Beauty and Fitness for Purpose: the Architecture
of Proteins, the Building Blocks of Life
ROBERT HUBER*
Abstract. Atomic views of protein structures have been determined
with increasing pace in the last twenty years by a rapid development
of methods and instrumentation of protein crystallography, electron
microscopy and nuclear magnetic resonance, allowing the determination
of very large and complex protein structures. These structures document
the beauty and unlimited versatility of the proteins’́ architecture, but
reveal also unexpected relationships, allowing views on biological
evolution far back in time. The structures are a basis for understanding
the proteins’ binding specificities and catalytic properties (chemistry),
their spectral and electron transfer properties (physics), and their roles
in physiological systems (biology and medicine). They allow design and
development of specific ligands of target proteins opening novel strategies
for therapeutic intervention and development of new medicaments as well
as plant protection.
The basis of the studies described and of molecular biology in general
is X–ray diffraction of crystals, discovered by Laue in 1912 in München. I
shall discuss the history and development of Laue’s discovery, which has
revolutionized many fields of science.
1. Introduction
We celebrated the Laue Centennial in 2012 and commemorate the
International Year of Crystallography in 2014. The year 2012 represented
the centennial of the first single crystal X-ray experiments, performed at the
Ludwig Maximilian Universität in Munich (Germany) by Paul Knipping and
Walter Friedrich under the supervision of Max Laue. The first photograph
by Max Von Laue of an X-ray diffraction by a crystal, even if today may
seem smeared and ugly, meant a revolution and represents the foundation of
crystallography, an analytical tool for chemistry enabling to see molecules at
atomic resolution starting from small molecules to proteins. This experiment
was also a revolution because it shows that X-rays are electromagnetic radiation
*
Max-Planck-Institut für Biochemie, Technische Universität München, Fakultät für Chemie,
Universität Duisburg-Essen, Zentrum für Medizinische Biotechnologie, Cardiff University,
School of Biosciences.
22 Robert Huber
and crystals are regular and
periodic lattices able to diffract
them. These findings had a huge
impact on different disciplines
such as chemistry, biochemistry,
material science and all the fields
where X-ray crystallography is
used.
Laue’s experiment, published
in 1912 was spread very quickly
to England, where William
Lawrence and William Henry
Bragg immediately understood
the importance of Laue’s
experiment and developed the
famous Bragg’s law and the
X-ray spectrometer.
Biological
crystallography
started in the 1960s and since
then there has been an exponential
growth in crystal structures of
biological macromolecules solved
so that nowadays we know many
basic protein folds. Such a growth
Figure 1. Crystal structure of photosynthetic reis mainly due to technological
action center. Nobel Prize in Chemistry, 1988.
advances as well as the interest
Deisenhofer, Huber, Michel.
in understanding biology at
molecular level and because
of the potential impact and application of structural biology to medicine
and biotechnology. For example, structural biology is of high interest for
pharmaceutical companies for structure-based drug design and development.
Since 60% of drug receptors are membrane proteins, in the 80s-90s the
crystallization of these proteins, that represent 25-30% of total proteins in a cell,
was a big problem. They were difficult to prepare in a pure form for crystallization
purposes. In 1985, we were able to obtain crystals from the photosynthetic
membrane protein from Rhodopseudomonas viridis. The crystal structure was
solved and showed the four subunits of this complex (Figure 1). Two of them are
embedded in the membrane bilayer and they contain chlorophyll. The crystal
structure offered the opportunity to understand how light energy is absorbed
Beauty and Fitness for Purpose: the Architecture of Proteins, the Building Blocks of Life 23
by chlorophyll and the electrons travel along redox centers. Furthermore,
the structure offered a model to physicists to calculate electron transfer rates
and to understand the basis of the fastest chemical reaction known at that
time, representing the primary event of photosynthesis. The impact of solving
the crystal structure of membrane proteins is really high because of their
importance in physiology. In 2012, Lefkowitz and Kobilka were awarded
the Nobel Prize in Chemistry for the crystallographic studies on G-protein
coupled receptors.
A
B
Figure 2. A) Protein crystals and B) diffraction of x-ray by a protein crystal recorded with
synchrotron radiation.
Achievements in protein crystallography were possible thanks to technical
progresses such as the production of recombinant proteins and the use of
crystallization robots to set up thousands of crystallization trials in a very
short time, improving the possibility to obtain high quality diffracting crystals
(Figure 2). The other important progress was the possibility to collect data
using synchrotrons radiation as powerful X-ray generators that allow to collect
a 3D dataset with thousands of intensity in few minutes (Figure 2). Another
technical advance is the development of software and computer graphics, first
developed in the 70s to visualize electron density maps.
2. Protein structure, synthesis and degradation
Protein structure studies are important because DNA does not give the
information that we need for understanding life. For example, mature butterfly
and larvae have exactly the same DNA, but they differ in the pattern of proteins
that are expressed at different developmental stages (one genome, different
24 Robert Huber
proteomes). Different 3D structures are determined from different sequences
and we use models to describe protein structures that are metaphors. An
example is proteasome that is constituted by 28 subunits. In order to understand
how it is made up, we have to reduce the complexity of its representation just
showing the polypeptide chain or the surface or the architecture. Then we use
metaphors for describing substructures (the subunits): for example we use the
terms propellers made up by 6 blades or barrels made up by 8 elements related
by a 8-fold symmetry axis. The representation of a protein in terms of subunits
architecture can also show that symmetrical arrangements can be present in
protein complexes. For example, rotational symmetry is present in proteins of
silica cell wall of diatoms.
Crystallography helped to understand the protein synthesis machinery, the
ribosome. Ribosomes walk along the message, mRNA, which is transcribed
from the genes, and they synthesize protein that spontaneously folds up.
Despite the progress in our understanding of protein folding, how this process
takes place is still a mystery. And this is the reason why our knowledge is not
enough to reliably predict protein structure starting from a sequence. Protein
folding is sometimes not fast enough, because other proteins are around in the
A
B
Figure 3. Architecture of A) archeal thermosome and eukaryotic CCT and B) bacterial chaperonine GroEL/S.
Beauty and Fitness for Purpose: the Architecture of Proteins, the Building Blocks of Life 25
cell, and therefore there is the danger that an unfolded polypeptide aggregates
with other proteins and does not find the native structure. For this reason, in
the cell there are folding helpers, folding catalysts that are protein complexes
that enclose an unfolded polypeptide inside a large container so that they
are isolated form the rest of the cell. One of the well known systems is the
bacterial protein folding catalyst called GroEL. We were able to solve the
crystal structure of the thermosome, the archaeal group II chaperonin from
T. acidophilum, that is the homolog of the eukaryotic chaperonin CCT/
TRiC. This structure showed that there is a structural relationship between
prokaryotic and eukaryotic proteasome since they have the same role, they
use energy (ATP) leading motions to the subunits and they give the enclosed
protein just more time to find the proper fold (Figure 3).
Proteins that are not properly folded as well as damaged proteins that are
not more functional must be removed from the cell. To this end, proteases are
essential for the degradation of the non-functional proteins.
Furthermore, proteases can also have a regulatory role as in the case of the
pancreatic enzymes. These proteins must be synthesized as inactive precursors
(pro-enzymes) otherwise they would start digesting other proteins as soon as
they are synthesized in pancreas and before reaching the intestine. For this
reason, they are secreted as inactive form and then in the digestive tract they
are activated by cleavage by other peptidases. We solved the crystal structure
of bovine trypsin in early 70s also in complex with a natural inhibitor, and this
represented the first example of a high resolution structure of the individual
components and of the protein-inhibitor showing a very strong protein-protein
interaction.
3. Protein evolution
Protein crystallography is also important for understanding protein
evolution. It is very difficult to compare insects and whales when considering
the whole organism but when we look at proteins, similarities can be found. In
the late 60s, the first structure I determined was heart erythrocruorin, a protein
involved in oxygen storage in insects. If we look at the fold, it is very similar to
that of sperm whale myoglobin (the first determined by Perutz and Kendrew)
even if the sequence similarity is very low (about 20%). This is an example
showing that, at protein level, we can see relationship among distant-related
organisms.
26 Robert Huber
Figure 4. A) Schematic organization of a phycobilisome and B) crystals from the individual
components.
4. Photosynthesis
Photosynthesis is the basic process for life. Sunlight gives the energy to
produce sugar from CO2 and water, and oxygen is produced at the same time.
In plants, the photosynthetic system is in chloroplasts and the 3D structure of
the basic components is known.
In cyanobacteria light collectors are protein complexes called
phycobilisomes that are anchored to the thylakoid membrane close to
photosystem II (Figure 4). Electron micrograph shows the fragility of these
protein complexes and their diversity and therefore it is not possible to
crystallize them. However, the individual components can be isolated and
crystallized (Figure 4) providing an example of how structural biology can
help to elucidate how efficient light collection and energy transduction can
occur only with biological material (proteins and cofactors). The strategy for
phycobilisome is to act as a focusing device: the outer components of the
collecting system have many more chromophores than the inner components
and, in this way light is concentrated.
5. Use of proteins in gene technology, medicine and plant protection
Pharmaceutical companies are interested in structural biology for drug
development. We solved the structure of thrombin in complex with a natural
inhibitor. In fact, there are natural inhibitors of blood coagulation targeting
this protein and therefore we can learn directly from natural strategies. Such
inhibitors are used for example by leech that eats blood and therefore has to
Beauty and Fitness for Purpose: the Architecture of Proteins, the Building Blocks of Life 27
produce blood coagulation inhibitors. Nowadays, the main thrombin inhibitors
in leech are approved anticoagulant drugs and the molecular scaffolds of these
compounds have been modified by chemical methods on the basis of the
structural information. Another application of naturally occurring strategies
for protein inhibition and drug development comes from plant biology. A
bean plant disease is caused by a bacterial pathogen (Pseudomonas syringae)
carrying a virulence factor, syringolin A. This factor is a proteasome inhibitor
therefore causing the plant to die, and the bacterium is no more a pathogen
without this factor. The molecule is now used as a scaffold for drug synthesis.