2008
MicroRNAs Repress Mainly through mRNA Decay
29.12.2008
Stephanie Esslinger,
Klaus Förstemann
Angew. Chem. Int. Ed.,
2009,
48,
853–855, doi:10.1002/anie.200805127
published on 29.12.2008
Angew. Chem. Int. Ed., online article
Inhibition or degradation? MicroRNAs have been considered primarily as inhibitors of translation, even though degradation of mRNAs also plays a role in their repressive potential. Two research groups have now quantified the extent to which each mechanism contributes to gene regulation by combining mass spectrometry with transcriptome profiling. The surprising conclusion is that translational inhibition plays only a minor role!
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RNA polymerase fidelity and transcriptional proofreading
18.12.2008
Jasmin F Sydow
, Patrick Cramer
Curr. Opin. Struct. Biol.,
2009,
19(6),
732-739, doi:10.1016/j.sbi.2009.10.009
published on 18.12.2008
Curr. Opin. Struct. Biol., online article
Whereas mechanisms underlying the fidelity of DNA polymerases (DNAPs) have been investigated in detail, RNA polymerase (RNAP) fidelity mechanisms remained poorly understood. New functional and structural studies now suggest how RNAPs select the correct nucleoside triphosphate (NTP) substrate to prevent transcription errors, and how the enzymes detect and remove a misincorporated nucleotide during proofreading. Proofreading begins with fraying of the misincorporated nucleotide away from the DNA template, which pauses transcription. Subsequent backtracking of RNAP by one position enables nucleolytic cleavage of an RNA dinucleotide that contains the misincorporated nucleotide. Since cleavage occurs at the same active site that is used for polymerization, the RNAP proofreading mechanism differs from that used by DNAPs, which contain a distinct nuclease specific active site.
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The archaeal RNA polymerase subunit P and the eukaryotic polymerase subunit Rpb12 are interchangeable in vivo and in vitro
18.12.2008
Christoph Reich, Mirijam Zeller, Philipp Milkereit, Winfried Hausner, Patrick Cramer, Herbert Tschochner, Michael Thomm
Mol. Microbiol.,
2009,
71(4),
989-1002, doi:10.1111/j.1365-2958.2008.06577.x
published on 18.12.2008
Molecular Microbiology, online article
The general subunit of all three eukaryotic RNA polymerases, Rpb12, and subunit P of the archaeal enzyme show sequence similarities in their N-terminal zinc ribbon and some highly conserved residues in the C-terminus. We report here that archaeal subunit P under the control of a strong yeast promoter could complement the lethal phenotype of a RPB12 deletion mutant and that subunit Rpb12 from yeast can functionally replace subunit P during reconstitution of the archaeal RNA polymerase. The ΔP enzyme is unable to form stable open complexes, but can efficiently extend a dinucleotide on a premelted template or RNA on an elongation scaffold. This suggests that subunit P is directly or indirectly involved in promoter opening. The activity of the ΔP enzyme can be rescued by the addition of Rpb12 or subunit P to transcription reactions. Mutation of cysteine residues in the zinc ribbon impair the activity of the enzyme in several assays and this mutated form of P is rapidly replaced by wild-type P in transcription reactions. The conserved zinc ribbon in the N-terminus seems to be important for proper interaction of the complete subunit with other RNA polymerase subunits and a 17-amino-acid C-terminal peptide is sufficient to support all basic RNA polymerase functions in vitro.
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Identification, structure, and functional requirement of the Mediator submodule Med7N/31
04.12.2008
Tobias Koschubs, Martin Seizl, Laurent Larivière, Fabian Kurth, Sonja Baumli, Dietmar E Martin ,
Patrick Cramer
The EMBO Journal,
2008,
doi:10.1038/emboj.2008.254,
69 - 80
published on 04.12.2008
The EMBO Journal, online article
Mediator is a modular multiprotein complex required for regulated transcription by RNA polymerase (Pol) II. Here, we show that the middle module of the Mediator core contains a submodule of unique structure and function that comprises the N-terminal part of subunit Med7 (Med7N) and the highly conserved subunit Med31
(Soh1). The Med7N/31 submodule shows a conserved novel fold, with two proline-rich stretches in Med7N wrapping around the right-handed four-helix bundle of Med31. In vitro, Med7N/31 is required for activated transcription and can act in trans when added exogenously. In vivo, Med7N/31 has a predominantly positive function on the expression of a specific subset of genes, including genes involved in methionine metabolism and iron transport. Comparative phenotyping and transcriptome profiling
identify specific and overlapping functions of different Mediator submodules.

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Cooperativity in Adhesion Cluster Formation during Initial Cell Adhesion
01.12.2008
Christine Selhuber-Unkel, Monica Lopez-Garcia,
Horst Kessler, Joachim P. Spatz
Biophysical Journal,
2008,
95, 11,
5424 - 31
published on 01.12.2008
Biophysical Journal, online article
We have studied the initial phase of cell adhesion as a function of the lateral organization of individual integrin
molecules with single-cell force microscopy. Nanostructures, consisting of hexagonally ordered gold dots, were prepared with
diblock-copolymer micelle lithography and functionalized with arginine- glycine-aspartate peptides, thus defining integrin position
with nanometer resolution. Adhesion strength was characterized with an atomic force microscope and both cell detachment forces
and work of detachment showed a reinforcement of adhesion if the distance between integrin molecules was ,70 nm. This
reinforcement had already occurred at cell-substrate contact times ,5 min. We believe our results show quantitatively the
relevance of the distance between adjacent integrin binding sites rather than their density. Furthermore, we propose a model
describing the cooperative stabilization of early integrin clusters as a function of receptor patterning at the nanoscale

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Phospholipid scramblases and Tubby-like proteins belong to a new superfamily of membrane tethered transcription factors
13.11.2008
Alex Bateman, Robert D. Finn, Peter J. Sims, Therese Wiedmer, Andreas Biegert,
Johannes Söding
Bioinformatics,
2009,
25(2),
159-162, doi:10.1093/bioinformatics/btn595
published on 13.11.2008
Bioinformatics, online article
Motivation: Phospholipid scramblases (PLSCRs) constitute a family of cytoplasmic membrane-associated proteins that were identified based upon their capacity to mediate a Ca2+-dependent bidirectional movement of phospholipids across membrane bilayers, thereby collapsing the normally asymmetric distribution of such lipids in cell membranes. The exact function and mechanism(s) of these proteins nevertheless remains obscure: data from several laboratories now suggest that in addition to their putative role in mediating transbilayer flip/flop of membrane lipids, the PLSCRs may also function to regulate diverse processes including signaling, apoptosis, cell proliferation and transcription. A major impediment to deducing the molecular details underlying the seemingly disparate biology of these proteins is the current absence of any representative molecular structures to provide guidance to the experimental investigation of their function.
Results: Here, we show that the enigmatic PLSCR family of proteins is directly related to another family of cellular proteins with a known structure. The Arabidopsis protein At5g01750 from the DUF567 family was solved by X-ray crystallography and provides the first structural model for this family. This model identifies that the presumed C-terminal transmembrane helix is buried within the core of the PLSCR structure, suggesting that palmitoylation may represent the principal membrane anchorage for these proteins. The fold of the PLSCR family is also shared by Tubby-like proteins. A search of the PDB with the HHpred server suggests a common evolutionary ancestry. Common functional features also suggest that tubby and PLSCR share a functional origin as membrane tethered transcription factors with capacity to modulate phosphoinositide-based signaling.
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Cell interactions with hierarchically structured nano-patterned adhesive surfaces
10.11.2008
Marco Arnold, Marco Schwieder, Jacques Blümmel, Elisabetta A. Cavalcanti-Adam, Mónica López-Garcia,
Horst Kessler, Benjamin Geiger, Joachim P. Spatz
Soft Matter,
2009,
5,
72- 77
published on 10.11.2008
Soft Matter,
online article
The activation of well-defined numbers of integrin molecules in predefined areas by adhesion of tissue cells to biofunctionalized micro-nanopatterned surfaces was used to determine the minimum number of activated integrins necessary to stimulate focal adhesion formation. This was realized by combining micellar and conventional e-beam lithography, which enabled deposition of 6 nm large gold nanoparticles on predefined geometries. Patterns with a lateral spacing of 58 nm and a number of gold nanoparticles, ranging from 6 to 3000 per adhesive patch, were used. For v3-integrin activation, gold nanoparticles were coated with c(-RGDfK-)-thiol peptides, and the remaining glass surface was passivated to prevent non-specific protein adsorption and cell adhesion. Results show that focal adhesion formation is dictated by the underlying hierarchical nanopattern. Adhesive patches with side lengths of 3000 nm and separated by 3000 nm, or with side lengths of 1000 nm and separated by 1000 nm, containing approximately 3007 ± 193 or 335 ± 65 adhesive gold nanoparticles, respectively, induced the formation of actin-associated, paxillin-rich focal adhesions, comparable in size and shape to classical focal adhesions. In contrast, adhesive patches with side lengths of 500, 250 or 100 nm, and separated from adjacent adhesive patches by their respective side lengths, containing 83 ± 11, 30 ± 4, or 6 ± 1 adhesive gold nanoparticles, respectively, showed a significant increase in paxillin domain length, caused by bridging the pattern gap through an actin bundle in order to mechanically, synergistically strengthen each single adhesion site. Neither paxillin accumulation nor adhesion formation was induced if less than 6 c(-RGDfK-)-thiol functionalised gold nanoparticles per adhesion site were presented to cells.

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A NMR strategy to unambiguously distinguish nucleic acid hairpin and duplex conformations applied to a Xist RNA A-repeat
05.11.2008
Malgorzata M. Duszczyk, Katia Zanier,
Michael Sattler
Nucleic Acids Research,
2008,
36, 22,
7068 - 77
published on 05.11.2008
Nucleic Acids Research, online article
Structural investigations are frequently hindered by difficulties in obtaining diffracting crystals of the target protein. Here, we report the crystallization and structure solution of the U2AF homology motif (UHM) domain of splicing factor Puf60 fused to Escherichia coli thioredoxin A. Both modules make extensive crystallographic contacts, contributing to a well-defined crystal lattice with clear electron density for both the thioredoxin and the Puf60-UHM module. We compare two short linker sequences between the two fusion domains, GSAM and GSPPM, for which only the GSAM-linked fusion protein yielded diffracting crystals. While specific interdomain contacts are not observed for both fusion proteins, NMR relaxation data in solution indicate reduced interdomain mobility between the Trx and Puf60-UHM modules. The GSPPM-linked fusion protein is significantly more flexible, albeit both linker sequences have the same number of degrees of torsional freedom. Our analysis provides a rationale for the crystallization of the GSAM-linked fusion protein and indicates that in this case, a four-residue linker between thioredoxin A and the fused target may represent the maximal length for crystallization purposes. Our data provide an experimental basis for the rational design of linker sequences in carrier-driven crystallization and identify thioredoxin A as a powerful fusion partner that can aid crystallization of difficult targets.

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Can N-methylated amino acids serve as substitutes for prolines in conformational design of cyclic pentapeptides?
04.11.2008
Burkhardt Laufer, Jayanta Chatterjee, Andreas O. Frank,
Horst Kessler,
Journal of Peptide Science,
2008,
15, 3,
141 - 46
published on 04.11.2008
Journal of Peptide Science online article
The incorporation of proline into cyclic peptides seems to be the most promising way to induce -turn structures. Recently, however, it was shown that N-methylated amino acids might be even better suited than proline for introducing turn structures. Another property of proline, the ability to effect cis-peptide bonds, has also been reported for N-methylated amino acids. These findings raise the question if it might be possible to replace a proline by an N-methylated amino acid without altering the desired conformational features. The most important benefit of replacing proline by an N-methylated residue is that one recovers the side-chain functionalities, which could be used for enhancing binding selectivity, or to tune a cyclic peptide concerning its pharmacological properties.
Here, we compare cyclic peptides containing one or two prolines or N-methylated alanines and a combination of both with respect to preferred conformations and cis-peptide bonds. In addition, the positions have been investigated where an N-alkylated amino acid has to be incorporated to mimic structural aspects usually introduced by proline residues.

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Dimerization and Protein Binding Specificity of the U2AF Homology Motif of the Splicing Factor Puf60*
29.10.2008
Lorenzo Corsini, Michael Hothorn, Gunter Stier, Vladimir Rybin, Klaus Scheffzek, Toby J. Gibson,
Michael Sattler
The Journal of Biological Chemistry,
2008,
284, 1,
630-9
published on 29.10.2008
jbc, online article
PUF60 is an essential splicing factor functionally related and homologous to U2AF65. Its C-terminal domain belongs to the family of U2AF (U2 auxiliary factor) homology motifs (UHM), a subgroup of RNA recognition motifs that bind to tryptophan-containing linear peptide motifs (UHM ligand motifs, ULMs) in several nuclear proteins. Here, we show that the Puf60 UHM is mainly monomeric in physiological buffer, whereas its dimerization is induced upon the addition of SDS. The crystal structure of PUF60-UHM at 2.2 Å resolution, NMR data, and mutational analysis reveal that the dimer interface is mediated by electrostatic interactions involving a flexible loop. Using glutathione S-transferase pulldown experiments, isothermal titration calorimetry, and NMR titrations, we find that Puf60-UHM binds to ULM sequences in the splicing factors SF1, U2AF65, and SF3b155. Compared with U2AF65-UHM, Puf60-UHM has distinct binding preferences to ULMs in the N terminus of SF3b155. Our data suggest that the functional cooperativity between U2AF65 and Puf60 may involve simultaneous interactions of the two proteins with SF3b155.

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Ulrike Gaul comes to CIPSM! Germany, Munich – the house is rocking.
17.10.2008
2008,
published on 17.10.2008
www.humboldt-foundation.de
We are very happy that Ulrike Gaul, professor of molecular biology at Rockefeller University New York, will join CIPSM at the Gene Center in 2009 as a newly established CIPSM full professor. This CIPSM professorship for molecular systems biology has been selected for the first Alexander von Humboldt professorship ever to be awarded and comes with an prize money of five million EUR. With Ulrike Gaul joins the second top scientist from the USA CIPSM this year. Just in August Dirk Trauner, professor for organic chemistry from the University of California at Berkeley, moved with his research group to CIPSM. This shows among other gravitys like newly established state of the art equipment platforms, that Germany’s research community, especially in Munich, is teaming up to become the hot spot of the scene.
Ulrike Gaul is an internationally leading development biologist whose work on the fruit fly drosophila has contributed enormously to our understanding of gene regulation during organismic development and the role of glial cells in the nervous system. Her laboratory has discovered many new genes that control the formation of the blood-brain barrier and the efficient elimination of dying neurons by glia. In recent years, Gaul has been increasingly interested in how to decrypt and make a quantitative description of the complex genetic networks underlying embryonic pattern formation. Her work on regulating gene transcription and translation in early development, which has often involved working with physicists and bioinformaticians, is pioneering for linking organismic biology with the quantitative analysis of systems biology. Gaul will use the award money associated with the Humboldt professorship to establish a new research focus in molecular systems biology at the Gene Center of CIPSM. This research focus will complement the existing strengths of the Gene Center. The newly elected laureate is exhilarated about the distinction. "Combined with this very attractive offer from LMU and CIPSM, the Humboldt professorship will give me working conditions that are very hard to find in the US.” But it is not just the financial backing that incites her to move to Germany. “LMU and Munich are an excellent environment for my work,” says Gaul. “I really like the hands-on, collegial spirit everyone has in Munich, the cosmopolitan, fun-loving character of the city and the general spirit of optimism you feel in German science.”
About Systems Biology
Systems biology is based to genome and proteome research, and will play a pivotal role in the biosciences in the future. The aim of this interdisciplinary research field is not only to create a catalogue of all molecules participating in specific cellular processes but also, and above all, to understand and quantify the interplay of molecules in complex networks. One aim is to develop models for making predictions of the behavior of living cells. By closely combining biochemical, genomic and mathematical methods, the researchers at the Gene Center in Munich intend to explain the principles of gene regulation in higher cells and organisms, which will give fresh impetus to cancer and dementia research as well as the development of stem cell therapies. The head of the Gene Center, Professor Patrick Cramer, is especially delighted that, with Ulrike Gaul, LMU has gained a top international researcher for the Gene Center and the national cluster of excellence for protein sciences CIPSM: “The appointment of Ulrike Gaul is our fifth appointment from the US since 2001.“ The Humboldt professorship is a special distinction for Gaul, but also for her future colleagues here in Munich: “It is very important," says Cramer, “to recruit only the very best. I am very grateful for everyone who helped make this appointment happen. Appointing Gaul is a central aspect of our strategy for expanding into the future field of systems biology and, with it, for further increasing the international visibility and scientific capacity of the Gene Center and the CIPSM cluster of excellence. We can once again assert our leading role in establishing innovative biological research within Europe.”
Vita Professor Ulrike Gaul
Professor Ulrike Gaul studied biochemistry and physics in Tübingen, and received her doctorate in 1988 at the Max Planck Institute for Developmental Biology under Professor Herbert Jäckle. After research visits to the University of Washington and University of California in Berkeley, she has been a professor since 1993 at Rockefeller University in New York, one of the world’s leading biomedical research centers. Gaul plans to move with her group to the Gene Center of CIPSM in the spring of 2009.
Alexander von Humboldt Professorships
The most valuable international award for research in Germany is being granted by the Alexander von Humboldt Foundation (www.humboldt-foundation.de) globally leading academics of all disciplines working abroad. The award is valued at up to five million EUR and is designed to allow the winners to spend five years working on ground-breaking research at German universities. The award ceremony will take place in Berlin in May 2009.
“I am delighted that we have been able to nominate outstanding candidates for the award. The Alexander von Humboldt Professorship is a strong argument for convincing cutting-edge researchers from all over the world to come to Germany,” Federal Minister of Research, Dr. Annette Schavan, commented in Berlin on Wednesday. “We are making an important contribution to maintaining a permanent position for Germany in the top international league,” Schavan said.

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Structure of the intracellular domain of the amyloid precursor protein in complex with Fe65-PTB2
01.11.2008
Jens Radzimanowski, Bernd Simon, Konrad Beyreuther, Irmgard Sinning, KlemensWild,
Michael Sattler
EMBO reports,
2008,
9,
1134-40
published on 17.10.2008
EMBO reports, online article
Viscotoxins are small cationic proteins found in European mistletoe Viscum album. They are highly toxic towards phytopathogenic fungi and cancer cells. Heterologous expression of viscotoxins would broaden the spectrum of methods to be applied for better understanding of their structure and function and satisfy possible biopharmaceutical needs. Here, we evaluated 13 different proteins as a fusion partners for expression in Escherichia coli cells: His6 tag and His6-tagged versions of GB1, ZZ tag, Z tag, maltose binding protein, NusA, glutathione S-transferase, thioredoxin, green fluorescent protein, as well as periplasmic and cytosolic versions of DsbC and DsbA. The fusion to thioredoxin gave the highest yield of soluble viscotoxin. The His6-tagged fusion protein was captured with Ni2+ affinity chromatography, subsequently cleaved with tobacco etch virus protease. Selective precipitation by acidification of the cleavage mixture was followed by cation exchange chromatography. This protocol yielded 5.2 mg of visctoxin A3 from 1 l of culture medium corresponding to a recovery rate of 68%. Mass spectrometry showed a high purity of the sample and the presence of three disulfide bridges in the recombinant viscotoxin. Proper folding of the protein was confirmed by heteronuclear NMR spectra recorded on a uniformly 15N-labeled sample. Recombinant viscotoxins prepared using this protocol are toxic to HeLa cells and preserve the activity differences between isoforms B and A3 found in native proteins.

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Screening of fusion partners for high yield expression and purification of bioactive viscotoxins
17.10.2008
Julius Bogomolovasa, Bernd Simona,
Michael Sattler, Gunter Stier
Protein Expression and Purification,
2008,
64, 1,
16-23
published on 17.10.2008
Protein Expression and Purification, online article
Viscotoxins are small cationic proteins found in European mistletoe Viscum album. They are highly toxic towards phytopathogenic fungi and cancer cells. Heterologous expression of viscotoxins would broaden the spectrum of methods to be applied for better understanding of their structure and function and satisfy possible biopharmaceutical needs. Here, we evaluated 13 different proteins as a fusion partners for expression in Escherichia coli cells: His6 tag and His6-tagged versions of GB1, ZZ tag, Z tag, maltose binding protein, NusA, glutathione S-transferase, thioredoxin, green fluorescent protein, as well as periplasmic and cytosolic versions of DsbC and DsbA. The fusion to thioredoxin gave the highest yield of soluble viscotoxin. The His6-tagged fusion protein was captured with Ni2+ affinity chromatography, subsequently cleaved with tobacco etch virus protease. Selective precipitation by acidification of the cleavage mixture was followed by cation exchange chromatography. This protocol yielded 5.2 mg of visctoxin A3 from 1 l of culture medium corresponding to a recovery rate of 68%. Mass spectrometry showed a high purity of the sample and the presence of three disulfide bridges in the recombinant viscotoxin. Proper folding of the protein was confirmed by heteronuclear NMR spectra recorded on a uniformly 15N-labeled sample. Recombinant viscotoxins prepared using this protocol are toxic to HeLa cells and preserve the activity differences between isoforms B and A3 found in native proteins.

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A nano-positioning system for macromolecular structural analysis
12.10.2008
Adam Muschielok, Joanna Andrecka, Anass Jawhari, Florian Brückner,
Patrick Cramer.,
Jens Michaelis
Nature Methods,
2011,
doi:10.1038/nmeth.1259,
965-971
published on 12.10.2008
Nature Methods, online article
Very often, the positions of flexible domains within macromolecules as well as within macromolecular complexes
cannot be determined by standard structural biology methods.
To overcome this problem, we developed a method that uses
probabilistic data analysis to combine single-molecule
measurements with X-ray crystallography data. The method
determines not only the most likely position of a fluorescent dye molecule attached to the domain but also the complete three-dimensional probability distribution depicting the experimental uncertainty. With this approach, single-pair fluorescence resonance energy transfer measurements can now be used as a quantitative tool for investigating the position and dynamics of flexible domains within macromolecular complexes. We applied this method to find the position of the 5¢ end of the nascent RNA exiting transcription elongation complexes of yeast (Saccharomyces cerevisiae) RNA polymerase II and studied the influence of transcription factor IIB on the position of the RNA.

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Deuterated polymer gels for measuring anisotropic NMR parameters with strongly reduced artefacts
06.10.2008
Grit Kummerlöwe, Sebastian Knör, Andreas O. Frank, Thomas Paululat,
Horst Kessler, Burkhard Luy
ChemComm,
2008,
10.1039/b812905c,
5722-4
published on 06.10.2008
ChemComm,
online article
Perdeuterated poly(styrene) is introduced as an almost artefactfree
and arbitrarily scalable alignment medium for measuring residual dipolar couplings and other anisotropic NMR parameters; the spectral quality achievable in this new medium is demonstrated for HSQC spectra leading to the conformational analysis of staurosporine and homonuclear TOCSY-type experiments.

The oxazolidinone antibiotics perturb the ribosomal peptidyl-transferase center and effect tRNA positioning
30.08.2008
Daniel N Wilson, Frank Schluenzen, Joerg M. Harms, Agata L. Starosta, Sean R. Connell, Paola Fucini
PNAS,
2008,
published on 30.08.2008
PNAS, online article
The oxazolidinones represent the first new class of antibiotics to
enter into clinical usage within the past 30 years, but their binding site and mechanism of action has not been fully characterized. We
have determined the crystal structure of the oxazolidinone linezolid
bound to the Deinococcus radiodurans 50S ribosomal subunit.
Linezolid binds in the A site pocket at the peptidyltransferase center of the ribosome overlapping the aminoacyl moiety of an A-site bound tRNA as well as many clinically important antibiotics. Binding of linezolid stabilizes a distinct conformation of the universally conserved 23S rRNA nucleotide U2585 that would be
nonproductive for peptide bond formation. In conjunction with
available biochemical data, we present a model whereby oxazolidinones impart their inhibitory effect by perturbing the correct
positioning of tRNAs on the ribosome.

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Perspectives on NMR in drug discovery: a technique comes of age
26.08.2008
Maurizio Pellecchia, Ivano Bertini, David Cowburn, Claudio Dalvit, Ernest Giralt, Wolfgang Jahnke, Thomas L. James, Steve W. Homans,
Horst Kessler, Claudio Luchinat, Bernd Meyer, Hartmut Oschkinat, Jeff Peng, Harald Schwalbe, Gregg Siegal
Nature Reviews - Drug Disc,
2008,
7,
738-45
published on 26.08.2008
Nature Reviews - Drug discovery,
online article
in the past decade, the potential of harnessing the ability of nuclear
magnetic resonance (NMr) spectroscopy to monitor intermolecular interactions as a tool for drug discovery has been increasingly appreciated in academia and industry. in this Perspective, we highlight some of the major applications of NMR in drug discovery, focusing on hit and lead generation, and provide a critical
analysis of its current and potential utility.

CIPSM Coordinator Horst Kessler wins Josef Rudinger Award
22.08.2008
2008,
published on 22.08.2008
http://www.eurpepsoc.com
CIPSM Coordinator Horst Kessler wins 2008 Joseph Rudinger Award of the European Peptide Society for his lifetime achievements in peptide chemistry!
The Joseph Rudinger Award award is presented „in commemoration of Josef Rudinger’s role in the foundation of the European Peptide Symposia and of the diverse contributions he made to peptide chemistry.” We thank and congratulate Horst Kessler! Well done!

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YEAH! CIPSM-Junior-Group Leader Katja Strässer receives big €-EU-Grant
19.08.2008
2008,
published on 19.08.2008
ERC
CIPSM-Junior-Group Leader Katja Sträßer has done it! She received a grant of nearly one million Euros from the European Research Council for the next five years. She will explore the understanding of a new aspect of how genetic information is read out by a cell. Many of the individual steps examined separately in the past are coupled with each other to allow for highly efficient regulation and control of gene expression.

Searching for the Builder of the Bridge from Gene to Protein
The European Research Council (ERC) promotes pioneering basic research by supporting excellent and unusually creative scientists. CIPSM-Junior-Group Leader Dr. Katja Sträßer from the Gene Center at Ludwig-Maximilians-Universität (LMU) München successfully took part in the “ERC Starting Grant Scheme”, a new EU program for junior scientists. Dr. Sträßer is one of only 300 applicants whose projects were selected from among 10,000 project proposals throughout Europe. Sträßer will now receive about one million Euros over the next five years. The biologist’s research elucidates the mechanisms of gene expression or, in other words, the conversion of genetic information into proteins. Genes are sections of DNA, the molecule that governs heredity and contains the “blueprint” for building proteins. “What interests me most is how the individual steps of the gene expression are coupled,” explains Sträßer, “The reason being that this explains how the conversion of genetic information is controlled and regulated.” In a past project she was able to demonstrate that the Ctk1 protein plays a role during different temporally as well as spatially separated phases of gene expression. In the newly funded project she therefore intends to examine the function of this molecule more closely and identify other factors that connect different steps of gene expression. The individual steps of gene expression are already well understood. The coupling of these different steps, however, is a completely new mechanism for regulation – and also occurs in humans.
In higher organisms, gene expression entails a variety of different steps: First, during transcription the genetic information is copied into an RNA molecule. RNA is a nucleic acid chemically similar to DNA. This messenger RNA or mRNA is then processed, that is chemically altered, and packaged by different proteins in a complex that is then exported out of the nucleus. Within the cell interior or cytoplasm, the genetic information contained within the mRNA is finally converted into a protein during translation. “These processes, which are for the most part temporally and spatially separated, are already well understood,” explains Sträßer. “Only in recent years, it has been shown that some of these processes do not operate independently of each other but are interconnected.” This is especially true for processes that take place in the nucleus or in cytoplasm. The main objective of the newly funded project is to demonstrate a coupling of spatially separate processes or, more specifically, the coupling of transcription to translation.”
A first step in this direction is already taken: Sträßer recently demonstrated that one well-known protein mediates such a wide-ranging coupling. The Ctk1 protein in yeast ensures that transcription is not prematurely terminated and also plays an important role in the processing of the mRNA. “We demonstrated that Ctk1 interacts with the TREX complex, which couples transcription with the nuclear export of the mRNA,” reports the biologist. “Our new results show that Ctk1 is also important for efficient translation or, in other words, for the synthesis of proteins in the cytoplasm. It is highly likely that the human counterpart of Ctk1, namely the CDK9 protein, also performs this very surprising function. We suspect that Ctk1 binds to the mRNA after the mRNA is processed and is transported to the cytoplasm along with the mRNA where it can then enhance translation.” This protein would therefore be one of the first examples of a connection between gene expression processes taking place in the nucleus and the cytoplasm.
One objective of the planned project is therefore a detailed analysis of the various functions of Ctk1. Furthermore, additional factors with a similarly broad range of functions in gene expression shall be identified. Sträßer is also interested in ribosomes, large complexes made up of proteins and nucleic acids. Ribosomes are responsible for the synthesis of the encoded proteins using mRNAs as a template. “We want to examine how and which ribosomal proteins are chemically modified and which factors participate in this process,” explains Sträßer. “These modifications are important for the regulation of many cellular processes. Yet to date very little is known about the modification of ribosomes, which are so important for protein synthesis. This makes the modifications alone a very exciting object to study. In addition, these modifications could also be important for the coupling of translation with transcription. Finally, we will examine whether our results obtained with the yeast S. cerevisiae as a model system also hold true for mammal cells. Thus, we aim to identify mechanisms that connect gene expression processes in nucleus and cytoplasm in all higher organisms.”
Since January 2003, Dr. Katja Sträßer is the head of an independent research group at the Gene Center at LMU Munich. The 36-year-old biotechnologist received the prestigious EMBO Young Investigator Award in 2004 and the “Habilitation” Prize of the Dr. Klaus Römer Endowment at the LMU Department of Chemistry and Biochemistry in 2007. The ERC's decision was solely based on the scientific excellence of the applicant and the proposed project. Projects eligible for funding must be highly innovative – risky but, when successful, opening new and important scientific horizons. The project is expected to involve cooperations as well as a high degree of interdisciplinarity. This EU program for the advancement of basic research was established in 2007.
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A new tRNA intermediate revealed on the ribosome during EF4-mediated back-translocation
17.08.2008
Sean R Connell, Maya Topf, Yan Qin,
Daniel N Wilson, Thorsten Mielke, Paola Fucini, Knud H Nierhaus, Christian M T Spahn
Nature structural & molecular biology,
2008,
doi:10.1038/nsmb.1469,
published on 17.08.2008
Nature structural & molecular biology, online article
EF4 (LepA) is an almost universally conserved translational GTPase in eubacteria. It seems to be essential under environmental stress conditions and has previously been shown to back-translocate the tRNAs on the ribosome, thereby reverting the canonical
translocation reaction. In the current work, EF4 was directly visualized in the process of back-translocating tRNAs by singleparticle cryo-EM. Using flexible fitting methods, we built a model of ribosome-bound EF4 based on the cryo-EM map and a recently published unbound EF4 X-ray structure. The cryo-EM map establishes EF4 as a noncanonical elongation factor that interacts not only with the elongating ribosome, but also with the back-translocated tRNA in the A-site region, which is present in a previously unseen, intermediate state and deviates markedly from the position of a canonical A-tRNA. Our results, therefore, provide insight into the underlying structural principles governing back-translocation.

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A structure refinement protocol combining NMR residual dipolar couplings and small angle scattering restraints
01.08.2008
F. Gabel, B. Simon, M.Nilges, M. Petoukhov, D.Svergun,
Michael Sattler
Biomol NMR Assign,
2008,
41, 4,
199-208
published on 01.08.2008
Biomol NMR Assign, online article
We present the implementation of a target function based on Small Angle Scattering data (Gabel et al. Eur Biophys J 35(4):313–327, 2006) into the Crystallography and NMR Systems (CNS) and demonstrate its utility in NMR structure calculations by simultaneous application of small angle scattering (SAS) and residual dipolar coupling (RDC) restraints. The efficiency and stability of the approach are demonstrated by reconstructing the structure of a two domain region of the 31 kDa nuclear export factor TAP (TIP-associated protein). Starting with the high resolution X-ray structures of the two individual TAP domains, the translational and orientational domain arrangement is refined simultaneously. We tested the stability of the protocol against variations of the SAS target parameters and the number of RDCs and their uncertainties. The activation of SAS restraints results in an improved translational clustering of the domain positions and lifts part of the fourfold degeneracy of their orientations (associated with a single alignment tensor). The resulting ensemble of structures reflects the conformational space that is consistent with the experimental SAS and RDC data. The SAS target function is computationally very efficient. SAS restraints can be activated at different levels of precision and only a limited SAS angular range is required. When combined with additional data from chemical shift perturbation, paramagnetic relaxation enhancement or mutational analysis the SAS refinement is an efficient approach for defining the topology of multi-domain and/or multimeric biomolecular complexes in solution based on available high resolution structures (NMR or X-ray) of the individual domains.

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Genome-associated RNA Polymerase II Includes the Dissociable Rpb4/7 Subcomplex*
30.07.2008
Anna J. Jasiak, Holger Hartmann, Elena Karakasili, Marian Kalocsay, Andrew Flatley, Elisabeth Kremmer, Katja Strässer, Dietmar E. Martin, Johannes Söding,
Patrick Cramer
Journal of Biological Chemistry,
2008,
10.1074/jbc.M803237200,
26423-26427
published on 30.07.2008
jbc,
online article
Yeast RNA polymerase (Pol) II consists of a 10-subunit core enzyme and the Rpb4/7 subcomplex, which is dispensable for catalytic activity and dissociates in vitro. To investigate whether Rpb4/7 is an integral part of DNA-associated Pol II in vivo, we used chromatin immunoprecipitation coupled to high resolution tiling microarray analysis. We show that the genome-wide occupancy profiles for Rpb7 and the core subunit Rpb3 are essentially identical. Thus, the complete Pol II associates with DNA in vivo, consistent with functional roles of Rpb4/7 throughout the transcription cycle.

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Backbone assignment of the UHM domain of Puf60 free and bound to five ligands
22.07.2008
Lorenzo Corsini,
Michael Sattler
Biomol NMR Assign,
2008,
2,
published on 22.07.2008
Biomol NMR Assign, online article
U2AF homology motifs (UHM) are protein domains that bind peptidic UHM ligand motifs (ULM) and thus form an intricate network of interactions involved in splicing regulation. Here, we report the backbone assignment of the UHM domain of the splicing factor Puf60 as well as 1H, 15N chemical shifts upon binding of the ULM
peptides U2AF65 (85–112), SF1 (1–25), SF3b155 (194–
229), SF3b155 (317–357), and Prp16 (201–238).

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N-Methylation of Peptides: A New Perspective in Medicinal Chemistry
18.07.2008
Jayanta Chatterjee, Chaim Gilon, Amnon Hoffmann,
Horst Kessler
Accounts of Chemical Research,
2008,
DOI: 10.1021/ar8000603,
published on 18.07.2008
http://pubs.acs.org , online article
The potential of peptides as drug candidates is limited by their poor pharmacokinetic properties. Many peptides have a
short half-life in vivo and a lack of oral availability. Inspired by the excellent pharmacokinetic profile of cyclosporine, a natural, multiply N-methylated cyclic peptide, we envisioned multiple N-methylation as a promising way to rationally improve
key pharmacokinetic characteristics. In this Account, we summarize our efforts toward modulating the properties of peptides by multiple N-methylation. As a first step, we simplified the synthesis of N-methylated amino acids in solution, by employing very mild conditions that could be tolerated by the diverse protecting groups required when working with naturally occurring amino acids. We also report the rapid and inexpensive syntheses of N-methylated peptides on a solid support; this facilitated the N-methyl scanning of bioactive peptides. Because of a lack of information regarding the conformational behavior of multiply N-methylated peptides, a complete library of N-methylated cyclic alanine pentapeptides was synthesized. The library provided
valuable insight into the conformational modulation of cyclic peptides by N-methylation. This information is extremely valuable
for the design of bioactive peptides and spatial screening of cyclic N-methylated peptides. To demonstrate the applicability of N-methylation to highly active but poorly bioavailable peptides, we performed a full N-methyl scan of the cyclopeptidic somatostatin analog cyclo(-PFwKTF-), known as the Veber-Hirschmann peptide. We show here for the first time that the simple approach of multiple N-methylation can drastically improve the metabolic stability
and intestinal permeability of peptides, for example, resulting in 10% oral bioavailability for a tri-N-methylated Veber-Hirschmann peptide analog. In addition, we also describe a designed approach to N-methylated peptide library synthesis, which can accelerate the screening of N-methylated bioactive peptides. Finally, we find that multiple N-methylation of a cyclic hexapeptide integrin antagonist of GPIIb-IIIa (RIIb_3 integrin), cyclo(-GRGDfL-), increases the selectivity of this peptide toward different integrin subtypes. This result demonstrates the utility of multiple N-methylation in elucidating the bioactive conformation of peptides.

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The structure of a folding intermediate provides insight into differences in immunoglobulin amyloidogenicity
02.07.2008
Matthias J. Feige, Sandra Groscurth, Moritz Marcinowski, Zu Thur Yew, Vincent Truffault, Emanuele Paci,
Horst Kessler, Johannes Buchner
PNAS,
2008,
105, no.36,
13373-8
published on 02.07.2008
PNAS,
online article
Folding intermediates play a key role in defining protein folding
and assembly pathways as well as those of misfolding and aggregation. Yet, due to their transient nature, they are poorly accessible to high-resolution techniques. Here, we made use of the intrinsically slow folding reaction of an antibody domain to characterize its major folding intermediate in detail. Furthermore, by a single point mutation we were able to trap the intermediate in equilibrium and characterize it at atomic resolution. The intermediate
exhibits the basic b-barrel topology, yet some strands are distorted.
Surprisingly, two short strand-connecting helices conserved
in constant antibody domains assume their completely
native structure already in the intermediate, thus providing a
scaffold for adjacent strands. By transplanting these helical elements into b2-microglobulin, a highly homologous member of the
same superfamily, we drastically reduced its amyloidogenicity.
Thus, minor structural differences in an intermediate can shape the
folding landscape decisively to favor either folding or misfolding.

Induction of Cell Polarization and Migration by a Gradient of Nanoscale Variations in Adhesive Ligand Spacing
18.06.2008
Marco Arnold, Vera C. Hirschfeld-Warneken, Theobald Lohmüller, Patrick Heil, Jacques Blümmel, Elisabetta A. Cavalcanti-Adam, Monica Lopez-Garcia, Paul Walther,
Horst Kessler, Benjamin Geiger, Joachim P. Spatz
NANO Letters,
2008,
DOI: 10.1021/nl801483w,
published on 18.06.2008
NANO Letters, online article
Cell interactions with adhesive surfaces play a vital role in the regulation of cell proliferation, viability, and differentiation, and affect multiple biological processes. Since cell adhesion depends mainly on the nature and density of the adhesive ligand molecules, spatial molecular patterning, which enables the modulation of adhesion receptor clustering, might affect both the structural and the signaling activities of the adhesive interaction. We herein show that cells plated on surfaces that present a molecularly defined spacing gradient of an integrin RGD ligand can sense small but consistent differences in adhesive ligand spacing of about 1 nm across the cell diameter, which is approximately 61 μm when the spacing includes 70 nm. Consequently, these positional cues induce cell polarization and initiate cell migration and signaling. We propose that differential positional clustering of the integrin transmembrane receptors is used by cells for exploring and interpreting their environment, at high spatial sensitivity.

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Structural basis of transcription inhibition by alpha-amanitin and implications for RNA polymerase II translocation
13.06.2008
Florian Brueckner &
Patrick Cramer.
Nature Structural & Molecular Biology,
2011,
doi:10.1038/nsmb.1458,
811-818
published on 13.06.2008
Nature Structural & Molecular Biology, online article
To study how RNA polymerase II translocates after nucleotide incorporation, we prepared elongation complex crystals in which pre- and post-translocation states interconvert. Crystal soaking with the inhibitor alpha-amanitin locked the elongation complex in a new state, which was refined at 3.4-Å resolution and identified as a possible translocation intermediate. The DNA base entering the active site occupies a 'pretemplating' position above the
central bridge helix, which is shifted and occludes the templating position. A leucine residue in the trigger loop forms a wedge at the shifted bridge helix, but moves by 13 Å to close the active site during nucleotide incorporation. Our results support a Brownian ratchet mechanism that involves swinging of the trigger loop between open, wedged and closed positions, and suggest that alpha-amanitin impairs nucleotide incorporation and translocation by trapping the trigger loop and bridge helix.

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Rational Design of Highly Active and Selective Ligands for the alpha-5-beta-1 Integrin Receptor
09.06.2008
Dominik Heckmann, Axel Meyer, Burkhardt Laufer; Grit Zahn, Roland Stragies,
H. Kessler
ChemBioChem,
2008,
9,
1397-1407
published on 09.06.2008
ChemBioChem; online article
The inhibition of integrin function is a major challenge in medicinal
chemistry. Potent ligands are currently in different stages of clinical trials for the antiangiogenic therapy of cancer and agerelated
macula degeneration (AMD). The subtype a5b1has recently
been drawn into the focus of research because of its genuine role in angiogenesis. In our previous work we could demonstrate that the lack of structural information about the receptor could be overcome by a homology model based on the X-ray structure of the avb3 integrin subtype and the sequence similarities between both receptors. In this work, we describe the rational design and synthesis of high affinity alpha-5-beta-1binders, and the optimisation of selectivity against avb3 by means of extensive SAR
studies and docking experiments. A first series of compounds
based on the tyrosine scaffold resulted in affinities in the low and
even subnanomolar range and selectivities of 400-fold against avb3. The insights about the structure–activity relationship gained from tyrosine-based ligands could be successfully transferred to ligands that bear an aza-glycine scaffold to yield alpha-5-beta-1 ligands with affinities of ~1nm and selectivities that exceed 104-
fold. The ligands have already been successfully employed as
selective alpha-5-beta-1ligand s in biological research and might serve as lead structures for antiangiogenic cancer therapy.

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Crystallization of RNA polymerase I subcomplex A14/A43 by iterative prediction, probing and removal of flexible regions
09.05.2008
Sebastian R. Geiger, Claus.-D. Kuhn, Christoph Leidig, Jörg Renkawitz,
Patrick Cramer
Acta Crystallographica,
2008,
DOI: 10.1107/S174430910800972X,
Volume 64, Issue 5, pages 413–418
published on 09.05.2008
Acta Crystallographica,
online article
The removal of flexible protein regions is generally used to promote crystallization, but advanced strategies to quickly remove multiple flexible regions from proteins or protein complexes are lacking. Here, it is shown how a protein heterodimer with multiple flexibilities, the RNA polymerase I subcomplex A14/A43, could be crystallized with the use of an iterative procedure of predicting flexible regions, experimentally testing and improving these predictions and combining deletions of flexible regions in a stepwise manner. This strategy should enable the crystallization of other proteins and subcomplexes with multiple flexibilities, as required for hybrid structure solution of large macromolecular assemblies.

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The Role of 23S Ribosomal RNA Residue A2451 in Peptide Bond Synthesis Revealed by Atomic Mutagenesis
24.04.2008
Kathrin Lang; Matthias Erlacher;
Daniel N. Wilson; Ronald Micura; Norbert Polacek
Chemistry & Biology,
2008,
Vol. 15,
DOI: 10.1016/j.chembiol.2008.03.014
published on 24.04.2008
Chemistry & Biology;
online article
Peptide bond formation is a fundamental reaction in biology, catalyzed by the ribosomal peptidyl-transferase ribozyme. Although all active-site 23S ribosomal RNA nucleotides are universally conserved, atomic mutagenesis suggests that these nucleobases
do not carry functional groups directly involved in peptide bond formation. Instead, a single ribose 20-hydroxyl group at A2451 was identified to be of pivotal importance. Here, we altered the chemical
characteristics by replacing its 20-hydroxyl with selected functional groups and demonstrate that hydrogen donor capability is essential for transpeptidation. We propose that the A2451-20-hydroxyl directly
hydrogen bonds to the P-site tRNA-A76 ribose. This promotes an effective A76 ribose C20-endo conformation to support amide synthesis via a proton shuttle mechanism. Simultaneously, the direct interaction of A2451 with A76 renders the intramolecular transesterification of the peptide from the 30- to 20-oxygen unfeasible, thus promoting effective peptide bond synthesis.

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Isothiocyanate-functionalized RGD peptides for tailoring cell-adhesive surface patterns
22.04.2008
Sviatlana Kalinina, Hartmut Gliemann, Monica Lopez-Garcia, Andre Petershans, Jörg Auernheimer, Thomas Schimmel, Michael Bruns, Alexandra Schambony,
Horst Kessler, Doris Wedlich
Biomaterials,
2008,
Vol. 29,
3004-13
published on 22.04.2008
Biomaterials,
online article
With the advances made in surface patterning by micro- and nanotechnology, alternative methods to immobilize biomolecules for different purposes are highly desired. RGD peptides are commonly used to create cell-attractive surfaces for cell-biological and also medical applications. We have developed a fast,
one-step method to bind RGD peptides covalently to surfaces by thiourea formation, which can be applied to structured and unstructured materials. RGD peptides were fused to an isothiocyanate anchor during synthesis and directly immobilized on amino-terminated surfaces. The spreading behavior of
fibroblasts and the formation of focal contacts served to prove the applicability of the coupling method. Two different linear peptides and one cyclic peptide were compared. All the peptides induced spreading behavior and the formation of focal contacts in murine fibroblasts. Adhesion was specific as cells neither recognized the corresponding negative control peptides nor spread in the presence of soluble H-RGDSOH peptide. We successfully applied our coupling method to functionalize surface patterns created by
microcontact printing (mCP) and chemical etching. Cells recognize areas selectively coated with RGDcontaining
peptides, proliferate and maintain this preference during long-term cultivation. Our method significantly facilitates surface modification with any kind of peptide – even for the preparation of
peptide-functionalized small surface areas.

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Multiplexed Proteomics Mapping of Yeast RNA Polymerase II and III Allows Near-Complete Sequence Coverage and Reveals Several Novel Phosphorylation Sites
17.04.2008
Shabaz Mohammed, Kristina Lorenzen, Robert Kerkhoven, Bas van Breukelen, Alessandro Vannini,
Patrick Cramer, Albert J. R. Heck
Analytical Chemistry,
2008,
DOI: 10.1021/ac7024283,
pp 3584–3592
published on 17.04.2008
Analytical Chemistry,
online article
The multisubunit RNA polymerases (Pols) II and III
synthesize mainly eukaryotic mRNAs and tRNAs, respectively.
Pol II and Pol III are protein complexes consisting of 12 and 17 subunits. Here we analyzed both yeast Pol II and Pol III by multiplexed mass spectrometric analysis using various proteases and both collision induced and electron transfer dissociation. The cumulative data obtained from using the various proteases (trypsin, chymotrypsin, Glu-C and Lys-C) and the two peptide fragmentation approaches allowed us to map nearly the complete sequences of all constituents of both Pol II and III. Notably, chymotrypsin behaved equally well as and in certain circumstances better than trypsin in the context
of protein coverage. Although the available high resolution
structures have exposed extensive mechanistic insights
into transcription, the role of post-translational modification in these processes has been addressed to a lesser extent. In our analysis of Pol II and III we detected 19 phosphorylation sites, of which 12 have not been previously reported. Identified phosphosites were mapped on the Pol II structure which provided indications that they might play a role in regulating the conformation of the clamp region and, as a consequence, interaction of Pol II with nucleic acids. The described multiplexed proteomics approach is generic and reveals that it is possible to map a protein complex to near completion while applying less than 5 μg (approximately 10 pmol) of total starting
material.

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The sweetest candy for the virus
16.04.2008
Andreas O. Frank;
H. Kessler
Nature,
2008,
452,
822-23
published on 16.04.2008
nature; online article
A few weeks ago, many of our friends suffered from ‘winter vomiting disease’, a form of gastroenteritis that swept epidemically across
Germany. This unpleasant disease is caused by the highly contagious norovirus, a member of the Caliciviridae family of RNA viruses. Caliciviruses are unusual because they dock to sugar
residues on the surfaces of cells to be invaded, rather than to proteins. The chemical details of this process were unknown, but reporting in the Journal of the American Chemical Society, Rademacher et al.1 present a nuclear magnetic resonance (NMR) study that determines which sugar residues are involved in the binding of another calicivirus, rabbit haemorrhagic disease
virus (RHDV), to cells. Their technique could be used to develop drugs that block the entry of viruses into cells.

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Introduction of Functional Groups into Peptides via N-Alkylation
12.04.2008
O. Demmer, I. Dijkgraaf, M. Schottelius, H.-J. Wester,
H. Kessler
Organic Letters,
2008,
10,
2015-18
published on 12.04.2008
organic letters; online article
An optimized protocol for the mild and selective Fukuyama−Mitsunobu reaction was used for mono- and di-N-alkylation on solid support. Thereby, nonfunctionalized aliphatic and aromatic residues are quickly introduced into transiently protected, primary amines of a linear peptide. N-Alkylation can also be used to implement alkyl chains carrying (protected) functionalities suited for subsequent modification. Applicability of this method is demonstrated by various N-alkylated analogues of a cyclic CXCR4 receptor antagonist originally developed by Fujii et. al.

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Translational Regulation via L11: Molecular Switches on the Ribosome Turned On and Off by Thiostrepton and Micrococcin
11.04.2008
Joerg M. Harms,
Daniel N. Wilson, Frank Schluenzen, Sean R. Connell, Torsten Stachelhaus, Zaneta Zaborowska, Christian M.T. Spahn, Paola Fucini
MolCell,
2008,
30,
26–38
published on 11.04.2008
MolCell,
online article
The thiopeptide class of antibiotics targets the GTPase-associated center (GAC) of the ribosome to inhibit translation factor function. Using X-ray crystallography, we have determined the binding sites of thiostrepton (Thio), nosiheptide (Nosi), and micrococcin (Micro), on the Deinococcus radiodurans large ribosomal subunit. The thiopeptides, by binding within a cleft located between the ribosomal protein L11 and helices 43 and 44 of the 23S rRNA, overlap
with the position of domain V of EF-G, thus explaining how this class of drugs perturbs translation factor binding to the ribosome. The presence of Micro leads to additional density for the C-terminal domain (CTD) of L7, adjacent to and interacting with L11. The results
suggest that L11 acts as a molecular switch to control L7 binding and plays a pivotal role in positioning one L7-CTD monomer on the G0 subdomain of EF-G to regulate EF-G turnover during protein synthesis.


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1H, 13C, and 15N chemical shift assignments for the Eps15-EH2-stonin 2 complex
28.03.2008
Julia Rumpf, Bernd Simon, Yvonne Groemping,
Michael Sattler
Biomol NMR Assign,
2008,
2,
55-58
published on 28.03.2008
Biomol NMR Assign, online article
EH domains are protein–protein interaction domains that function in vesicular trafficking and endocytosis. Here, we report the NMR spectral assignments of the high-affinity complex between the second EH domain of Eps15 and a stonin 2 peptide—providing the basis for the characterization of a two-site binding mode.

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The CLIP/CLAP-HSQC: Pure absorptive spectra for the measurement of one-bond couplings
21.03.2008
Andreas Enthart, J. Christoph Freudenberger, Julien Furrer,
Horst Kessler, Burkhard Luy
Journal of Magnetic Resonance,
2008,
192,
314-322
published on 21.03.2008
Journal of Magnetic Resonance, online article
Heteronuclear residual dipolar one-bond couplings of organic molecules at natural abundance are most easily measured using t2 coupled HSQC spectra. However, inevitably mismatched transfer delays result in phase distortions due to residual dispersive antiphase coherences in such experiments. In this article, slightly modified t2 coupled HSQC experiments with clean inphase (CLIP) multiplets are introduced which also reduce the intensities of undesired long-range cross peaks. With the corresponding antiphase (CLAP) experiment, situations where a and b components overlap can be resolved for all multiplicities in an IPAP manner. A comparison of the experiments using hard pulses and shaped broadband excitation and inversion pulses on the heteronucleus is given and potential spectral artefacts are discussed in detail.

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Phosphorus NMR Spectroscopy as a Versatile Tool for Compound Library Screening
29.02.2008
Florian Manzenrieder, Andreas O. Frank,
Horst Kessler
Angew. Ch. Int. Ed.,
2008,
Vol. 47,
2608-11
published on 29.02.2008
Angew. Chem. Int. Ed.,
online article
NMR spectroscopy is a well-established technique for the screening of compound libraries. One of the biggest advantages of NMR spectroscopy in relation to other methods is that it directly detects even weakinteractions between ligand and target molecules, which makes it ideally suited for fragment-based ligand design. In addition, the number of false-positive hits, often obtained in bioassays, is
minimized. Among the variety of NMR screening approaches, methodologies based on exclusively tracing ligand signals are the most powerful tools to identify binders in compound libraries. Standard and groupselective saturation-transfer difference (STD) spectroscopy or fluorine NMR screening are prominent
examples. Together with recently developed highthroughput
techniques, for example, target-immobilized NMR screening (TINS), ligand-based NMR screening is a potent technology in the field of drug science.

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Improving Oral Bioavailability of Peptides by Multiple N-Methylation: Somatostatin Analogues
26.02.2008
Eric Biron, Jayanta Chatterjee, Oded Ovadia, Daniel Langenegger, Joseph Brueggen, Daniel Hoyer, Herbert A. Schmid, Raz Jelinek, Chaim Gilon, Amnon Hoffman,
Horst Kessler
Angewandte Chemie International Edition,
2008,
47 (early view),
1-6
published on 26.02.2008
Angewandte Chemie, online article
Full methyl jacket? A complete library of the N-methylated somatostatin cyclopeptidic analogue Veber–Hirschmann peptide cyclo(-PFwKTF-) is performed with the aim of improving its bioavailability. Several analogues from the library were found to bind to the somatostatin receptor in the nanomolar range and one of them shows a significant oral bioavailability of 10%. Conformational analysis shows that N-methylation is allowed at specific positions without affecting the bioactive conformation.

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Role of Ser-340 and Thr-341 in Transmembrane Domain IX of the Na+/Proline Transporter PutP of Escherichia coli in Ligand Binding Transport
22.02.2008
Daniel Hilger, Maret Böhm, Alexandra Hackmann,
Kirsten Jung
Journal of Biological Chemistry,
2008,
283 (8),
4921-9
published on 22.02.2008
Journal of Biological Chemistry,
online article
The Na+/solute symporter family comprises more than 400 members of pro- and eukaryotic origin. Using the Na+/proline transporter PutP of Escherichia coli as a model, the role of two conserved residues, Ser-340 and Thr-341, is investigated to obtain insights into the mechanism of transport catalyzed by members of this family. Substitution of these amino acids alters the transport kinetics of cells and proteoliposomes containing the PutP variants significantly. In particular, the apparent affinities for Na+ and Li+ are reduced by 2 orders of magnitude or
more. Also proline binding is affected, albeit to a lesser extent than ion binding. Thereby, the presence of a hydroxyl group at position 341 is essential for high affinity ligand binding. Furthermore, Cys placed at position 340 or 341 reacts with sulfhydryl reagents of different polarity, indicating accessibility from the water phase. In addition, Cys cross-linking suggests proximity of the residues to other amino acids previously shown to be crucial for ligand binding. For these reasons it is suggested that Ser-340 and Thr-341 are located in a ligand translocation pathway. Furthermore, it is proposed that the side chain of Thr-341 directly participates in Na+ binding. The Na+/solute symporter family comprises more than 400 members of pro- and eukaryotic origin. Using the Na+/proline transporter PutP of Escherichia coli as a model, the role of two conserved residues, Ser-340 and Thr-341, is investigated to obtain insights into the mechanism of transport catalyzed by members of this family. Substitution of these amino acids alters the transport kinetics of cells and proteoliposomes containing the PutP variants significantly. In particular, the apparent affinities for Na+ and Li+ are reduced by 2 orders of magnitude or more. Also proline binding is affected, albeit to a lesser extent than ion binding. Thereby, the presence of a hydroxyl group at position 341 is essential for high affinity ligand binding. Furthermore, Cys placed at position 340 or 341 reacts with sulfhydryl reagents of different polarity, indicating accessibility from the water phase. In addition, Cys cross-linking suggests proximity of the residues to other amino acids previously shown to be crucial for ligand binding. For these reasons it is suggested that Ser-340 and Thr-341 are located in a ligand translocation pathway. Furthermore, it is proposed that the side chain of Thr-341 directly participates in Na+ binding.

Structure of an Archaeal RNA Polymerase
15.02.2008
Anselm G. Kusser, Michela G. Bertero, Souad Naji, Thomas Becker, Michael Thomm,
Roland Beckmann, Patrick Cramer
Journal of Molecular Biology,
2008,
doi:10.1016/j.jmb.2007.08.066,
Volume 376, Issue 2, Pages 303-307
published on 15.02.2008
Journal of Molecular Biology, online article
Related multisubunit RNA polymerases (RNAPs) carry out gene transcription in all kingdoms of life. Since structural information is limited to bacterial and eukaryotic RNAPs, we determined the cryo-electron microscopic structure of the RNAP from the thermophilic archaeon Pyrococcus furiosus at 13 Å resolution. Comparison with eukaryotic RNAP II reveals a conserved architecture, no homologues for subunits Rpb8 and Rpb9, and significant deviation in the polymerase foot, jaws, pore, and protrusion. The structural organization of the archaeal RNA polymerase serves as a reference for future structure–function analysis of the transcription mechanism and allows for evolutionary comparisons.

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Conformational Preference and Potential Templates of N-Methylated Cyclic Pentaalanine Peptides
08.02.2008
Jayanta Chatterjee, Dale F. Mierke,
Horst Kessler
Chemistry a European Journal,
2008,
14,
1508-17
published on 08.02.2008
Chemistry a European Journal, online article
Systematic N-methylation of all peptide bonds in the cyclic pentapeptide cyclo(-d-Ala-Ala4-) has been performed yielding 30 different N-methylated derivatives, of which only seven displayed a single conformation on the NMR time scale. The conformation
of these differentially N-methylated peptides was recently reported by us (J. Am. Chem. Soc. 2006, 128, 15 164–15 172). Here we present the conformational characterization of nine additional N-methylated peptides from the previous library which are not homogeneous but exist as a mixture in which at least one conformation is preferred by over 80%. The structures of
these peptides are investigated employing various 2D-NMR techniques, distance geometry calculations and further
refined by molecular dynamics simulations in explicit DMSO. The
comparison of the conformation of these nine peptides and the seven conformationally homogeneous peptides allow us to draw conclusions regarding the influence of N-methylation on the
peptide backbone of cyclic pentapeptide of the class cyclo(-d-Ala-Ala4-).
Here we present the different conformational
classes of the peptides arising from the definitive pattern of N-methylation which can eventually serve as templates for the design of bioactive peptides.

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Structure of Eukaryotic RNA Polymerases
07.02.2008
P. Cramer, K.-J. Armache, S. Baumli, S. Benkert, F. Brueckner, C. Buchen, G.E. Damsma, S. Dengl, S.R. Geiger, A.J. Jasiak, A. Jawhari, S. Jennebach, T. Kamenski, H. Kettenberger, C.-D. Kuhn, E. Lehmann, K. Leike, J.F. Sydow, and A. Vannini
Biophysics,
2008,
DOI: 10.1146/annurev.biophys.37.032807.130008,
337-352
published on 07.02.2008
Biophysics,
online article
The eukaryotic RNA polymerases Pol I, Pol II, and Pol III are the central multiprotein machines that synthesize ribosomal, messenger, and transfer RNA, respectively. Here we provide a catalog of available structural information for these three enzymes. Most structural data have been accumulated for Pol II and its functional complexes. These studies have provided insights into many aspects of the transcription mechanism, including initiation at promoter DNA, elongation of the mRNA chain, tunability of the polymerase active site, which supports RNA synthesis and cleavage, and the response of Pol II to DNA lesions. Detailed structural studies of Pol I and Pol III were reported recently and showed that the active center region and core enzymes are similar to Pol II and that strong structural differences on the surfaces account for gene class-specific functions.

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Assessment of αvβ3 Integrin Expression after Myocarcdial Infarction by Positron Emission Tomography
06.02.2008
Takahiro Higuchi; Frank M. Bengel; Stefan Seidl; Petra Watzlowik;
Horst Kessler; Renate Hegenloh; Sybille Reder; Stephan G. Nekolla; Hans J. Wester; Markus Schwaiger
Cardiovascular Research,
2008,
doi:10.1093/cvr/cvn033,
1-37
published on 06.02.2008
Cardiovascular Research, online article
The purpose of this study was to determine the feasibility of a new positron emission tomography (PET) imaging approach using a 18F-labeled αvβ3 integrin antagonist (18F-Galacto-RGD) to monitor the integrin expression after myocardial infarction.
METHODS: Male Wister rats were subjected to 20 min of transient left coronary artery occlusion followed by reperfusion.
Autoradiographic analysis and in-vivo PET imaging were used to determine myocardial 18F-Galacto-RGD uptake at different time points following reperfusion.
RESULTS: PET imaging and autoradiography demonstrated no significant focal myocardial 18F-Galacto-RGD uptake in non-operated control rats and at day 1 after reperfusion. However, focal accumulation in the infarct area started at day 3 (uptake ratio = 1.91 ± 0.22 vs remote myocardium), peaked between 1 (3.43 ± 0.57) and 3 weeks (3.43 ± 0.95), and decreased to 1.96 ± 0.40 at 6 months after reperfusion. Pretreatment with αvβ3 integrin antagonist c(-RGDfV-) significantly decreased tracer uptake, indicating the specificity of tracer uptake. The time course of focal tracer uptake paralleled vascular density as measured by CD31 immunohistochemical analysis.
CONCLUSION: Regional 18F-Galacto-RGD accumulation suggests up-regulation of αvβ3 integrin expression after myocardial infarction, which peaks between 1 and 3 weeks and remains detectable until 6 months after reperfusion. This new PET tracer is promising for the monitoring of myocardial repair processes.

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Structure–function analysis of the RNA polymerase cleft loops elucidates initial transcription, DNA unwinding and RNA displacement
01.02.2008
Souad Naji, Michela G. Bertero, Patrizia Spitalny,
Patrick Cramer, Michael Thomm
Nucleic Acids Research,
2008,
doi:10.1093/nar/gkm1086,
Volume 36, Issue 2, Pages 676-687
published on 01.02.2008
Nucleic Acids Research,
online article
The active center clefts of RNA polymerase (RNAP) from the archaeon Pyrococcus furiosus (Pfu) and of yeast RNAP II are nearly identical, including four protruding loops, the lid, rudder, fork 1 and fork 2. Here we present a structure-function analysis of recombinant Pfu RNAP variants lacking these cleft loops, and analyze the function of each loop at different stages of the transcription cycle. All cleft loops except fork 1 were required for promoter-directed transcription and efficient elongation. Unprimed de novo transcription required fork 2, the lid was necessary for primed initial transcription. Analysis of templates containing a pre-melted bubble showed that rewinding of upstream DNA drives RNA separation from the template. During elongation, downstream DNA strand separation required template strand binding to an invariant arginine in switch 2, and apparently interaction of an invariant arginine in fork 2 with the non-template strand.

Structure–system correlation identifies a gene regulatory Mediator submodule
01.02.2008
Laurent Larivière, Martin Seizl, Sake van Wageningen, Susanne Röther, Loes van de Pasch, Heidi Feldmann, Katja Sträßer, Steve Hahn, Frank C.P. Holstege,
Patrick Cramer
Genes & Development,
2008,
10.1101/gad.465108,
872-877
published on 01.02.2008
Genes & Development,
online article
A combination of crystallography, biochemistry, and gene expression analysis identifies the coactivator subcomplex Med8C/18/20 as a functionally distinct submodule of the Mediator head module. Med8C forms a conserved α-helix that tethers Med18/20 to the Mediator. Deletion of Med8C in vivo results in dissociation of Med18/20 from Mediator and in loss of transcription activity of extracts. Deletion of med8C, med18, or med20 causes similar changes in the yeast transcriptome, establishing Med8C/18/20 as a predominantly positive, gene-specific submodule required for low transcription levels of nonactivated genes, including conjugation genes. The presented structure-based system perturbation is superior to gene deletion analysis of gene regulation.

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The C-Terminal Regulatory Domain Is the RNA 5`-Triphosphate Sensor of RIG-I
01.02.2008
Sheng Cui, Katharina Eisenächer, Axel Kirchhofer, Krzysztof Brzozka, Alfred Lammens, Katja Lammens,
Takashi Fujita, Karl-Klaus Conzelmann, Anne Krug, Karl-Peter Hopfner
Molecular Cell,
2008,
Vol. 29,
169-79
published on 01.02.2008
Molecular Cell;
online article
The ATPase RIG-I senses viral RNAs that contain 5`triphosphates in the cytoplasm. It initiates a signaling cascade that activates innate immune response by interferon and cytokine production, providing essential antiviral protection for the host. The mode of RNA
50-triphosphate sensing by RIG-I remains elusive. We show that the C-terminal regulatory domain RD of RIG-I binds viral RNA in a 50-triphosphate-dependent manner and activates the RIG-I ATPase by RNAdependent dimerization. The crystal structure of RD reveals a zinc-binding domain that is structurally related to GDP/GTP exchange factors of Rab-like GTPases. The zinc coordination site is essential for RIG-I signaling and is also conserved in MDA5 and LGP2, suggesting related RD domains in all three enzymes. Structure-guided mutagenesis identifies a positively charged groove as likely 5`-triphosphate-binding site of RIG-I. This groove is distinct
in MDA5 and LGP2, raising the possibility that RD confers ligand specificity.

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Structure of the Eps15–stonin2 complex provides a molecular explanation for EH-domain ligand specificity
17.01.2008
Julia Rumpf, Bernd Simon, Nadja Jung, Tanja Maritzen, Volker Haucke,
Michael Sattler, Yvonne Groemping
The EMBO Journal,
2008,
27 (3),
558-69
published on 17.01.2008
The EMBO Journal,
online article
Eps15 homology (EH) domain-containing proteins play a key regulatory role in intracellular membrane trafficking and cell signalling. EH domains serve as interaction platforms for short peptide motifs comprising the residues NPF within natively unstructured regions of accessory proteins. The EH–NPF interactions described thus far are of very low affinity and specificity. Here, we identify the presynaptic endocytic sorting adaptor stonin2 as a highaffinity ligand for the second EH domain (EH2) of the clathrin accessory protein Eps15. Calorimetric data indicate that both NPF motifs within stonin2 interact with EH2 simultaneously and with sub-micromolar affinity. The solution structure of this complex reveals that the first NPF motif binds to the conserved site on the EH domain,
whereas the second motif inserts into a novel hydrophobic pocket. Our data show how combination of two EH-attachment sites provides a means for modulating specificity and allows discrimination from a large pool of potential binding partners containing NPF motifs.

Efficient factor VIII affinity purification using a small synthetic ligand
09.01.2008
S. Knör, A. Khrenov, B. Laufer, A. Benhida, S.C. Grailly, R. Schwaab, J. Oldenburg, N. Beaufort, V. Magdolen, J.-M.R. Saint-Remy, E.L. Saenko, C.A.E. Hauser,
H. Kessler
Journal of Thrombosis and Haemostasis,
2008,
Vol. 6,
470-7
published on 09.01.2008
Journal of Thrombosis and Haemostasis, online article
BACKGROUND: Hemophilia A is currently treated by infusions of the coagulation factor (F) VIII, of which production and purification remain a challenging task. Current purification procedures using immunoaffinity chromatography are cumbersome, expensive, and suffer from the instability of the applied antibody ligands, which elute along with the product and contaminate it. Recently, FVIII was purified using octapeptide ligands, but their use is limited due to the low resistance to proteases. OBJECTIVE: Our goal was to develop and evaluate a novel ligand for FVIII purification, overcoming the drawbacks of current procedures. METHODS: Peptide ligands were screened for binding of (125)I-plasma-derived-FVIII (pdFVIII) in a microbead assay. A selected ligand-coated Toyopearl resin was then used for pdFVIII purification from cell-conditioned Delbucco's modified Eagle's medium (DMEM) containing fetal bovine serum. The proteolytic stability of ligand was measured by incubating with human serum and proteinase K, and its cytotoxicity towards human OV-MZ-6 cells was assayed. RESULTS: A high-affinity octapeptidic FVIII ligand was modified into the small, highly stable and non-toxic peptidomimetic ligand L4 by rational and combinatorial design without affecting its affinity for FVIII. Using ligand L4-coated Toyopearl resin, pdFVIII was isolated from cell-conditioned medium with high purity and 89% column retention after elution with a mild buffer containing 0.6 m NaCl at pH 6.8. CONCLUSIONS: Ligand L4 offers a valuable alternative to antibody-based procedures for laboratory and industrial production. Its synthesis by established solid-phase procedures is straightforward and considerably cheaper than the biotechnological production of antibodies, and safety concerns associated with the use of biological material are overcome.

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Single-molecule tracking of mRNA exiting from RNA polymerase II
08.01.2008
Joanna Andrecka, Robert Lewis, Florian Brückner, Elisabeth Lehmann,
Patrick Cramer,
Jens Michaelis
PNAS,
2008,
105,
135-40
published on 08.01.2008
www.pnas.org,
online article
Single-pair fluorescence resonance energy transfer was used to track RNA exiting from RNA polymerase II (Pol II) in elongation complexes. Measuring the distance between the RNA 5´ end and three known locations within the elongation complex allows us determine its position by means of triangulation. RNA leaves the polymerase active center cleft via the previously proposed exit tunnel and then disengages from the enzyme surface. When the RNA reaches lengths of 26 and 29 nt, its 5´ end associates with Pol II at the base of the dock domain. Because the initiation factor TFIIB binds to the dock domain and exit tunnel, exiting RNA may prevent TFIIB reassociation during elongation. RNA further extends toward the linker connecting to the polymerase C-terminal repeat domain (CTD), which binds the 5´-capping enzyme and other RNA processing factors.

The Solution Structure of BMPR-IA Reveals a Local Disorder-to-Order Transition upon BMP-2 Binding
21.10.2008
Jochen Klages, Alexander Kotzsch, Murray Coles, Walter Sebald, Joachim Nickel, Thomas Müller,
Horst Kessler
Biochemistry,
2008,
47,
11930-9
published on 21.10.2007
Biochemistry,
online article
The structure of the extracellular domain of BMP receptor IA was determined in solution by NMR spectroscopy and compared to its structure when bound to its ligand BMP-2. While most parts of
the secondary structure are highly conserved between the bound and unbound forms, large conformational rearrangements can be observed in the b4b5 loop of BMPR-IA, which is in contact with BMP-2 and harbors the main binding determinants for the BMPR-IA-BMP-2 interaction. In its unbound form, helix R1 in BMPR-IA, which is in the center of the binding epitope for BMP-2, is missing. Since BMP-2 also
shows conformational changes in the type I receptor epitope upon binding to BMPR-IA, both binding partners pass through an induced fit mechanism to adapt their binding interfaces to a given interaction
surface. The inherent flexibility of both partners possibly explains the promiscuous ligand-receptor interaction observed in the BMP protein superfamily.

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