ayout 1 - EMBL Grenoble

EMBL Hamburg
Disease-related proteins and the high-throughput
crystallisation facility
Previous and current research
Disease-related proteins from Mycobacterium tuberculosis and Schistosoma mansoni: Tuberculosis
remains the single most infectious disease, killing over 2 million individuals every year. The
disease is curable; treatment, however, is prolonged and requires the combination of several drugs.
At the same time multidrug-resistant strains of M. tuberculosis (Mtb), the bacilli that cause TB, have
been detected in virtually all 109 countries surveyed in 2006.
We have focussed on targets which were identified by comparing the expression and transcription
profiles of Mtb during different life cycles or under different growth conditions. These differences
often indicate that the corresponding proteins are involved in and important for the persistence
or pathogenicity of Mtb. The picture below shows the structure of iso-citrate dehydrogenase II
(Rv0066c). This rare dehydrogenase displays unexpected oligomeric characteristics and we are
currently in the process to determine its functional significance.
The trematode S. mansoni, a parasitic worm, is the causing agent of schistosomiasis. Also known
as bilharzia or snail fever, it is second only to malaria in socio-economic and public health importance.
Infection with S. mansoni triggers a specific reaction by the immune system and we have
recently determined the structure of this novel immune modulator.
Jochen Müller-
Dieckmann
PhD 199, Albert-Ludwigs-
Universität, Freiburg.
Postdoctoral research at the
University of California,
Berkeley.
Associate Director, SGX, San
Diego, until 200.
Team leader at EMBL
Hamburg since 200.
High-throughput crystallisation: A complete understanding of the function of biological macromolecules
requires knowledge of their 3D structures. X-ray crystallography has emerged as the method
of choice in structural biology due to its ability to resolve structures at atomic resolution without size
limitation. Current bottlenecks of this technique are the production of pure and soluble sample material
and the necessity to crystallise it. As a consequence, both aspects of macromolecular crystallography
have been automated.
The high-throughput crystallisation facility in Hamburg, which is open to the general user community,
has been in operation since 2005. The automated set up of initial screens with vapour diffusion or free
interface diffusion experiments has resulted in over 1,000,000 individual tests over the last years. In
2008, we have also developed and implemented procedures for the automated set-up of optimisation experiments.
More information is available at www.embl-hamburg.de/services/crystallisation.
Ethylene perception in plants: Ethylene is gaseous hormone in plants which regulates a multitude of
processes, ranging from seed germination and fruit ripening to leaf abscission and organ senescence. Signal
transduction involving ethylene is initiated by five receptors in Arabidopsis thaliana. Their domain
structure is related to bacterial two-component systems. In plants, however, the immediate downstream
target of this system is CTR1, a Raf-like Ser/Thr protein kinase which initiates a MAP kinase-like cascade.
The mechanism of signal transduction of eukaryotic two-component systems remains unclear. We have
produced a variety of constructs of the different signal receptors as well as of the cognate protein kinase.
These domains and their complexes are analysed by X-ray crystallography and X-ray small angle scattering with the goal to functionally characterise
the early steps of ethylene signalling through inter- and cross-domain activation of its components.
Future projects and goals
Ribbon representation of Rv0066c
(Icd2) of M. tuberculosis. Shown
is a dimer of Icd2 with a two-fold
axis in the image plane. α-helices
are in red and β-strands are in
blue. Each monomer consists of
745 amino acids. Substrate and
NADPH are bound in the large
cleft between domains.
The importance of construct design on protein expression, solubility and crystallisability has been widely accepted. In collaboration with
Darren Hart in Grenoble (page 91) we will evaluate the possibility to rapidly screen a large number (~40) of protein constructs and to identify
those which are more amenable to crystallisation using the Fluidigm technology. For a proof of principle study we used 7 different constructs
of NFκB which were identified with ESPRIT. All 7 constructs were expressed and purified in a one step procedure in Hamburg and
subject to crystallisation with Fluidigm’s Topaz chips. Small crystals were obtained under conditions that are very similar to published conditions
for one construct. We are now planning to demonstrate the general applicability of this process by using de novo protein samples. This
project is funded under the EU’s FP7 initiative.
Selected references
Hatzopoulos, G.N., Kefala, G. & Mueller-Dieckmann, J. (2008).
Cloning, expression, purification, crystallization and preliminary X-ray
crystallographic analysis of isocitrate dehydrogenase 2 (Rv0066c)
from Mycobacterium tuberculosis. Acta Crystallogr. Sect F Struct.
Biol. Cryst. Commun., 6, 1139-112
Groves, M.R., Muller, I.B., Kreplin, X. & Muller-Dieckmann, J. (2007).
A method for the general identification of protein crystals in
crystallization experiments using a noncovalent fluorescent dye. Acta
Crystallogr. D Biol. Crystallogr., 63, 526-535
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