Research Report 2010 2011 - Helmholtz-Zentrum für ...

SCIENTIFIC REPORTS | Infection and Immunity | Microbial Pathogenesis 69
01.1 Structural Analysis of Virulence Factors
PROJECT LEADER | Prof. Dr. Dirk Heinz | Department of Molecular Structural Biology | dih@helmholtz-hzi.de
PROJECT MEMBERS | Davide Ferraris | Thomas Heidler | Dr. Christian Kambach | Dr. Björn Klink |
Dr. Jörn Krauße | Dr. Anja Menzel | Nick Quade | Dr. Stefan Schmelz | Ulrich Wiesand
Pathogenic bacteria use an arsenal of virulence factors to
breach natural host barriers, circumvent or block defense
mechanisms or reprogramme host molecular processes
for their own benefit. As most of these bacterial factors are
missing in the host cell, they represent potential targets for
novel antibiotics. The aim of this project is to gain a precise
understanding of pathogen-host interactions during infection
by X-ray crystallographic structural analysis of microbial
virulence proteins in complex with their host target proteins.
Listeria InlB activates the human receptor tyrosine kinase
Met through dimerization L. monocytogenes is a harmful
food pathogen which can cause listeriosis, a systemic disease
with high mortality rates in neonates, the elderly and other
immuno-compromized individuals. The unique capability of
the bacterium to breach several important host-cell barriers
critically depends on two invasins, internalin A and B (InlB),
located at the bacterial surface. InlB interacts with the receptor
tyrosine kinase Met, leading to bacterial uptake. Met
is the receptor for hepatocyte growth factor (HGF), making
it a key player in cell growth, cell migration, wound healing
and cancer metastasis. Growth factor-mediated receptor
activation typically involves dimerization of the receptor by
the ligand, in this case as InlB2/Met2. In 2007, we determined
the crystal structure of the InlB/Met complex. Like in
the InlB structure, the complex displays a head-to-tail InlB
dimer interface leading to a Met/InlB/InlB/Met-arrangement
compatible with Met function, tyrosine kinase cross-phosphorylation
and receptor activation. To confirm the putative
role of InlB-mediated dimerization for Met activation, we
crosslinked InlB by two symmetrical inter-InlB disulfide
bridges like in the InlB dimers (Fig. 1). Strikingly, this covalent
InlB dimer leads to strong Met activation outperforming
even HGF. Conversely, weakening the InlB dimer interface
by introducing opposing positively charged amino acids
completely abrogates Met signalling.
A new class of bacterial guanine nucleotide exchange
factors for human GTPases Actin cytoskeleton remodelling
is essential for cell division, motility or cell-cell communication.
It is tightly regulated by multiple signalling pathways
employing many different actin-binding proteins. Most if not
all pathways converge on small Rho family GTPases which
act as switches inducing different actin-filament structures.
GTPase signalling requires control by guanine nucleotide exchange
factors (GEFs). Rho-GTPases are key targets of pathogens
modifying cell signalling or actin remodelling for their
own needs. Pathogens frequently bypass or activate Rho-
GTPase pathways by producing virulence factors mimicking
Rho-interacting proteins. This allows them to induce tight
adherence to or entry into host cells. Shigella flexneri IpgB1
and IpgB2 and Map from pathogenic E. coli are prototypic
members of an intriguing novel family of bacterial effectors.
These were originally classified as GTPase mimics, despite
the lack of sequence homology or GTP binding. They induce
filopodia (Map) or lamellipodia and stress fibers indicative
of Rac1 and RhoA activity for IpgB1 and IpgB2, respectively.
We solved the crystal structures of IpgB2 and its complex
with human RhoA (Fig. 2), unambiguously identifying IpgB2
as a bacterial RhoA-GEF not as a functional mimic. Structures
of the complex in various nucleotide bound states revealed
the molecular mechanism of GDP release, an essential
prerequisite for GTP binding.
Fig. 2. Structure of the complex between IpgB2 (blue) and
RhoA (brown). GDP is shown in orange and the GDP-coordinating
Mg 2+ is a yellow sphere. Red spheres are water molecules
coordinating Mg 2+ . The switch I region of RhoA is shown as a
yellow loop, switch II in green. The „catalytic loop“ of IpgB2 is
shown in red.
Fig. 1. Structure of the InlB dimer (blue) covalently cross-linked
by the introduction of two disulfide bridges (yellow sticks,
with electron density).
Ferraris, D. M., Gherardi, E., Di, Y., Heinz, D. W. & Niemann, H. N. (2010). Ligand-mediated
dimerization of the Met receptor tyrosine kinase by the bacterial invasion protein
InlB. Journal of Molecular Biology 395, 522-532.
Klink, B. U., Barden, S., Heidler, T. V., Borchers, C., Ladwein, M., Stradal, T. E., Rottner,
K. & Heinz, D. W. (2010). Structure of Shigella IpgB2 in complex with human RhoA: Implications
for the mechanism of bacterial GEF-mimicry. Journal of Biological Chemistry
285, 17197-17208.