-myocardial infarction (MI) heart, we treated adult male Sprague-Dawley rats with either placebo (PL) or C16, a selective ACE2 inhibitor, after permanent coronary artery ligation or sham operation. METHODS AND RESULTS: Coronary artery ligation resulting in MI between 25% to 50% of the left ventricular (LV) circumference caused substantial cardiac remodeling. Daily C16 administration from postoperative days 2 to 28 at a dose that inhibited myocardial ACE2 activity was associated with a significant increase in MI size and reduction in LV % fractional shortening. Treatment with C16 did not significantly affect post-MI increases in LV end-diastolic dimension but did inhibit increases in wall thickness and fibrosis in non-infarcted LV. On postoperative day 7, C16 had no significant effect on the increased level of apoptosis in the infarct and border zones nor did it significantly affect capillary density surrounding the MI. It did, however, significantly reduce the number of c-kit(+) cells in the border region. CONCLUSIONS: These findings support the notion that ACE2 exerts cardioprotective effects by preserving jeopardized cardiomyocytes in the border zone. The reduction in hypertrophy and fibrosis with C16, however, suggests that ACE2 activity has diverse effects on post-MI remodeling.

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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
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