Analysis of the HlyA toxin secretion system of Escherichia coli [Elektronische Ressource] / vorgelegt von Thorsten Jumpertz

Thorsten

Informations

• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2010
• Nombre de lectures: 30
• Langue: Deutsch
• Poids de l'ouvrage: 10 Mo

Extrait

Analysis of the HlyA toxin secretion
system of Escherichia coli



Inaugural-Dissertation


zur Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine Universität Düsseldorf



vorgelegt von

Thorsten Jumpertz
aus Mannheim



Düsseldorf, Juni 2010






Aus dem Institut für Biochemie
der Heinrich-Heine-Universität Düsseldorf











Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen
Fakultät der Heinrich-Heine-Universität Düsseldorf.


Referent: Prof. Dr. Lutz Schmitt
Koreferent: PD Dr. Ulrich Schulte


Tag der mündlichen Prüfung: 28. Juni 2010
TABLE OF CONTENTS

Table of contents...................................................................................................................... I

1. Introduction .......................................................................................................................... 1
1.1 Architecture of the Type I secretion complex.................................................................. 1
1.2 ABC transporters – the import/export business of cells................................................... 5

2. ABC transporters/Haemolysin B ........................................................................................ 8
2.1 ABC transporters – a smart example of molecular machineries...................................... 8
2.2 Structure and function of the nucleotide binding domain of Haemolysin B.................... 8
2.3 The catalytic cycle and the molecular basis of allostery in the HlyB-NBD .................. 10

3. Haemolysin A – cell lysis and beyond ................................................................................ 13
3.1 A secretion signal influences folding of a protein.......................................................... 13

4. Biophysical Methods .......................................................................................................... 16
4.1 A method to determine the CMC of biologically important detergents......................... 16

Paper I ..................................................................................................................................... 19
Paper II..... 62
Paper III... 70
Paper IV... 88
Paper V... 131

5. Summary ........................................................................................................................... 154

6. Zusammenfassung............................................................................................................ 155

7. Literature .......................................................................................................................... 157


INTRODUCTION
1. Introduction

The present work contains scientific publications that I prepared (as first or co-author) during
my PhD thesis at the Institute of Biochemistry at the Heinrich Heine University Duesseldorf.
The thesis deals with the Type I secretion system of an enterohaemorragic strain of E.
coli. During infection with this strain a toxin (Haemolysin A, (HlyA)) is secreted via a Type I
system which was identified due to its ability to lyse red blood cells. Untreated, this infection
can lead to a systemic disease and to the death of persons suffering from this infection. Apart
from lysing red blood cells it has been shown that HlyA is able to induce calcium spikes and
therefore might somehow interact with subsequent signal transduction pathways (Uhlen et al.,
2000). However, the underlying molecular mechanism of transport and the mode of action of
the toxin are still not understood.


1.1 Architecture of the Type I secretion complex

The Type I secretion system which is responsible for transporting the toxin HlyA
consists of various proteins – since an E. coli cell possesses an inner and outer membrane
substrates have to cross both lipid bilayers to reach the extracellular space. The 107 kDa toxin
HlyA has never been detected in the periplasmic space and therefore a continuous
channel/tunnel was proposed to exist that spans the periplasmic space (Holland et al., 2005).
This rather complex molecular machine designated for secretion of HlyA consists of an inner
membrane complex which includes the ABC transporter Haemolysin B (HlyB) and the so-
called membrane fusion protein Haemolysin D (MFP, HlyD). Together with the porin-like
protein TolC, which is located in the outer membrane, a ternary complex for secretion of the
toxin is assembled (Figure 01) (Zaitseva et al., 2005b). The periplasmic parts of HlyD and
TolC most likely interact extensively with each other to form a tunnel-like structure guiding
HlyA through the periplasm.







INTRODUCTION
A B
Figure 01: Panel A, schematic representation of a Type I secretion system of E. coli. HlyB (in blue) resides in
the inner membrane. The nucleotide-binding domain (NBD) of HlyB is shown with ATP (yellow) bound. The
second inner membrane component HlyD shown in green. HlyD has a huge periplasmic part and interacts with
the ABC transporter HlyB and the outer membrane porin-like protein TolC (orange) to form a continuous
channel/tunnel across the periplasm. The toxin HlyA which is the substrate of the Type I secretion system is
2+shown in red during the transport process. The grey spheres in the extracellular space represent Ca ions that
bind to calcium-binding motifs in HlyA. The right part of panel A shows a cross section of the secretion complex
to highlight the continuity of the channel and illustrates the idea that the 107 kDa substrate HlyA is probably
transported in an unfolded state. The picture was taken from Zaitseva et al., 2005b.
Panel B shows a modelling approach to visualize a similar intact tripartite complex derived from a different
transport system that involves TolC. The AcrAB-TolC comple is a multidrug-resistance pump (MDR) that was
identified due to its ability to transport a wide range of substrates, preferentially some beta-lactams. Subsequent
experiments showed that AcrB is a drug/proton antiporter (Nikaido & Takatsuka, 2009). In contrast to HlyB,
AcrB has a larger periplasmic domain and AcrA lacks the membrane spanning part which anchors HlyD in the
inner membrane. The right part of panel B illustrates how all three components interact tightly and how AcrA
wraps around the AcrB/TolC interface. A similar role is proposed for the membrane fusion protein HlyD. Panel
B was taken from http://www.csb.bit.uni-bonn.de/ with courtesy of Dr. Christian Kandt.

The crystal structure of TolC in its closed conformation was solved in the year 2000
(Koronakis et al., 2000) and clearly shows that a gating mechanism exists. This mechanism
was proven two years later by disrupting the hydrogen bonds and salt bridges responsible for
arresting TolC in the closed conformation (Andersen et al., 2002). Since TolC is a ubiquitous
outer membrane protein and interacts with different components of the inner membrane it is
not yet clear if this gating mechanism is directly responsible for the secretion of HlyA.
Probably, the interaction of TolC with HlyB/HlyD triggers TolC to open and guarantees
access to the extracellular space.

Not much is known about the membrane fusion protein HlyD. It is thought to connect HlyB
and TolC and therefore seal the channel/tunnel protruding through the periplasm (Figure 01)
(Zaitseva et al., 2005b). With its quite huge periplasmic domain it is perfectly suited to fulfill
this task. Certain mutations in HlyD cause a lysis-deficient HlyA but after unfolding and
refolding of the toxin it gains its usual activity (Pimenta et al., 2005). These mutations might
interfere with recognition or transport processes. Maybe the substrate is inserted differently
into the secretion complex or the mutations in HlyD disturb the assembly of the complex

INTRODUCTION
itself in a rather subtle manner. But what exactly happens during this process is not clear at
all.
Interestingly, there is evidence that HlyD is more than a mere protein-glue that holds the
ternary complex together – it can compensate a symmetry break in the secretion complex. The
crystal structure of TolC unveiled that this outer membrane protein acts as a trimer.
Unpublished results from our laboratory propose a dimer for a functional HlyB which is in
agreement with the available crystal structures of other ABC transporters (Kos & Ford, 2009,
Jumpertz & al., 2009). In this context HlyD appears probably as a hexamer so that a TolC
monomer interacts with two