Towards a structural understanding of pathogenesis.
Introduction
Knowledge of the way in which an invading pathogen interacts with its
host at a molecular level is an essential aid to understanding the
nature and extent of disease caused. My group aims to use a variety of
techniques to probe the interactions that characterise different
disease processes. Central to this approach is the use of X-ray
crystallography to determine the structures of individual host or
pathogen components, with a view in the longer term to examining the
atomic structure of important host- pathogen complexes. The major
targets are protein based but we are also involved in projects where
folded RNAs provide the structural target. To aid understanding of
biochemical and structural data we use a variety of other biophysical
techniques (including surface plasmon resonance) to further
characterise the biological systems under study. The main systems
studied during 2002-2003 are summarised below.
Decay Accelerating Factor (CD55)
Petra Lukacik, Pietro Roversi, Jason Billington with the groups of
Dr. D.J Evans (University of Glasgow) and Dr. R. Smith (AdProTech Ltd)
Decay Accelerating Factor (DAF, CD55) is a membrane associated
regulator of complement activation that contains four ~60 amino acid
long consensus sequences termed complement control protein repeats
(CCPs) or short consensus repeats (SCRs). The four SCRs comprise the
functional portion of the protein and are linked to the plasma membrane
via a heavily glycosylated serine/threonine rich linker and a
glycophophyditdylinositol (GPI) anchor. CD55 is a
multi-functional molecule accelerating the decay of both the classical
and alternative pathway C3 convertases, binding CD97 a member of the
EGF-TM7 family whose expression is rapidly upregulated on T and B cells
following activation, and acting as the receptor for a variety of viral
and bacterial pathogens. Domain swapping studies for a variety of CD55
interactions have shown that multiple domains are essential for
biological function and the precise arrangement of any one SCR domain
with respect to the others is therefore crucial to a full understanding
of the biology. This year we have solved the structure of all four SCR
domains giving insight into both natural and pathogenic activities
(Lukacik et al. 2004, White et al submitted, Williams
et al 2003, Williams et al 2004).
Shigella flexnerii Type Three Secretion System
Frank Cordes with the group of Dr. A. Blocker (Sir William Dunn
School of Pathology)
Gram-negative bacteria commonly interact with animal and plant hosts
using type III secretion systems (TTSSs) for translocation of proteins
into eukaryotic cells during infection. Ten of the twenty-five TTSS
encoding-genes are homologous to components of the bacterial flagellar
basal body which the TTSS needle complex morphologically resembles.
This indicates a common ancestry although no TTSS sequence homologues
for the genes encoding the flagellum are found. We are studying a
variety of proteins involved in this system ranging from structural
components of the secretion system (using X-ray fibre diffraction and
EM reconstruction) to the ATPase that powers the system.
This year we have published work describing a low resolution structure
for the needle component of the system, revealing the underlying
helical architecture (Cordes et al 2003). We now seek to
understand how activation of the complex occurs by use of mutants
trapped in active and inactive states and also by high resolution
structures of the component proteins.
Bacterial Adhesins
Jason Billington, David Pettigrew & Pietro Roversi in
collaboration with Dr. C. Le Bouguenec (Insitute Pasteur) and Dr S.
Matthews (Imperial College)
Bacterial adhesins are important virulence factors that allow
colonisation of the human urogenital tract by Eschericia coli.
The observation that many E. coli would haemagluttinate human
erythrocytes led eventually to the realisation that a large number of
these adhesins recognised and bound to CD55. These so-called Dr
haemagluttinins include the fimbrial Dr, X and diarrhoea-associated
F1845 adhesins and the afimbrial Afa adhesins. This family of
molecules share a common genetic organisation and have similar
nucleotide sequences but detailed analysis of the interaction
determinants shows that they are dependent on different portions of
CD55 for recognition and binding. This year we have solved
structures of Dr and Afa adhesins revealing a shared architecture and
collaborated with the group of Prof. S. Matthews to use NMR to map
DAF-binding onto the adhesin structures (Anderson et al
submitted).
Echoviruses
David Pettigrew & Pietro Roversi with the groups of Dr. D. Brown
(University of Cambridge), Dr. D. Evans (University of Glasgow) and Dr.
D. Bhella (MRC Virology Unit, Glasgow)
Picornaviruses and their receptors have been much studied by the
crystallographic community. We have determined the structure of a
clinical isolate of one of these viruses that is known to use CD55 as
the receptor for cellular infection. We now wish to extend these
studies to attempt to determine the structure of the virus in complex
with truncated forms of the receptor.
This year we have determined a low resolution structure for a viral-DAF
complex (Bhella et al 2004) and have shown that the DAF
conformation seen in our crystal structures (Williams et al
2003; Lukacik et al 2004) is the same as that seen bound to the
virus surface (Figure 1). This has allowed
an atomic interpretation of
the low resolution structure of the complex and leads to insights into
the way in which this class of viruses has evolved to bind the receptor.
Figure 1. Atomic model for the complex between Echovirus 12 and
domains 3 and 4 of CD55 (Bhella et al 2004). The virus is shown in blue
and the receptor in green as space filling representations.
EGF-TM7 family proteins
Saskia Neudek, Rachel Abbott & Pietro Roversi in collaboration
with the groups of Dr. P. Handford (Department of Biochemistry), Dr. S.
Gordon (Sir William Dunn School of Pathology) & Dr. J.M. Mc Donnell
The EGF-TM7 family is a group of cell-surface molecules characterised
by a unique chimaeric structure in which tandem EGF (Epidermal Growth
Factor-like) repeats are coupled to a G-protein coupled receptor moiety
via a mucin-like stalk. They are implicated in a range of biological
function but are or particular interest to us due to the
indentification of one of these proteins (CD97) as a T-cell ligand for
CD55. To date we have grown crystals of a natural variant of CD97
termed EMR2. Using a Ba co-crystal we have solved the structure of EMR2
at 1.9Å and shown that the molecule adopts an extended, 'banana'
shape. Via a collaboration with Dr. J. McDonnell we have now collected
NMR data that should allow mapping of the DAF-binding sites onto EMR2.
Complement System Components
Pietro Roversi & Jason Billington in collaboration with the
groups of Dr. P. Morgan (University of Wales College of Medicine) and
Dr. R. Sim (MRC Immunochemistry Unit)
The complement system is a highly evolved system of proteins that
together constitute a major element of host defences, functioning in
both innate and adaptive immunity. Activation of complement by
bacterial or other pathogens proceeds through enzymatic amplification
steps (which are tightly regulated by specific proteins) to generate
protein fragments and complexes that mediate acute inflammatory
reactions, clearance of foreign cells and killing of invading
pathogenic organisms. Conditions that result in misguided, excessive or
uncontrolled activation of complement lead to human disease. We have
several complement proteins in crystallisation trials at present.
RNA Structure
Antu Dey in collaboration with the groups of Dr. W. James (Sir
William Dunn School of Pathology) and Prof. A. Lever (University of
Cambridge)
Folded RNAs are important for the life cycles of many viral pathogens
and have provided us with long-term crystallographic challenges. Our
work at present is focussed on RNAs derived from HIV (from which we
have small crystals) and synthetic RNA aptamers designed to block
binding of HIV gp120 to CD4. This work is currently at the stage of
design of suitable crystallographic targets and determination of the
affinities and kinetics that characterise aptamer-gp120 binding. In
collaboration with Prof. Lever we have collected surface plasmon
resonance data to determine the binding affinity of the HIV Rev protein
for a novel piece of viral RNA (Gallego et al 2003) and hope
that this will lead to further structural work.
References:
Bhella, D., Goodfellow, I., Roversi, P., Pettigrew, D., Chaudhry, Y.,
Evans, D.J. & Lea, S.M. (2004) The structure of echovirus type 12
bound to a two-domain fragment of its cellular attachment protein
decay-accelerating factor (CD55) J. Biol. Chem. In the press
Cordes, F.S., Komoriya, K., Larquet, E., Yang, S., Egelman, E.,
Blocker, A. & Lea, S.M. (2003) Helical Structure of the Needle of
the Type III Secretion System of Shigella Flexneri J. Biol.
Chem. 278:17103-7.
Gallego, J., Greatorex, J., Zhang, H., Yang, B., Arunachalam, S., Fang,
J., Lea, S.M., Pomerantz, R.J. & Lever, A.M.L. (2003) Rev binds
specifically to a purine loop in the SL1 region of the HIV-1 leader
RNA J. Biol. Chem. 278:40385-40391
Lukacik, P., Roversi, P. , White, J., Esser, D., Smith G.P.,
Billington, J., Williams, P., Rudd, P.M., Wormald, M.R., Crispin,
M.D.M., Radcliffe, C.M., Dwek, R.A. , Evans, D.J., Morgan, B.P., Smith,
R.A.G. & Lea, S.M. (2004) Complement regulation at the molecular
level: the structure of Decay Accelerating Factor. Proc. Natl.
Acad. Sci. USA In the press
Williams, D.T., Chaudhry, Y., Goodfellow, I., Lea, S.M. &
Evans, D. (2004) Interaction of Decay Accelerating Factor (DAF) with
hemagluttinating human enteroviruses: Utilising variation in primate
DAF to map virus binding sites. J. Gen. Virol. In the press
Williams, P., Chaudhry, Y., Goodfellow, I., Billington, J., Spiller,
B., Evans, D. & Lea, S.M. (2003) Mapping CD55 function: the
structure of two pathogen-binding domains at 1.7A. J.Biol.Chem.
278:10691-6
