Future directions and interactions between teams
All groups are dedicated to take their projects to the next level of an approach of molecular systems biology by integrating structural biology with functional protein networks, a process that will depend on technology transfer and collaborations between labs at many points.
The Cramer lab will extend their structural studies but also will in parallel begin a functional analysis of the structurally investigated systems in vitro and in vivo, with the aim of gaining a comprehensive understanding of gene regulation. The structural studies of RNA polymerase II will be extended to the two other eukaryotic RNA polymerases, RNA polymerase I and III, which are even larger, and consist of 14 and 17 subunits, respectively. Collaborations with the Hopfner, Niessing and Conti labs will be very valuable during these projects. This part of the project will strongly benefit from the groups working on DNA protein complexes in areas D of the Cluster. The in vivo significance and interactions of specific structural domains will be discovered and analyzed in collaboration with the Jansen, Sträßer and Jentsch labs using yeast genetics and genome-wide yeast two-hybrid analysis. A second line of research will be to unravel the structure and function of multiprotein coactivator complexes that are essential for gene regulation in eukaryotes. In particular, the 25-subunit Mediator complex will be analyzed structurally in a bottom-up approach by reconstitution of subcomplexes. It will be analyzed functionally with the use of yeast genetics and biochemistry. The resulting complexes are now analyzed biophysically and with the use of cryo-electron microscopy (collaboration Beckmann). A successful collaboration with the Sattler lab will be continued, to analyze a Mediator subunit-activation domain complex by NMR. Taken together, a structural biology hybrid approach must be taken and will be integrated with biochemical and genetic studies, to successfully tackle the longstanding problem of gene regulation in eukaryotes. Since other groups in this research area move in the same direction, common platforms will be established, including a crystallization/visualization facility, a diffraction data collection facility, and a bioanalytics (Jung) and yeast functional genomics platform (Jentsch).
The Hopfner lab will ask how multiprotein complexes and dynamic molecular assemblies initiate the DNA damage-signaling network and initiate repair in chromatin. For instance, RNA polymerases stalled at DNA lesions are a major problem for the cell. They not only block the transcription of particular genes, but also prevent repair of the damage, which is typically buried in the active site of the polymerase. To remove polymerases from DNA lesions and initiate repair, cells possess the transcription coupled repair enzyme Cockayne Syndrome protein B (CSB). They will reconstitute a trapped Rad26/polymerase complex, in collaboration with Cramer and Beckmann groups, and study this complex with electron microscopy and X-ray crystallography. The DNA lesions needed for these studies come form the lab of Carell in organic chemistry (research area E). A second focus of the future research is the mechanism of DNA double-strand break (DSB) repair. Given the recent investments in electron microscopy, in collaboration with Beckman and Baumeister, they will attempt to obtain low to medium resolution structures of the Rad50/Mre11/Nbs1/ATM complex (DNA damage signalosome). Hybrid approaches will then allow the reconstitution of a model of the DNA damage signalosom. For several projects, they will continue to collaborate with the Jansen lab and use yeast genetics to identify functional protein interactions. Finally they will establish mass spectrometry combined with full complex limited proteolysis and D2O/H2O isotope labeling to identify flexible, solvent accessible regions and peripheral domains that may prevent crystallization.
The Beckmann lab will try and study membrane proteins in their natural environment. All the membrane protein complexes studied so far by cryo-EM have been visualized in detergent, but not within lipid bilayers. They will therefore try to establish a reconstituted system on the basis of small membrane discs or micro-vesicles, which are suitable for single particle analysis. They will study translocons of different species in order to identify the complexes with potential for higher resolution in electron microscopic analysis. In addition to the SecYEG/Sec61 translocon a conserved alternative translocon exists, the YidC/Oxa1 system. Macromolecular complexes for which no structural information is available, such as the Mediator (Cramer), RNA transport complexes (Jansen) or TREX (Sträßer) will be analyzed by cryo-EM in collaboration. Technically, they aim at improving the 3D reconstruction methods with the goal of reaching molecular resolution (4 Å or better).
The Conti lab plans to tackle by X-ray crystallography machineries such as the SURF complex in NMD, the SKI and TRAMP complexes in RNA degradation, and the TREX complex at the connection between transcription and export. The macromolecular complexes consist of a stable core of 3 to 10 proteins. The cores dynamically associate with additional components to carry out multiple functions. They aim to understand this complex network of interactions at the atomic level, not only the stable cores but also the more dynamic interactions and subcomplexes. Multicistronic expression cloning and large-scale fermentation will be employed and developed in exchange with the Cramer lab. Prescreening of the integrity of multicomponent complexes will be done by EM in collaboration with the Baumeister and Beckmann labs.
The Baumeister lab will develop their techniques and systems in various directions. Given its potential for studying large and structurally variable protein complexes, EM single particle analysis could contribute to template libraries in a major way. Therefore, they are currently establishing a high-throughput pipeline for single particle analysis. These developments are closely coordinated with the establishment of the high-end EM facility at the LMU (Beckmann). Once the challenges of obtaining cryo-ET with sufficiently good resolution and of creating efficient data-mining algorithms are met, and comprehensive libraries of template structures become available, they will be able to map the supramolecular landscape of cells in a systematic manner.
The Kessler group will study the interaction of HSP90 with p53 and other molecules to understand the influence of this chaperone in cancer. The high dynamics and the complicated intra- and intermolecular interaction of HSP90 and its regulation by small organic molecules (geldanamycin) make this system ideally suited for NMR-investigations. All this work will be carried out in close collaboration with research area B (Buchner). Another project is the structure determination of the folded spider silk N-terminal domain to understand the role of protein-protein interaction for the silk formation (with Scheibel/Buchner)
The Sattler lab (supposed successor of Prof. Kessler) will be interested in studying proteins and protein complexes that are involved in RNA-based regulation of gene expression, i.e. in the regulation of splicing and alternative splicing and RNA interference. They aim to contribute to an understanding of RNA interference by characterizing structural and dynamical aspects involved in RNAi. There will be close interactions with the Foerstemann lab, which provides biochemical assays on RNAi and a suited model organism to go in vivo (Drosophila). NMR methodology will be implemented to monitor and screen the interaction with small molecules and/or metabolites, and will be used in collaboration with the Conti and Cramer labs for screening for binders to several target proteins, including targets in the NMD pathway.
The Jung lab will expand their studies to a combined experimental, theoretical and computational approach to analyze prokaryotic signalling systems quantitatively. The lab already has close interactions with the Oesterhelt department that will be further strengthened. The lab will focus on the elucidation of the interacting protein partners of the Usp proteins in E. coli, determination of the kinetics of the interactions, analyses of the impact of chemical protein modification on stress-dependent alterations of the proteom, in vivo cross-linking studies to follow the time-dependent alterations of complex formation, in vitro reconstruction of the affected signaling cascades, and on structural analysis of the Usp proteins within multi-component complexes.. For structural analysis of small Usp proteins, NMR spectroscopy (Kessler, Sattler) is applicable. The larger proteins and the protein complexes will be studied by X-ray crystallography (Cramer, Hopfner, Conti), and by cryo-electron microscopy (Beckmann). Protein dynamics will be analyzed using site-directed spin-labeling and subsequent EPR spectroscopy.
The Jentsch laboratory will follow mainly two lines of investigation. First, they are interested in mechanistic aspects of ubiquitylation and degradation. Second, they employ the powerful genetic tools available for yeast to identify new functions, new components, and new pathways. Recent findings that have been expanded also to human cells, are of great medical importance because the ubiquitin-dependent PCNA pathway is responsible for mutagenesis induced e.g. by UV-light and is thus relevant for tumour biology. The research will nicely complement activities of the Cramer, Jansen, Hopfner, and Sträßer labs, since a major focus of future research in the Jentsch lab will be on chromatin-associated events (DNA replication, repair, recombination, transcription, genome stability) that are regulated by ubiquitin and SUMO. In addition, the Jentsch lab has set up a platform for automated robot-based yeast screens for the identification of interacting proteins, which will be very useful for many partners, including the Jansen and Cramer labs.
The Mann laboratory will on one hand continue to develop mass spectrometric technology for proteomics upon which all groups of the cluster will benefit from. The proteomics area will strongly profit from the small organic molecules created by Sieber and Carell to decipher protein function in networks of cells (area E). Proteomics crucially depends on instrumentation for measuring endogenous proteins, which occur in small amounts and in very complex mixtures. They wish to further develop proteomics technology with a view to increasing certainty of protein identification, dynamic range and completeness of analysis. In particular, they will focus on the analysis of post-translational modifications. The technology developed in the group for proteome profiling is employed in various proteome mapping experiments. In particular they are interested in growth factor signaling and in the insulin signalling/ diabetes area.
The Oesterhelt lab currently embarks fully into systems biology. Next to the level of protein structural research, protein interactions are systematically searched by combining high throughput techniques with in vitro translation systems to provide a platform to analyse networks. These activities are supported by one of the largest Sixth Framework EU projects, Interaction Proteome, specifically for certain cellular modules which are defined by manually annotated genomes, and all available data from experimental work, either by own efforts or by literature, are stored in a central data base, Halolex, and are used to design the baits and the prey to get a comprehensive view on the interactome of certain cellular modules. Technologies and know-how will be shared with the Jung laboratory.
Future efforts in the Niessing lab aim at a mechanistic understanding of directional transport of cytoplasmic mRNA and subsequent localized expression of transcripts for the generation of cellular asymmetry, guidance of cell differentiation, and development of multi-cellular organisms. By using a combination of X-ray crystallography (facilities provided by Cramer, Hopfner), biophysical (such as SPR by Jung), and biochemical techniques, the Niessing group now aims to characterize the molecular basis for motor-protein-dependent cargo transport at atomic resolution, but together with the Jansen and Jentsch groups it will be extended towards in vivo cell biology. With such a multidisciplinary approach, the Niessing group attempts to understand how the core factors of the ASH1 mRNP interact to detect their cargo mRNA, assemble into a cargo-specific, functional complex in response to ASH1 mRNA recognition by She2p, and translocate ASH1 mRNA through the cytoplasm.
The Sträßer lab focuses on the mechanisms how TREX and other multiprotein complexes involved in mRNA processing and export or transcription elongation interact with the transcription machinery as well as with each other. It is their goal to understand the intricate network between different protein complexes involved gene expression and mRNA biogenesis. To this end, large-scale genetic screens will be carried out with the Jentsch lab, and structural studies will be initiated together with the Beckmann lab.
The Foerstemann lab will focus on the proteins that are interpreting the structural code contained in the miRNA/miRNA* duplex and escort the miRNA through a series of complexes into a specific mature RISC. In Drosophila, this network consists of at least six proteins and Dicer-1 and Dicer-2, RNaseIII-like nucleases important for maturation, Loquacious and R2D2, double-stranded RNA binding domain (dsRBD) proteins that potentially bind and orient the small RNA duplex and finally Ago-1 and Ago-2, the Argonaute proteins that accept the mature, single-stranded miRNA or siRNA. They will also gain a structural perspective on the activity of this protein network by close interaction with the Sattler and Conti labs.