Macromolecular complexes (Werner Kühlbrandt and Elisa Izaurralde)

1. Definition All cells of all living organisms consist of the same basic building blocks of proteins, nucleic acids, carbohydrates and lipids. Yet in the crowded conditions of the cytoplasm or the cell membrane, few if any of these components work alone. On the contrary, they usually assemble into larger functional units, consisting of anything from a few to a few hundreds or even thousands of individual macromolecular components. Many of these macromolecular complexes can be thought of as molecular machines, in the sense that they are modular, complex, have moving parts that carry out the same step many times over, and consume energy. They perform essential tasks in the cell, such as reading out and translating the genetic code; generating or converting metabolic energy; generating force to enable the cell to move; taking up, synthesizing or secreting metabolites or other macromolecules; recognizing and reacting to signals from the outside world; to name but a few. Ultimately, the sum of all these assemblies defines the uniqueness of a given cell, an organism or an individual. Understanding these natural nano-machines and how they work is a fundamental and fascinating, but also one of the most challenging tasks for the future of basic biomedical research in the MPG.

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2. Present position Scientists in the Max Planck Society have made numerous distinguished contributions to this key area of the molecular life sciences, and have indeed in many cases laid its foundations. A classic example is the fatty acid synthase, one of the first macromolecular machines to be characterized, initially by Fedor Lynen at the MPI of Biochemistry in the 1950s and 60s, then by his student Dieter Oesterhelt, later himself a director at this institute. In the 1970s, Walter Hoppe at the MPI of Biochemistry used the fatty acid synthase complex to develop his method of 3D reconstruction from individual electron micrographs. Several more decades passed before the atomic structure was resolved, or the molecular mechanisms were unravelled by a combination of biochemistry, X-ray crystallography (Johansson et al, 2008) and electron cryo-microscopy (Figure 1). The long gestation period from discovery to a detailed understanding of molecular structure and mechanisms is typical for macromolecular assemblies of this size and complexity.

The proteasome (Figure 2) is a large cellular machinery that breaks down proteins earmarked for degradation, working hand in hand with the immune system that produces antibodies against the fragments. The structure of the 20S proteasome core was determined at the MPI of Biochemistry (Löwe et al, 1995), showing that it was a complex of 14 copies each of two different subunits, with two times 7 active protease sites. A three-dimensional map of the even larger and more flexible 26S proteasome obtained by electron cryo-

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microscopy revealed a modular buildup consisting of the 20S core, an ATPase module and a multiubiquitin receptor (Nickell et al, 2009). Another wellknown example of such a molecular nanomachine is the chaperonin complex GroEL/ES, discussed in detail in the article by Ulrich Hartl in this issue, which helps newly synthetized proteins in the cell to fold.

Cells respond to environmental changes by altering gene expression efficiently and accurately on both the transcriptional and the posttranscriptional levels. While the regulation of gene expression at the transcriptional level has been the focus of much attention, post-transcriptional processes have only recently been recognized as key mechanisms by which the expression of many genes can be changed rapidly. Post-transcriptional processes, such as splicing, nuclear export, translation and mRNA degradation, involve the activity of molecular machines including the spliceosome, the nuclear pore complex, the ribosome, and the exosome, which act on mRNAs in a precise order of events.

The spliceosome (Figure 3) catalyzes the removal of introns from precursor mRNAs and ligates together exonic sequences that may be more than tens of thousands of base pairs apart. The spliceosome contains five small nuclear ribonucleoprotein particles and about 150 proteins that assemble transiently on pre-mRNAs. Studies in the department of R. Lührmann at the MPI for Biophysical Chemistry revealed that the spliceosome is highly dynamic and its

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composition changes during the splicing process. Nevertheless, a nearly complete inventory of the components of the spliceosome and initial insights into its structure are available. However, we still do not have high-resolution structural information into spliceosome assembly and catalysis.

One of the largest macromolecular assemblies is the nuclear pore complex (Figure 4), a massive structure built from multiple copies of 500-700 individual polypeptides and a total mass of 50-120 MDa. NPCs act as barriers that prevent the uncontrolled intermixing of the contents of the cell nucleus with the cytoplasm. They are extremely efficient sorting devices that each perform up to 1000 facilitated transport events per second. The nuclear pore complex, its structure, components and molecular mechanisms are investigated at the MPIs of Biochemistry (Beck et al, 2007; Cook et al, 2009) and the MPI of Biophysical Chemistry (Frey & Goerlich, 2007). Recent evidence obtained at the MPI of Biophysical Chemistry suggests that the NPC barrier consists of a sieve-like hydrogel. Elucidating the underlying mechanism in atomic detail will be a tremendous challenge, because the amorphous and insoluble hydrogels are not accessible to the standard tools of structural analysis. A second fundamental open question concerns the insertion of NPCs into the nuclear envelope. For topological reasons, it cannot be performed by the standard cellular fusion machinery. It seems clear that the identification of the luminal fusion machinery will require really unconventional approaches.

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Protein

synthesis

in

the

cell

is

performed

by

ribosomes,

large

ribonucleoprotein particles that consist of several RNA molecules and more than 50 proteins (Figure 3). Ribosomes translate the genetic information into the sequence of amino acids in a protein in a cyclic process. The ribosome is a molecular machine that selects its substrates, aminoacyl-tRNAs, rapidly and accurately according to the codon presented on the mRNA. It is also a ribozyme, an RNA-based enzyme that catalyzes peptide bond formation. The award of this year's Nobel Prize in Chemistry to Ada Yonath, Venki Ramakrishnan, and Thomas Steitz for determining the structures of the ribosome (Ban et al, 2000; Schluenzen et al, 2000; Wimberly et al, 2000). The crystallization of the ribosome, first achieved by Ada Yonath as a visitor at the MPI of Molecular Genetics in Berlin, and later as the head of the Max Planck Ribosome Structure Group at DESY in Hamburg from 1986 to 2004, laid the foundations for these groundbreaking achievements.

The exosome (Figure 3) plays a critical role in eukaryotic RNA metabolism. Since its discovery more than ten years ago, the exosome degrades mRNAs and is responsible for the maturation of stable RNAs and quality control, both in the nucleus and the cytoplasm (Lorentzen & Conti, 2005) The nuclear and cytoplasmic forms of the eukaryotic exosome share the same core of nine conserved proteins organized in a ring. The activity of the exosome resides in two additional associated proteins. However, even in the presence of these

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proteins, the exosome displays little enzymatic activity in isolation and requires additional cofactors. Each of the cofactors represents a separate molecular machine with its own catalytic function. How the exosome interacts with these complexes and how it is recruited to specific RNAs remains largely unknown.

A special class of macromolecular complexes are those that reside in the lipid bilayer of the membrane, which surrounds every cell or its compartments, such as the nucleus, mitochondria, peroxisomes or chloroplasts. Because of their amphipathic nature - their exterior surface is part hydrophilic and part hydrophobic - membrane proteins are difficult to deal with, and this is especially true of their complex assemblies. Nevertheless, Max Planck scientists were first to determine the detailed atomic structure the large pigment-protein complexes that harvest and convert solar energy in photosynthetic bacteria (Deisenhofer et al, 1985) and plants, recognized by the 1988 Nobel Prize in Chemistry to Huber, Deisenhofer and Michel. Working together, the photosynthetic membrane protein complexes generate molecular oxygen and supply the energy for the synthesis of organic compounds that provide the basis for all life on earth. A key area of research at the MPI of Biophysics is focused on the large membrane protein complexes of the respiratory chain in mitochondria or bacteria, which consist of up to 40 different protein components.

In a process of controlled oxidation, they

transfer electrons from organic substrates to molecular oxygen, generating a

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proton gradient across the membrane which is then utilized by the ATP synthase, itself a complex of 20 or more subunits, to produce ATP by rotary catalysis (Figure 5) (Pogoryelov et al, 2009), providing all animal cells with the energy to live. The atomic structures of several of the electron transport chain complexes have been determined in the department of Hartmut Michel (Hunte et al, 2000; Iwata et al, 1995). More recently, scientists at this institute have discovered by electron cryo-tomography that the mitochondrial ATP synthase is not distributed randomly in the membrane, but is arranged in long rows of complex dimers, which give rise to high local membrane curvature (Strauss et al, 2008) (Figure 6). Mitochondria also play a key role in ageing and cell death. Many diseases, notably neurodegenerative disorders such as Parkinson’s or Alzheimer’s, are related to mitochondrial dysfunction, which in turn may be associated with a breakdown of membrane organization.

Synapses are the sites of information processing in the nervous system at the interface between neurons. Essentially all aspects of synaptic function are governed by dynamic protein-protein interactions. These include the recycling of synaptic vesicles by the endocytotic machinery, the presynaptic release sites where protein complexes mediate synaptic vesicle docking and fusion, the presynaptic neurotransmitter transporters and receptor complexes, and the postsynaptic density and the cytoskeleton. The complex assemblies at the synapse control essentially all functions of the higher nervous system, including motor coordination, the perception of pain, anxiety, memory and

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learning, which are central research topics at the MPIs of Neurobiology, Brain Research, and Experimental Medicine. Mutation or deletion of individual protein components often results in severe neurological disorders, such as those investigated at the MPI of Neurological Research.

3. Key scientific questions Although the molecular machines described above all perform different cellular functions, a number of key questions must be addressed in the coming decade to understand how they operate. Firstly, what is the identity of all components and how do they interact with each other to assemble into macromolecular complexes? Secondly, how do molecular machines interact with their substrates or additional regulatory complexes? Finally, how and when do the composition, structure and interactions of these machines change as they perform their functions?

Precise information about the three-dimensional architecture, ideally at the atomic scale, of any macromolecular assembly in the cell is the first prerequisite for understanding how they work. Given the large size, fragility and often, scarcity of these complexes, this information is difficult to come by. X-ray crystallography at a number of Max Planck Institutes (Biochemistry, Biophysics, Molecular Physiology, and the Max Planck Groups at the DESY in Hamburg) has been highly successful in unravelling the structure of several of the more stable and plentiful complexes, such as the ribosome, the

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photosynthetic and the respiratory chain complexes, the proteasome, and the fatty acid synthase. The structure of even larger, more flexible or more rarefied assemblies is the domain of electron cryo-microscopy and singleparticle averaging methods, practiced at the MPIs of Biochemistry, Biophysics and Biophysical Chemistry in Göttingen. Even larger assemblies, such as the centrosomes, investigated at the MPI of Molecular Cell Biology in Dresden, can be studied in detail at present only by electron tomography (Pelletier et al, 2006).

Apart from their sheer size, the fact that most macromolecular complexes undergo elaborate changes of conformation in doing their work makes it particularly challenging to study them. Capturing the same machinery in different states is a difficult task that usually requires ingenious methods to trigger or trap particular reactions or intermediate stages. Very often the mechanism of action of such a molecular machine requires a series of such snapshots combined into a “molecular movie”. For example, the mechanism of muscle contraction by the sliding-filament mechanism can only be understood from the high-resolution structures of the key components, actin and myosin, fitted to low-resolution envelopes of this linear motor obtained by electron microscopy (Holmes et al, 2003) (Figure 7).

With an increasing number of complex structures unravelled, and the growing success in understanding how they work, questions of how many of each of

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them there are in the cell, and where exactly they are located are now coming into focus. The cell is anything but a random collection of macromolecules or macromolecular assemblies. With every new look inside it, primarily by the new techniques of electron tomography and high-resolution light microscopy, described by Hell and Baumeister in this issue, there is a growing realization that each cell is a microcosm in which every component has its place, and the function of each relates to their copy number and position. In such a highly organized environment it is likely that the relative position and distribution of macromolecular complexes governs their potential interactions in space and time, in a controlled ensemble of processes that form the essence of what we think of as life.

The investigation of these interactions and controlled

processes is a highly promising new field of research where very little is known. This information will be essential for putting the processes that happen in the cell onto a quantitative foundation, and will offer new opportunities for therapeutic intervention.

4. Looking ahead Understanding how molecular machines perform their cellular functions provides research opportunities in many virtually all areas of the chemical, physical, engineering and life sciences to address common challenges at multiple levels for the next 5 years and beyond. Purifying macromolecular complexes in homogeneous functional states and in sufficient quantities for biochemical or structural studies is a challenge in itself that will occupy a

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number of our institutes in the medium term. Even the exact composition and stoichiometry of larger complexes is often unknown, and needs to be resolved as a first step towards understanding how each complex functions. Also, it is becoming increasingly important to find out how they interact in the cell or in vitro, in cooperative or mutually exclusive ways.

New and more powerful methods for investigating the structure and workings of macromolecular complexes are needed. Indeed such methods are already being developed in the institutes of the Max Planck Society, and will become available within the next five years. Mass spectrometry is reaching an extraordinary level of accuracy at which the exact molecular composition of large assemblies, including membrane protein complexes, of several hundred thousand atoms can be determined. More sensitive techniques for solution and solid state NMR, devised at the MPI of Biophysical Chemistry, will be able to gain detailed structural information about ever larger complexes and their dynamics.

The brighter PETRA III beamlines at the German Electron

Synchrotron facility (DESY) in Hamburg will enable smaller crystals of larger assemblies to be measured more accurately. The new free electron laser on the same site, scheduled to come on line in 2013, may make it possible to examine the structure of large, non-crystalline macromolecular assemblies with single, ultra-short but extremely powerful X-ray pulses.

In both

developments, and especially in their applications to the life sciences, the Max Planck Society has made major investments and is taking a leading role.

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A new, highly promising generation of electron microscopes with demonstrated capability of delivering near-atomic resolution structures (Yu et al, 2008) of large, non-crystalline complexes such as viruses is being installed at several of our institutes (Biochemistry, Biophysical Chemistry, Biophysics). In combination with phase plates for electron microscopy, an exciting new development at the MPI of Biophysics (Majorovits et al, 2007) in collaboration with caesar (Bonn), plus the new direct electron image detectors, they will soon deliver tomograms and micrographs of individual macromolecular assemblies of unprecedented quality. The combination of fluorescence light microscopy and electron tomography will make it easier to target regions of special interest in the cell, such as synapses. An important interface between the new cellular electron tomography and high-resolution light microscopy will offer new opportunities for localizing and investigating the workings of macromolecular assemblies in the cell. Last but not least, this field offers a rich and so far largely unexplored ground for computational approaches to enhance and extend structural studies, as computers become ever more powerful, can handle larger assemblies and simulate longer time trajectories.

5. Expected outcome and benefits The study of molecular machines demands communication across the traditional scientific boundaries and will stimulate many areas of the synthetic,

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the analytical, and the physical sciences in the Max Planck Society. Any detailed mechanistic understanding of the diverse molecular machines in the cell has important biomedical implications. For example, the availability of the structure of the ribosome makes it now possible to design new antimicrobials with improved antibiotic properties. More detailed structural information is necessary to find out how mutations or mis-expression of components of these machineries contribute to disease and to develop rational therapeutic strategies.

Figures

Figure 1 Structure of yeast fatty acid synthase. Three-dimensional map at 5.9 Å resolution determined by electron cryo-microscopy (transparent surface), with the fitted X-ray structure (colour). Detailed view of the alpha-6 wheel in the centre of the 2.6 MDa complex. Courtesy of Martin Grininger and Dieter Oesterhelt, MPI of Biochemistry; Preeti Gipson, Janet Vonck and Werner Kühlbrandt, MPI of Biophysics.

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Figure 2 The 26 S proteasome. Three-dimensional map at 5.9 Å resolution determined by electron cryo-microscopy (transparent surface), with the fitted X-ray structure of the core protease (colour). Courtesy of Wolfgang Baumeister, MPI of Biochemistry.

Figure 3 Three macromolecular complexes in RNA biology, drawn to the same scale. Three-dimensional maps obtained by electron cryo-microscopy of the HeLa C spliceosome complex at ~25 Å resolution (left), of the 70 S ribosome at ~9 Å resolution (centre), and atomic structure of the exosome determined by X-ray crystallography (right). Courtesy of Elena Conti, MPI of Biochemistry (exosome); Reinhard Lührmann and Holger Stark, MPI of Biophysical Chemistry (spliceosome, ribosome).

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Figure 4 The nuclear pore complex. Averaged three-dimensional map (purple) superposed on an electron tomogram of the nuclear pore membrane (yellow). Courtesy of Wolfgang Baumeister, MPI of Biochemistry.

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Figure 5 High-resolution X-ray structure of the ATP synthase rotor ring complex from the cyanobacterium Spirulina platensis. The ATP synthase uses the electrochemical membrane potential for producing ATP by rotary catalysis. The rotor ring, consisting in this case of 15 individual c-subunits shown in different colours, generates torque by transporting protons across the membrane. The amino acid sidechains forming the proton binding site of each subunit are shown in ball-and-stick representation. Courtesy of Denys Pogoryelov and Thomas Meier, MPI of Biophysics.

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Figure 6 Section through the volume of a small mitochondrion from the yeast, Yarrowia lipolytica, obtained by electron cryo-tomography. The ATP synthase (yellow) forms long rows of complex dimers at the highly curved edges of cristae membranes (grey). The inner and outer mitochondrial membrane (blue and purple, respectively) are also visible. Courtesy of Mike Strauss, Bertram Daum and Werner Kühlbrandt, MPI of Biophysics.

Figure 7 Structure of the actin-myosin complex. Three-dimensional map (transparent surface) obtained by electron cryo-microscopy of actin filaments decorated with myosin heads. The fitted X-ray structures (coloured) show how each actin molecule in the central filament binds one myosin S1 head. Courtesy of Rasmus Schröder and Ken Holmes, MPI - 17 -

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