Research Highlights

Deutsch

PELDOR in high magnetic fields: Structure determination of protein complexes

The stable tyrosyl radical in ribonucleotide reductase was used to characterize the dimer structure at low temperature. Using Pulsed Electron Double Resonance (PELDOR) at high frequency (180 GHz) and high field (6.4 T), not only the distance but also the relative orientation between the two radicals in the dimer could be determined. The position of the tyrosyl radicals in the monomer were taken from the crystal structure. The self-developed PELDOR measurement setup in Frankfurt has the highest frequency worldwide. Denysenkov, V. P., Prisner, T. F., Stubbe, J.& Bennati, M. High-field pulsed electron-electron double resonance spectroscopy to determine the orientation of the tyrosyl radicals in ribonucleotide reductase. Proc. Nat. Acad. Sci. 103, 13386-13390 (2006) Denysenkov, V. P., Biglino, D., Lubitz, W., Prisner, T. F. & Bennati, M. Structure of the tyrosyl biradical in mouse R2 ribonucleotide reductase from high-field PELDOR. Angew. Chem. Int. Edit. 47, 1224-1227 (2008) Bennati, M.& Prisner, T. F. New developments in high field electron paramagnetic resonance with applications in structural biology. Rep. Prog. Phys. 68, 411-448 (2005)

Solution NMR DNP in high magnetic fields: A new method for the solution NMR spectroscopy

A globally unique Dynamic Nuclear Polarization (DNP) spectrometer was constructed in Frankfurt, which operates at an electron Larmor frequency of 260 GHz and a proton frequency of 400 MHz. The microwave excitation of probes in liquid water is achieved by separating the E and B fields in the double-resonance setup. Denysenkov, V. P., Prandolini, M. J., Krahn, A., Gafurov, M., Endeward, B. & Prisner, T. F. High-field DNP spectrometer for liquids. Appl. Magn. Reson. 34, 289-299 (2008) Denysenkov. V. & Prisner, T. F. German Patent Application N10 2008 017 135.2 Mett, R., Hyde, J., Sibradas, J. & Prisner, T. F. US Patent 2007 Prandolini, M. J., Denysenkov, V. P., Gafurov, M., Endeward, B. & Prisner, T. F. High-Field Dynamic Nuclear Polarization in Aqueous Solutions. J. Am. Chem. Soc. 131, 6090-6092 (2009)

NMR structure determination of an RNA-protein-antibiotic complex

One of so far the biggest complexes of the ribosomal protein L11, ribosomal RNA, and the antibiotic thiostreptone was solved. This complex provides valuable indications on the mode of action of the antibiotic. Jonker, H. R. A., Ilin, S., Grimm, S. K., Wöhnert, J. & Schwalbe, H. L11 domain rearrangement upon binding to RNA and thiostrepton studied by NMR spectroscopy. Nucleic Acids Res. 35, 441-454 (2007)

Development of a lanthanide binding tag for NMR und X-ray based protein structure determination

We developed together with B. Imperiali (MIT) a universal lanthanide binding tag that can improve NMR protein structures by the measurement of residual dipolar couplings (RDCs) and X-ray structures by heavy metal derivatives. Silvaggi, N. R., Martin, L. J., Schwalbe, H., Imperiali, B. & Allen, K. N. Double-lanthanide-binding tags for macromolecular crystallographic structure determination. J. Am. Chem. Soc. 129, 7114-7120 (2007) Martin, L. J., Hähnke, M. J., Nitz, M., Wöhnert, J., Silvaggi, N. R., Allen, K. N., Schwalbe, H. & Imperiali, B. Double-lanthanide-binding tags: Design, photophysical properties, and NMR applications. J. Am. Chem. Soc. 129, 7106-7113 (2007)

Structure elucidation of the Hsp90-Cdc37 complex

The “cell division cycle” protein 37 (Cdc37) and the 90 kDa heat shock protein (Hsp90) are molecular chaperones, which play important roles in the protein signal transduction. Cdc37 and Hsp90 are chaperones for proteinkinase. We determined the crystal structure of the middle domain of Cdc37 with a resolution of 1.88 Å and the NMR structure of its complex with the 23 kDa N-terminal domain of human Hsp90. Sreeramulu, S., Jonker, H. R. A., Langer, T., Richter, C., Lancaster, C. R. D. & Schwalbe, H. The human Cdc37 center dot Hsp90 complex studied by heteronuclear NMR spectroscopy. J. Biol. Chem. 284, 3885-3896 (2009) Molecular mechanism of inhibition of the human protein complex Cdc37-Hsp90, a kinome chaperone-cochaperone, by triterpene celastrol. Angew. Chem. 48, 5853-5855 (2009)

Riboswitch-folding by means of time-resolved NMR spectroscopy

The change between alternative conformations are important elements in RNA regulations. For example, riboswitch-RNAs can be found in the 5’-nontranslated region of messenger RNAs (mRNA). They control the gene expression by allosteric conformational transformations triggered by ligand binding. NMR spectroscopy is used in order to analyse the real time change. Buck, J., Fürtig, B., Noeske, J., Wöhnert, J. & Schwalbe, H. Time-resolved NMR methods resolving ligand-induced RNA folding at atomic resolution. Proc. Natl. Acad. Sci. USA 104, 15699-15704 (2007)

RNA-Folding by time-resolved NMR spectroscopy

For the first time by means of time-resolved NMR spectroscopy, suggestions on conformational transofrmations of RNAs could be collected and their general rules could be derived. Fürtig, B., Wenter, P., Reymond, L., Richter, C., Pitsch, S. & Schwalbe, H. Conformational dynamics of bistable RNAs studied by time-resolved NMR spectroscopy.J. Am. Chem. Soc. 129, 16222-16229 (2007)

Development of New NMR Techniques for Monitoring of Protein Folding with Atomic Resolution

Experimental setup for the study of light induced reactions by high resolution NMR We have developed two independent methods to study structural transitions and reactions by time-resolved NMR spectroscopy with millisecond dead-time. Therefore, on one hand, we use a rapid mixing system inside the active volume in the probe, which we developed in cooperation with BRUKER. In addition, we have developed a method to laser-trigger cofactor dependent reactions inside the NMR tube which we established. These studies are supported by a wide variety of other spectroscopic techniques including stopped-flow fluorescence, CD, FTIR and EPR to gain further insight in structural characteristics during the folding process. At the moment we are focusing on the structural characterization of transient intermediates in protein folding. We are studying the kinetics of the natural structural changes of a-lactalbumine and calmoduline upon ion addition in cellular conditions, the binding kinetics of calmoduline to the calcium-pump receptor peptides and the refolding from denaturating reagents of calmodulin, a-lactalbumine, lysozyme and ubiquitin. Kühn, T. & Schwalbe, H. Monitoring the kinetics of ion-dependent protein folding by time-resolved NMR spectroscopy at atomic resolution.J. Am. Chem. Soc. 122, 6169-6174 (2000)

NMR studies of the structure and dynamics of the ribosomal protein L11 from Thermotoga maritima

Overlay of the backbones of the 20 best structures out of 50. a) Backbone atoms of residues 5-70 that define N-terminus were fit by a least-square method. The r.m.s.d. for this superimposition is 0.28 Å for backbone atoms and 0.77 Å for all heavy atoms. b) Backbone atoms of resides 75-140 that define C-terminus. The r.m.s.d. for the superimposition of the well defined regions 97-111 and 121-140 is 0.295 Å for the backbone atoms and 0.82 Å for all heavy atoms. The beta-sheets and alpha-helices are color-coded and their first and last residues are indicated. L11 is highly conserved ribosomal protein and its interaction with the ribosomal RNA segment from 23S subunit is considered to undergo an „induced fit“-conformational adjustment of both the protein and the RNA with respect to their conformations in the unbound state (Leulliot and Varani, 2001; Draper et al., 1996). Furthermore, the conformational dynamics of L11 are thought to play an important role in the binding process. Binding of the C-terminal domain of L11 stabilizes the tertiary structure of a compactly folded RNA domain whereas the N-terminus is implicated in the binding to the antibiotic thiostrepton (Xing and Draper, 1996). The conformation of the RNA-L11-complex is structurally well characterized (Wimberly et al., 1999, Conn et al., 1999). In addition, the conformation of the C-terminal domain of L11 from Bacillus stearothermophilus has been characterized in its RNA bound and free form by NMR (Hinck et al., 1997, Markus et al., 1997). Yet, to obtain a more detailed picture of the dynamic processes accompanying RNA-protein interactions we characterize in detail the conformation and the dynamics of the full-length protein free in solution. In particular, the relative orientation of the N- and C-terminal domain of the protein in its free form is being investigated by NMR-spectroscopy. By analysis of long-range structural information derived from heteronuclear relaxation rates and residual dipolar couplings, it can be shown that the two domains are connected by a relatively rigid poly-Proline helix, their relative flexibility is low and the prominent conformation in solution preorganizes the correct conformation in complex with RNA.

Comparison of the NMR Spectroscopy Solution Structure of Pyranosyl-RNA and Its Nucleo-delta-peptide Analogue

Structure of pRNA and NDP For all biological systems, nature has chosen ribo- and deoxyribonucleic acids as its genetic building block. In order to understand this selectivity, the structures of the potential alternatives to the natural nucleic acids have to be investigated. We have determined the solution structures of pRNA and Nucleo-d-peptides by NMR spectroscopy. The structures pose important questions about the origin of helicity, stacking and inclination of these oligomers. Ilin, S., Schlönvogt, I., Ebert, M.O., Jaun, B. & Schwalbe, H. Comparison of the NMR solution structures of pyranosyl-RNA and its nucleo-δ-peptides analogues. ChemBioChem, 3, 93-99 (2002) Schwalbe, H., Wermuth, J., Richter, C., Szalma, S., Eschenmoser, A. & Quinkert, G. δ-Peptide analogues of pyranosyl-RNA, Part 2, Nucleo-δ-peptides derived from conformationally constrained nucleo-δ-amino acids: NMR study of the duplex formed by self-pairing of the (1'S, 2'S, 4'S)-(phba)-nucleo-δ-peptide-(AATAT). Helv. Chim. Acta 83, 1079-1107 (2000)

Structure of calmodulin complexed with the target peptide C20W from the Ca-ATPase pump NMR spectroscopy is used to determine the structure of proteins, of RNA and DNA in order to provide structural insight into intermolecular interactions. Elshorst, B., Hennig, M., Försterling, H., Diener, A., Maurer, M., Schulte, P., Schwalbe, H., Griesinger, C., Krebs, J., Schmid, H., Vorherr, T. & Carafoli, E. NMR solution structure of a complex of calmodulin with a binding peptide of the Ca2+ pump. Biochemistry 38, 12320-12332 (1999)

Structure, enzymatic function and RNA-binding of ribosome assembly factor Nep1

Nep1 is an essential factor for the ribosome biogenesis in eukaryotes. A mutation in the human Nep1 is the cause of the Bowen-Conradi syndrome, one of the heavy developmental disorder, which is mortal already in the infancy. Our structure determination and subsequent NMR-based enzymatic assays show that Nep1 is a pseudouridine-specific N3-methyltransferase, which introduces a specific modification in the 18S ribosomal RNA. By solution NMR the RNA-binding site of Nep1 was identified and the RNA-specificity of Nep1 was analysed. The mutation responsible for the Bowen-Conradi syndrome is located in this RNA-binding site. Taylor, A. B., Meyer, B., Leal, B. Z., Köttter, P., Schirf, V., Demeler, B., Hart, P. J., Entian, K. D. & Wöhnert, J. The crystal structure of Nep1 reveals an extended SPOUT-class methyltransferase fold and a pre-organized SAM-binding site. Nucl. Acids Res. 36, 1542-1554 (2008)

Stereo-array isotope labeling (SAIL)

The 20 standard amino acids are labeled such that each CHn group carries at most a single NMR-visible 1H nucleus, the others being replaced by NMR-invisible 2H. The remaining 1H nuclei, shown as lights in the Figure, provide data that allows the NMR structure determination of proteins about twice as large as by conventional NMR approaches. The structure of the 42 kDa maltodextrin-binding protein MBP that is shown in the center of the Figure was solved in collaboration with the laboratory of Prof. Masatsune Kainosho at Tokyo Metropolitan University, Japan, using SAIL in conjunction with the structure calculation program CYANA. Kainosho, M., Torizawa, T., Iwashita, Y., Terauchi, T., Ono, A. M. & Güntert, P. Optimal isotope labeling for NMR protein structure determinations. Nature 440, 52–57 (2006)

Protein structure determination in living cells

The first three-dimensional protein structure calculated exclusively on the basis of information obtained in living cells was solved by in-cell NMR for the putative heavy metal-binding protein TTHA1718 from Thermus thermophilus HB8 overexpressed in E. coli cells. A major hurdle for determining in-cell NMR structures is the limited lifetime of the cells inside the NMR sample tube. Standard NMR experiments usually require 1–2 days of data collection, which is an unacceptably long time for live cells. This time could be shortened to 2–3 hours by preparing a fresh sample for each experiment and by applying a nonlinear sampling scheme in combination with maximum entropy processing for the indirectly acquired dimensions. Sakakibara, D., Sasaki, A., Ikeya, T., Hamatsu, J., Hanashima, T., Mishima, M., Yoshimasu, M., Hayashi, N., Mikawa, T., Wälchli, M., Smith, B. O., Shirakawa, M., Güntert, P. & Ito, Y. Protein structure determination in living cells by in-cell NMR spectroscopy. Nature 458, 102-105 (2009)

Fully automated NMR structure determination of proteins

(A) ENTH domain At3g16270(9–135) from Arabidopsis thaliana. (B) Rhodanese homology domain At4g01050(175–295) Arabidopsis thaliana. (C) Src homology domain 2 (SH2) from the human feline sarcoma oncogene Fes. Protein structures obtained by fully automated structure determination with the FLYA algorithm (blue) are virtually identical to the corresponding NMR structures determined by conventional methods (red). Güntert, P. Automated structure determination from NMR Spectra. Eur. Biophys. J. 38, 129-143 (2009)

Cell-free expression of membrane proteins

Fig.1 Introduction Cell-free (CF) expression systems have emerged in recent times as promising tools in order to accelerate and to streamline MP expression approaches. The elimination of a living host environment during protein overexpression in combination with the open accessibility of the reaction offers a variety of valuable advantages. It is evident that problems with toxic or inhibitory effects of the recombinant MPs to the host cell physiology are minimized or even completely eliminated. The expression reaction is not enclosed by membranes and any compound can thus be directly added without considering transport or metabolic conversion problems. Protease inhibitors, ligands, cofactors or substrates can be considered as possible additives that might be helpful in stabilizing the freshly translated MPs (Fig. 1). CF extracts are devoid of native membranes and complicated transportation or translocation systems for the synthesized MPs are therefore no longer necessary. In contrast, the MPs can be maintained soluble in artificial hydrophobic environments like detergent micelles. Furthermore, CF reactions are carried out in small volumes of few millilitres or even microlitres and incubation times of few hrs are already sufficient for the production of mg amounts of MPs. Altogether, these properties make CF systems interesting for the preparative scale expression of MPs as well as for throughput screening and proteomics approaches. Junge, F., Schneider, B., Reckel, S., Schwarz, D., Dötsch, V. & Bernhard, F. Large-scale production of functional membrane proteins.Cell. Mol. Life Sci., 65, 1729-1755 (2008) Keller, T., Schwarz, D., Bernhard, F., Dötsch, V., Hunte, C., Gorboulev, V. & Koepsell, H. Cell free expression and functional reconstitution of eukaryotic drug transporters.Biochemistry, 47, 4552-4564 (2008) Schwarz, D., Junge, F., Durst, F., Frölich, N., Schneider, B., Reckel, S., Sobhanifar, S., Dötsch, V. & Bernhard, F. Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems.Nature Protocols, 2, 2945-2957 (2007) Klammt, C., Schwarz, D., Eifler, N., Engel, A., Piehler, J., Haase, W., Hahn, S., Dötsch, V. & Bernhard, F. Cell-free production of G protein-coupled receptors for functional and structural studies.J. Struct. Biol., 158, 482-493 (2007) Klammt, C., Schwarz, D., Fendler, K., Haase, W., Dötsch, V. & Bernhard, F. Evaluation of detergents fort he soluble expression of α-helical and β-barrel-type integral membrane proteins by a preparative scale individual cell-free expression system.FEBS J., 272, 6024-6038 (2005) Klammt, C., Löhr, F., Schäfer, B., Haase, W., Dötsch, V., Rüterjans, H., Glaubitz, C. & Bernhard, F. High level cell-free expression and specific labeling of integral membrane proteins.[http://dx.doi.org/10.1111/j.1432-1033.2003.03959.x Eur. J. Biochem., 271, 568-580 (2004)

NMR investigations of membrane proteins

Fig.1 Membrane proteins pose a great challenge for structural biology. Structure determination of membrane proteins is difficult because many membrane proteins cannot be expressed in E. coli or other cellular systems in quantities necessary for structural investigations and because obtaining diffracting crystals or high resolution NMR spectra is problematic. Recently, we have shown that at least the expression problem can be solved for many membrane proteins by using cell-free expression systems. Based on these cell-free expression systems we have developed labelling methods with NMR active isotopes that reduce the peak overlap and enable us to obtain the backbone assignments of even larger membrane proteins. The first method, called TMS labelling (transmembrane segment enhanced labelling) is based on the observation that 60% of the amino acids found in the transmembrane helices belong to the following 6 amino acids: Ala, Leu, Ile, Phe, Gly and Val. TMS-labelled proteins contain these six amino acid types 15N/13C double labelled with all other amino acids 14N/12C labelled. This reduced labelling strategy significantly reduces the signal overlap, at the same time, however, allows for the assignment of large stretches of residues due to the clustering of these six amino acid types in the transmembrane regions. The figure shows the same plane taken from an HNCA of a uniformly double labelled sample (Fig.1, left) and a TMS-labelled sample (Fig.1, right). In the TMS-labelled sample connectivities can be identified which cannot be unambigously assigned due to severe overlap in the uniformly labelled sample. Fig.2 A second labelling method that is based on cell-free expression of membrane proteins is combinatorial selective labelling. In this scheme a double labelling strategy in which one amino acid type is labelled with 15N and a second one with 13C is used. In 2D HNCO experiments peaks will only appear for amino acids combinations in which the 13C-lablled amino acid type is directly followed by the 15N-lablled type31-35. Provided that this combination exists only once in the entire protein sequence the single HNCO peak can be assigned to one specific amino acid. To obtain more than one assignment per sample we have developed a combinatorial labelling strategy that allows us to obtain more than one assignment per sample. (Fig.2)

Reckel, S., Sobhanifar, S., Schneider, B., Junge, F., Schwarz, D., Durst, F., Löhr, F., Güntert, P., Bernhard, F. & Dötsch, V. Transmembrane segment enhanced labeling as a tool for the backbone assignment of α-helical membrane proteins. Proc. Natl. Acad. Sci. USA 105, 8262–8267 (2008) Trbovic, N., Klammt, C., Koglin, A., Löhr, F., Bernhard, F. & Dötsch, V. Efficient strategy fort the rapid backbone assignment of membrane proteins. J. Am. Chem. Soc., 127, 13504-13505 (2005) Koglin, A., Klammt, C., Trbovic, N., Schwarz, D., Schneider, B., Schäfer, B. Löhr, F., Bernhard, F. & Dötsch, V. Combination of cell-free expression and NMR spectroscopy as a new approach for structural investigation of membrane proteins. Magn. Reson. Chem., 44, 17-23 (2006)

Structural and functional investigations of p63

p63 belongs to the emerging family of proteins that are homologous to the tumor suppressor protein p53. The importance of p53 can be seen from the fact that more than half of all human primary tumors contain mutations that inactivate p53. In cell culture experiments p63 binds to p53 sites and induces apoptosis. However, despite these similarities knock-out mouse studies have demonstrated that both proteins have very different biological functions. While p53 is a key player in cell cycle control, p63 is involved in the development of epithelial tissue. These results are surprising based on the fact that the DNA-binding domain is 65% identical with all amino acids known to be important for p53 DNA binding being conserved. The key to understanding the different functions of both proteins lies in their C-termini. The C-terminus of p53 forms an open, protease-sensitive domain of 26 amino acids, while p63 exists in three C-terminal isoforms differing in the length of their C-termini between 6 kD and 27 kD. These three different forms show remarkably different characteristics in their transactivation potential. The largest version of p63 (p63 with the α C-terminus) shows only very low activity and seems to mainly act as a negative regulator of transactivating p63 isoforms and possibly p53. In contrast, the other two forms (p63α and p63α) are transcriptionally active. Because the differences in the biological activities between p53 and p63 appear to be linked to the specific regulatory mechanisms of both proteins, we want to investigate how the α C-terminus regulates the transcriptional activity of p63 using a combination of cell biology, biochemistry and structure determination. So far we have identified the domain in p63 that is responsible for transcriptional suppression (transcriptional inhibitory domain or TID). Moreover, we have shown that this domain binds to the transactivation domain. In this grant application we propose to investigate the exact mechanism of transcriptional regulation. In the three specific aims described below we outline a sequence of experiments that will provide increasingly detailed structural information about the largest form of p63, TAp63α which contains both the transactivation domain as well as the inhibitory domain. Starting with biochemical experiments we will map the exact domain boundaries as well as structurally important amino acids. In a second step we will use cryo-electron microscopy in combination with chemical cross-linking to obtain a low resolution model of the entire complex. In the final step we want to improve the quality of our current crystals of the entire TAp63α tetramer to obtain a high resolution structure. Yang, A., Kaghad, M., Wang, Y., Gillett, E., Fleming, M.D., Dötsch, V., Andrews, N.C., Caput, D. & McKeon, F. p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Molecular Cell, 2, 305-316 (1998) McGrath, J.A., Duijf, P., Kelly, A., Dötsch, V., Irvine, A.D., de Waal, R., Vanmolkot, K., Wessagowit, V., Atherton, D.J., Griffiths, W.A.D., Orlow, S.J., Yang, A., McKeon, F., Bamshad, M.A., Brunner, H.G., Hamel, B.C.J. & van Bokhoven, H. Hay-Wells syndrome is caused by heterozygous missense mutations in the SAM domain of p63. Human Molecular Genetics, 10, 221-229 (2001 Duijf, P.H.G., Vanmolkot, K.R.J., Propping, P., Friedl, W., Krieger, E., McKeon, F., Dötsch, V., Brunner, H.G. & van Bokhoven, H. Gain-of-function mutation in ADULT syndrome reveals the presence of a second transactivation domain in p63. Hum. Mol. Genet., 11, 799-804 (2002) Serber, Z., Lai, H.C., Yang, A., Yang, A., Ou, H.D., Sigal, M., Kelly, A.E., Darimont, B.D., Duijf, P.H.G., van Bokhoven, H., McKeon, F. & Dötsch, V. A C-terminal inhibitory domain controls the activity of p63 by an intramolecular mechanism. Molecular Cellular Biology, 22, 8601-8611 (2002) Der Ou, H., Löhr, F., Vogel, V., Mäntele, W. & Dötsch, V. Structural evolution of C-terminal domains in the p53 family. EMBO J. 26, 3463-3473 (2007)

Non-ribosomal peptide synthetase

Non-ribosomal peptide synthetases constitute a class of modular multi-domain enzymes found in the cytoplasm of bacteria and fungi that synthesize a large variety of highly diverse peptides. Many of these peptides have been used as antibiotics, anti-inflammatory, anti-tumor or immunosuppressive drugs, making the investigation of the structure and function of NRPS and the related polyketide synthetase (PKS) systems of high medical relevance. NRPS systems are organized in multi-subunit clusters and each subunit in turn is composed of modules, capable of carrying out one cycle of chain elongation. A typical module consists of an adenylation (A) domain, a peptidyl carrier protein (PCP) domain and a condensation(C) domain. A domains (~550 residues) and C domains (~450 residues) are responsible for loading PCP domains with the cognate amino acid and catalyzing the peptide bond formation between the upstream aminoacyl or peptidyl PCP and downstream peptidyl PCP, respectively. During the entire process, the growing peptide chain is covalently linked to a phosphopantetheine cofactor which itself is attached to a conserved serine by a dedicated Ppan transferase (Pptase). As an example the organization of the surfactin synthetase is shown below.

The modular design of NRPS assembly lines combined with the use of many of their products as antibiotics, anti-cancer or anit-inflammatory drugs has sparked hopes that new and potentially medically relevant peptides could be obtained by designing new assembly lines from the individual modules of naturally occurring NRPS systems. These attempts, however, so far have demonstrated that even small changes in the assembly lines lead to considerable reductions in product yield. One interpretation of these failures to design a new assembly line is that the individual domains are not merely beads on string which only pass the intermediates to different reaction centers, but rather that there must be specific inter domain or even inter subunit contacts within these huge clusters which are vital to the completion of the final products. We are investigating the interaction between the different components of several NRPS systems. In particular, we have found that the PCP domains adopt several conformations that are importnat for selecting the interaction partners. Currently, we are focusing on structural investigaton of teh interaction of PCPs with several other domains. Koglin, A., Mofid. M. R., Löhr, F., Schäfer. B., Rogov, V. V. , Blum, M. M., Mittag, T., Marahiel, M.A., Bernhard, F.& Dötsch, V. Conformational switches modulate protein interactions in peptide antibiotic synthetases. Science 312, 273–276 (2006) Koglin, A., Löhr, F., Bernhard, F., Rogov, V. R., Frueh, D. P., Strieter, E. R., Mofid, M. R., Güntert, P., Wagner, G., Walsh, C. T., Marahiel, M. A. & Dötsch, V. Structural basis for the selectivity of the external thioesterase of the surfactin synthetase. Nature 454, 907–911 (2008)

RcsC signalling pathway

The enterobacterial Rcs phosphorelay system is an advanced and multivalent prokaryotic signal transduction pathway that has originated from the classical two-component system. Its exceptional structural complexity involving accessory modulators, unique subclasses of phosphoreceiver domains and phosphorylation-induced structural rearrangements correlates with its participation in diverse regulation circuits affecting capsule biosynthesis, motility, virulence, cell-division and many others. We combine high resolution NMR techniques with biochemical and microbiological assays for the concerted structural and functional analysis of the individual Rcs signalling components. Our strategy focuses in particular on structural dynamic effects and on the modulation of protein binding interfaces by phosphorylation of individual interaction partners. Formation of protein complexes within the signalling chain will be analysed based on our NMR structures of individual interaction partners and we will further complete the molecular picture of the Rcs system by solving structures of still lacking components. The expected results will deliver a comprehensive view of Rcs phosphorelay mechanisms comprising structural details as well as insights into dynamic conformational rearrangements during the signalling processes. Rogov, V.V., Rogova, N.Y., Bernhard, F., Koglin, A., Löhr, F. & Dötsch, V. A new structural domain in the Escherichia coli RcsC hybrid sensor kinase connects histidine kinase and phosphoreceiver domains.J. Mol. Biol., 364, 68-79 (2006) Rogov, V.V., Bernhard, F., Löhr, F. & Dötsch, V. Solution structure of the Escherichia coli YojN histidine phosphotransferase domain and its interaction with cognate phosphoryl receiver domains.J. Mol. Biol., 343, 1035-1048 (2004) Rogov, V.V., Schmöe, K., Löhr, F., Rogova, N.Y., Bernhard, F. & Dötsch, V. Modulation of the Rcs-mediated signal transfer by conformational flexibility.Biochem. Soc. Trans., 36, 1427-1432(2008)

G-protein coupled receptors (GPCRs)

G-protein coupled receptors (GPCRs) are responsible for numerous physiological processes such as signal transduction, hormone regulation and cell-cell communication. The development of pharmacologically active ligands could be enhanced by structural data for the ligand binding site or the ligand itself. Using solid state NMR, we were able to determine the backbone structure of the neuropeptide bradykinine bound to the human G-protein coupled bradykinin-2 receptor. Lopez, J. J., Shukla, A. K., Reinhart, C., Schwalbe, H., Michel, H. & Glaubitz, C. The structure of the neuropeptide bradykinin bound to the human G-protein coupled receptor bradykinin B2 as determined by solid-state NMR spectroscopy. Angew. Chem. Int. Ed. 47, 1668-1671 (2008) Gieldon, A., Lopez, J. J., Glaubitz, C. & Schwalbe, H. Theoretical Study of the human bradykinin-bradykinin B2 receptor complex. ChemBioChem 9, 2487-2497 (2008)

Proteorhodopsin (PR)

(a) Proteorhodopsin forms in the membrane a ring-shaped, mainly hexameric complex. The protein has been analysed using cryo-EM and AFM in collaboration with W. Kühlbrandt, MPI Biophysik Frankfurt, and D. Müller, TU Dresden. (b) Solid-state NMR has been used to probe the active site of proteorhodopsin. For example, using 15N-1H HETCOR experiments, we were able to detect bound water close to the protonated Schiff base. Proteorhodopsin is a light-driven proton pump found in marine bacteria in the upper zone of the oceans. Its electrochemical gradient could provide an essential source of energy for these organisms. So far, 900 variants have been found, which show high sequence similarity but colors adapted to their environment. Proteorhodopsin forms a ring shaped complex of 40 Å diameter (Shastri et al. 2009). Using solid-state NMR, we have been able to investigate the photoactive centre in great detail. We were able to show that in the ground state the chromophore retinal is almost 100% all-trans and that the protonated Schiff base must be involved in a strong interaction network involving bound water (Pfleger et al. 2009). Shastri, S., Vonck, J., Pfleger, N., Haase, W., Kuehlbrandt, W. & Glaubitz, C. Proteorhodopsin: Characterisation of 2D crystals by electron microscopy and solid state NMR. Biochimica et Biophysica Acta-Biomembranes 1768, 3012-3019 (2007) Pfleger, N., Woerner, A.C., Yang, J., Shastri, S., Hellmich, U.A., Aslimovska, L., Maier, M.S.M., Glaubitz, C. (2009) Solid-state NMR and functional studies on proteorhodopsin. BBA Bioenergetics. 1787,697-705

Multidrug transport proteins

Multidrug transport proteins export a wide range of antibiotics out of the cell. We study both ABC transporters (LmrA) and secondary drug-proton antiporters of the small multidrug resistance (SMR) family. We are interested in key events and structural changes during the transport cycle, in characterizing the properties of the drug binding pockets, and in investigating the role of lipids and oligomerization for protein activity. For SMR proteins, we were able to show the existence of an occluded transport cycle intermediate state (Basting et al. 2008). Essential residues in the binding pocket have been analysed by solid state NMR (Lehner et al. 2008). For the ABC transporter LmrA, the molecular dynamics of the ATP bindings domains has been probed (Siarheyeva et al. 2007) and, for the first time, ATP hydrolysis was directly observed by ssNMR in real time (Hellmich et al. 2008). Basting, D., Lorch, M., Lehner, I. & Glaubitz, C. Transport cycle intermediate in small multidrug resistance protein is revealed by substrate fluorescence. FASEB J. 22, 365-373 (2008) Lehner, I., Basting, D., Meyer, B., Haase, W., Manolikas, T., Kaiser, C., Karas, M. & Glaubitz, C. The key residue for substrate transport (Glu(14)) in the EmrE dimer is asymmetric. J. Biol. Chem. 283, 3281-3288 (2008)