The Cluster of Excellence Frankfurt "Macromolecular Complexes" (CEF) was established in 2006 by Goethe University Frankfurt together with the Max Planck Institute of Biophysics and the Max Planck Institute for Brain Research in the context of the German Universities Excellence Initiative. Funding by the Deutsche Forschungsgemeinschaft (DFG) ended in October 2019. CEF grew out of the long-standing collaborative research on membrane proteins and RNA molecules and strengthened research efforts in these fields by recruiting further scientists to Frankfurt/Main. CEF brought together the research activities of up to 45 research groups, the majority of which were based on Riedberg Campus in Frankfurt/Main. CEF founded the Buchmann Institute for Molecular Life Sciences (BMLS).
Aims CEF scientists set out to investigate the structure and function of large macromolecular complexes, in particular membrane proteins and their assemblies, complexes involved in signal transduction and quality control, and RNA-protein complexes.
Research Important structures of macromolecular complexes were determined in CEF. Examples for important membrane complexes include the atomic structures of complex I and the ATP synthase of the mitochondrial respiratory chain and of the transporter associated with antigen processing (TAP). Research on RNA structure and function led to the definition of regulatory principles of temperature sensing riboswitches, the structure-function relationship of RNA polymerase I, the functions of microRNAs and the mechanisms of rRNA maturation and downstream processes during ribosome biogenesis and recycling. For instance, CEF scientists identified the receptors of ubiquitin chains on the proteasome, deciphered the role of linear ubiquitin chains and described macromolecules regulating mitophagy, xenophagy and ER-phagy. They delineated the role of sumoylation in ribosome quality control and characterized the process of genetic quality control in oocytes. The efforts in these three research areas were accompanied by approaches to design or reprogram macromolecular complexes and new methods developed to expand the already strong expertise. CEF scientists established and advanced the principles of optogenetics as well as biochemical methods for light regulation. They also developed biophysical techniques for the structural and functional characterization of macromolecules. Example include light-switchable molecules designed for in-cell applications and time-resolved techniques to study RNA folding. Light sheet fluorescence microscopy for the observation of development and LILBID mass spectrometry for the analysis of membrane complexes was improved. PELDOR-EPR was developed to a resolution that allows in-cell measurements. The Cluster promoted scientific exchange through a range of programmes as well as through workshops, international conferences and lecture series. Optogenetics and light sheet fluorescence microscopy were selected as the "Method of the Year" across all fields of science and engineering by the interdisciplinary research journal Nature Methods in 2010 and 2014, respectively.
The five research areas of CEF included: (A) Structure, mechanisms and dynamics of complexes in the membrane, (B) Composition and dynamics of macromolecular complexes in quality control and signalling, (C) Dynamics of ribonucleic acid-protein-complexes, (D) Design of macromolecular complexes, and (E) Methods for studying macromolecular complexes.
CEF Research Area A - Structure, mechanisms and dynamics of complexes in the membrane Biological membranes have a very important role in life processes as everything a cell needs to live, grow and respond has to either pass through or act on them. The energy conversion processes of cellular respiration and photosynthesis happen in membranes, every sensory stimulus and the information processing in the brain is mediated by them. This array of diverse actions is performed by a large number of different membrane proteins. In the crowded conditions of the cell membrane, most membrane proteins associate into complex dynamic assemblies to carry out their various tasks. For this reason, and because they are embedded in the lipid bilayer of the membrane, most membrane proteins are difficult to study and their functions have often been intractable. CEF scientists have done groundbreaking work to overcome some of these challenges and made major contributions to elucidating the structure, mechanisms and regulation of a number of important large complexes, including respiratory complex I, rotary ATPases, supercomplex I1III2IV1, cytochrome cbb3 oxidase, cytochrome bd oxidase, a sulfide:quinone oxidoreductase, a fungal TOM core complex, a bacterial double-pore K+ uptake system KtrAB, the Na+-independent carnitine/butyrobetaine antiporter CaiT, the betaine/Na+ symporter BetP, the multidrug efflux transporter AcrB and the chaperone and editing TAPBPR–MHC I complex and the human MHC-I peptide-loading complex. Antigenic peptide recognition on TAP was resolved by DNP-enhanced solid-state NMR spectroscopy. The conformational coupling and trans-inhibition in the human antigen transporter ortholog TmrAB was resolved with the aid of dipolar EPR spectroscopy. The progress in 3D structure determination of membrane proteins by X-ray crystallography and cryo electron microscopy has created an increasing demand and opportunity for in-depth mechanistic studies by magnetic resonance methods. Due to the challenges intrinsic to membrane proteins, progress relies on the availability of techniques at the forefront of method development. Especially solid-state (MAS) NMR enables bridging the gap between 'static' structures and biochemical data by probing membrane proteins directly within the bilayer environment. Such experiments are challenging and breakthroughs could only be achieved thanks to the availability of dynamic nuclear polarization for sensitivity enhancement and very high magnetic fields for spectral resolution. CEF scientists were able to provide new insights into the catalytic mechanism of ABC transporters. Based on real-time 31P-MAS-NMR they found that the homodimeric lipid A flippase MsbA is able to catalyze a reverse adenylate kinase-like reaction in addition to ATP hydrolysis. In addition, the ATP hydrolysis cycle of the ABC transporter LmrA was probed by site-directed spin labeling and pulsed electron–electron double resonance (PELDOR/DEER) spectroscopy. The secondary multidrug efflux pump EmrE from E. coli was extensively studied with 31P- and DNP-enhanced solid-state NMR. Also, a number of photoreceptors such as microbial rhodopsins are involved in trans-membrane transport processes. For example, fundamental contributions were made towards the structural and functional description of proteorhodopsin, a pentameric light-driven proton pump by groups within CEF. CEF researchers have developed mass spectrometry approaches specifically suitable for large membrane protein complexes. Laser induced liquid beam/bead ion desorption mass spectrometry (LILBID) enables mass analysis of whole membrane protein complexes of 1 MDa or more. A team of CEF scientists resolved the mechanism of the subtype selectivity of human bradykinin receptors for their peptide agonists by integrating DNP-enhanced solid-state nuclear magnetic resonance with advanced molecular modeling and docking
CEF Research Area B - Composition and dynamics of macromolecular complexes in quality control and signalling The characterization of function and structural composition of signalling complexes controlling cellular quality control programs was one of the major topics of CEF research. The view that proteins act as single entities has been replaced with the concept suggesting that dynamic reorganization of multimeric soluble complexes annotated as signalosomes is essential for signal transmission in the cell. Regulation of the activity of these complexes is achieved by their dynamic composition as well as by post-translational modifications (PTMs) of proteins. Domains that recognize these modifications play decisive roles in a cell's ability to respond to alterations in their microenvironment. Significant progress has been accomplished by CEF in characterizing several signalling pathways and their regulation by PTMs including ubiquitylation, phosphorylation and acetylation. A particular focus of research in CEF has been on protein quality control mechanisms that are the basis for the autophagic and the ubiquitin/proteasomal pathways, the two cellular systems used to degrade faulty or superfluous proteins, complexes and organelles. Additional foci of CEF research were genetic quality control in oocytes and epithelial stem cells by the p53 protein and the regulation of and by kinases.
Research into autophagy During selective autophagy, cargo is specifically targeted for degradation, and distinct cargo receptors have been described that regulate selectivity. This process is facilitated by autophagy receptors specifically recognizing and binding their cargo, and delivering it to the phagophore. In humans, there are six different LC3/GABARAP proteins, which play a central role by connecting nascent autophagosome membranes and cargo-loaded autophagy receptors to facilitate engulfment, sometimes mediated or supported by additional adaptor proteins. CEF scientists showed that GABARAP proteins are not only involved in autophagy but also in the ubiquitin-dependent degradation of TIAM1. Breakthroughs were achieved in how cells fight intracellular pathogens and how intracellular bacteria try to evade these counter measures. The kinase Tbk1 was identified as important for mediating optineurin based xenophagy to remove the bacteria from the infected cells. Using mass spectrometry, a global analysis of the ubiquitinome of Salmonella-infected cells was carried out, that enabled CEF scientists to identify specific targets of bacterial ligases that are secreted into the cellular cytoplasm by the pathogens. CEF scientists also revealed the molecular mechanism of a novel type of phosphoribosyl-linked serine ubiquitination by the effector SdeA of the pathogen Legionella, which is very different from the canonical lysine-based ubiquitination mechanism. They further showed that another effector of Legionella bacteria, SidJ, opposes the toxicity of SidE in yeast and mammalian cells. Mass spectrometry analysis revealed that SidJ is a glutamylase that modifies the catalytic glutamate in the mono-ADP ribosyl transferase domain of the SdeA, thus blocking the ubiquitin ligase activity of SdeA. They further discovered that reticulon-type proteins act as ER-specific autophagy receptors and simulated their effect on the membrane curvature.
Ubiquitination Ubiquitination plays a central role for marking proteins to be degraded either via the autophagy pathway or via the proteasome. Several groups of CEF have contributed to advances in understanding how ubiquitin signalling is not only used as a degradation signal but also involved in several other cellular processes
p63 Research on TP63, also known as p63, has shown that this protein plays essential roles both for the proliferation and differentiation of stratified epithelial tissues as well as for the surveillance of the genetic quality in female germ cells. Investigations by CEF scientists showed that a specific isoform of p63 is highly expressed in primordial oocytes which are arrested in prophase of meiosis I. This isoform adopts a closed, inactive and only dimeric conformation in which both, the interaction with the DNA as well as with the transcriptional machinery is significantly reduced The inhibition is achieved by blocking the tetramerization interface of the oligomerization domain with a six-stranded anti-parallel beta-sheet. Activation requires phosphorylation and follows a spring-loaded, irreversible activation mechanism.