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 well-

known 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 post-

transcriptional 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 single-

particle 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

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References

Ban N, Nissen P, Hansen J, Moore PB, Steitz TA (2000) The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. Science 289(5481): 905-920 Beck M, Lucic V, Foerster F, Baumeister W, Medalia O (2007) Snapshots of nuclear pore complexes in action captured by cryo-electron tomography. Nature 449(7162): 611-615 Cook AG, Fukuhara N, Jinek M, Conti E (2009) Structures of the tRNA export factor in the nuclear and cytosolic states. Nature 461(7260): 60-65 Deisenhofer J, Epp O, Miki K, Huber R, Michel H (1985) Structure of the Protein Subunits in the Photosynthetic Reaction Center of Rhodopseudomonas-Viridis at 3a Resolution. Nature 318(6047): 618-624 Frey S, Goerlich D (2007) A saturated FG-repeat hydrogel can reproduce the permeability properties of nuclear pore complexes. Cell 130(3): 512-523 Holmes KC, Angert I, Kull FJ, Jahn W, Schroder RR (2003) Electron cryo-microscopy shows how strong binding of myosin to actin releases nucleotide. Nature 425(6956): 423-427 Hunte C, Koepke J, Lange C, Rossmanith T, Michel H (2000) Structure at 2.3 A resolution of the cytochrome bc(1) complex from the yeast Saccharomyces cerevisiae co-crystallized with an antibody Fv fragment. Structure 8(6): 669-684 Iwata S, Ostermeier C, Ludwig B, Michel H (1995) Structure at 2.8 A resolution of cytochrome c oxidase from Paracoccus denitrificans. Nature 376(6542): 660-669 Johansson P, Wiltschi B, Kumari P, Kessler B, Vonrhein C, Vonck J, Oesterhelt D, Grininger M (2008) Inhibition of the fungal fatty acid synthase type I multienzyme complex. Proc Natl Acad Sci U S A 105(35): 12803-12808 Lorentzen E, Conti E (2005) Structural basis of 3' end RNA recognition and exoribonucleolytic cleavage by an exosome RNase PH core. Mol Cell 20(3): 473-481 Löwe J, Stock D, Jap B, Zwickl P, Baumeister W, Huber R (1995) Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 A resolution. Science 268(5210): 533-539 Majorovits E, Barton B, Schultheiss K, Perez-Willard F, Gerthsen D, Schroder RR (2007) Optimizing phase contrast in transmission electron microscopy with an electrostatic (Boersch) phase plate. Ultramicroscopy 107(2-3): 213-226 Nickell S, Beck F, Scheres SH, Korinek A, Forster F, Lasker K, Mihalache O, Sun N, Nagy I, Sali A, Plitzko JM, Carazo JM, Mann M, Baumeister W (2009) Insights into the molecular architecture of the 26S proteasome. Proc Natl Acad Sci U S A 106(29): 11943-11947 Pelletier L, O'Toole E, Schwager A, Hyman AA, Muller-Reichert T (2006) Centriole assembly in Caenorhabditis elegans. Nature 444(7119): 619-623 Pogoryelov D, Yildiz O, Faraldo-Gomez JD, Meier T (2009) High-resolution structure of the rotor ring of a proton-dependent ATP synthase. Nat Struct Mol Biol 16(10): 1068-1073

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Schluenzen F, Tocilj A, Zarivach R, Harms J, Gluehmann M, Janell D, Bashan A, Bartels H, Agmon I, Franceschi F, Yonath A (2000) Structure of functionally activated small ribosomal subunit at 3.3 angstroms resolution. Cell 102(5): 615-623 Strauss M, Hofhaus G, Schroder RR, Kuehlbrandt W (2008) Dimer ribbons of ATP synthase shape the inner mitochondrial membrane. Embo J 27(7): 1154-1160 Wimberly BT, Brodersen DE, Clemons WM, Jr., Morgan-Warren RJ, Carter AP, Vonrhein C, Hartsch T, Ramakrishnan V (2000) Structure of the 30S ribosomal subunit. Nature 407(6802): 327-339 Yu X, Jin L, Zhou ZH (2008) 3.88 A structure of cytoplasmic polyhedrosis virus by cryo-electron microscopy. Nature 453(7193): 415-419

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