ayout 1 - EMBL Grenoble

EMBL Research at a Glance 2009
Structural biology of macromolecular complexes
Matthias
Wilmanns
PhD 1990, University of
Basel.
Postdoctoral research at the
University of California, Los
Angeles.
Group leader at EMBL
Heidelberg 1993-1997. Head
of EMBL Hamburg since
1997.
Our central focus is on the structural characterisation of interactions in networks of biological
molecules. We are particularly interested how proteins are regulated either by post-translational
modification or by interactions with cellular ligands. We use synchrotron radiation to determine
molecular structures at high resolution by X-ray crystallography, complemented by other structural
biology methods such as NMR spectroscopy, small angle X-ray scattering, in vitro FRET for
distance measurements and in vivo imaging methods. We are interested in exploiting present and
future opportunities, provided by synchrotron and laser facilities at DESY (DORIS-III, PETRA III,
FLASH, X-FEL), to develop new methods and to apply them to biology-driven projects. Some of
our specific interests are highlighted below (more information can be found at
www.embl-hamburg.de/~wilmanns/home.html).
Protein-protein complexes of the muscle sarcomere, including titin, myomesin and binding partners.
Titin is the largest gene product of the human genome, and it comprises up to 38,000 residues
in its largest isoform. It is known as
the third filament of the muscle sarcomere
and is involved in multiple
functions, such as acting as a ‘molecular
ruler’ keeping major components
of the sarcomere in place,
muscle development, passive elasticity
of the muscle sarcomere and muscle signalling. Titin is inter-connected to other
long filament proteins, such as nebulin, myomesin, M-protein and obscurin. Recently,
we have been able to determine the structures of the N-terminal assembly Figure 1: C-terminal myomesin model as M-band crosslinker.
The shape of the C-terminal myomesin filament been
complex of titin in the Z-disk (Zou et al., 2006) and the C-terminal assembly complex
of myomesin in the M-band (Pinotsis et al., 2008). In the latter complex, we of the M1, M4, and M4’ bands and some of the N-terminal
fit into previous immuno-EM data. The approximate locations
have identified a novel type of helical linker, connecting neighbouring Ig domains.
myomesin domains are indicated schematically.
By making use of a series of additional high resolution structures of different parts
of the C-terminal myomesin filament and small angle X-ray scattering data, we have been
able to build a model of the complete filament (figure 1). Our future work will concentrate
on protein-ligand complexes from sarcomeric filament proteins.
The architecture of the translocon of peroxisomes. Peroxisome are cell organelles that
allow sequestered metabolic processes that would interfere with other processes that, for
instance, take place in the cytosol. Those proteins that are involved in these processes are
generally translocated as active and folded targets. We have been able, the first time, to unravel
the mechanism of the recognition of peroxisome protein targets by the peroxisome
import receptor Pex5p, by determining the structure of the cargo binding domain of the
receptor in the absence and presence of the cargo protein sterol carrier protein 2 (Stanley
et al., 2006). Our present focus is on structural/functional studies of several other protein
components of the peroxisomal translocation machinery.
Figure 2: Work flow of the X-MTB structural
proteomics project (www.xmtb.org).
Structural proteomics on Mycobacterium tuberculosis targets. During the last three years we have determined the X-ray structures of about
ten protein targets, some of them with an already known function and others of unknown function. For instance, we have were able to identity
Rv2217 as a novel cysteine/lysine dyad acyltransferase, which allows activation of several important protein complexes by lipoylation (Ma
et al., 2006). With the help of the high resolution structures of a total of six protein-ligand complexes, we have been able to unravel the catalytic
mechanism of Rv1603 (PriA) as a bifunctional isomerase (Kuper et al., unpublished) and, by screening a large compound library, we have been
able to find an inhibitor that specifically blocks the bifunctional enzyme in vitro and in vivo (Due et al., unpublished). Our future interest will
focus on the human host/M. tuberculosis interactome, with the aim to quantitatively analyse protein-ligand interactions, where the ligands may
range from protein, lipids, metabolites and other compounds. The workflow of our projects on M. tuberculosis targets is outlined in figure 2.
Selected references
Lamber, E.P., Vanhille, L., Textor, L.C., Kachalova, G.S., Sieweke,
M.H. & Wilmanns, M. (2008). Regulation of the transcription factor
Ets-1 by DNA-mediated homo-dimerization. EMBO J., 27, 2006-2017
Pinotsis, N., Lange, S., Perriard, J.C., Svergun, D.I. & Wilmanns, M.
(2008). Molecular basis of the C-terminal tail-to-tail assembly of the
sarcomeric filament protein myomesin. EMBO J., 27, 253-26
98
Stanley, W.A., Filipp, F.V., Kursula, P., Schuller, N., Erdmann, R.,
Schliebs, W., Sattler, M. & Wilmanns, M. (2006). Recognition of a
functional peroxisome type 1 target by the dynamic import receptor
pex5p. Mol. Cell, 2, 653-663
Zou, P., Pinotsis, N., Lange, S., Song, Y.H., Popov, A., Mavridis, I.,
Mayans, O.M., Gautel, M. & Wilmanns, M. (2006). Palindromic
assembly of the giant muscle protein titin in the sarcomeric Z-disk.
Nature, 39, 229-233