Towards a structural understanding of pathogenesis.
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 2000-2001 are summarised below.
1. Decay Accelerating Factor (CD55)
Jason Billington, Petra Lukacik with the groups of Dr. D. 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.
Using data collected from ESRF and Daresbury we have solved the structure
(at 1.7A) of the lower two SCR domains of CD55 in several crystal forms
using MIRAS (Figure 1 - Williams et al in the press).
This structure suggests that pathogen binding of CD55 has arisen via convergent
evolution, but a better understanding of the natural, complement regulating,
functions of the molecule requires structural information about the other
SCR domains. We have collected various data from P1 crystals of the all
four extracellular SCR domains including native, potentially Au and Pt derivatised
crystals and SeMet crystals. The crystals are fairly poorly ordered and it
has required much optimisation of crystal growth and freezing conditions to
collect data to a moderate resolution. None of the data collected have allowed
phasing of the structure to date.
References
1. Williams, P., Chaudhry, Y., Goodfellow, I., Billington, J., Spiller,
B., Evans, D. & Lea, S. (2003) Mapping CD55 function: the structure of
two pathogen-binding domains at 1.7A. J.Biol.Chem. (in the press)
2. Lea, S. (2002) Interactions of CD55 with non-C ligands. Biochem. Soc.
Trans (in the press)
3. Lea, S., Powell, R., and Evans, D. (1999). Crystallization and preliminary
X-ray diffraction analysis of a biologically active fragment of CD55, Acta-Crystallogr-D-Biol-Crystallogr
55, 1198-200.
4. Lea, S., Powell, R. M., McKee, T., Evans, D. J., Brown, D., Stuart,
D. I., and van der Merwe, P. A. (1998). Determination of the affinity and
kinetic constants for the interaction between the human virus echovirus 11
and its cellular receptor, CD55, J-Biol-Chem 273, 30443-7.
2. 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.
We have used X-ray fibre diffraction (Figure 2)
to determine the helical arrangement of the needle component of the TTSS
(~5.6 subunits per turn, 24Å helical pitch) and negative stain EM reconstruction
to yield a structure at ~16A resolution (Figure 3).
This structure has confirmed the analogies to the flagellar system and allows
us to speculate about the structural basis of activation of secretion. Further
studies of the needle will include attempts to purified needles in different
activation states and X-ray crystallographic studies of the needle component
and other isolated proteins from the TTSS complex.
Reference
1. Cordes, F., Komoriya, K., Larquet, E., Yang, S., Egelman, E., Blocker,
A. & Lea, S. Helical Structure of the Needle of the Type III Secretion
System of Shigella Flexneri (manuscript submitted)
3. Bacterial Adhesins
Jason Billington & David Pettigrew
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. To date we have no further information
about the structures of these molecules or a more detailed characterisation
of the interaction but recent work has begun to demonstrate a role for pathogen-CD55
interactions that goes beyond simply providing a convenient hook for the
invading pathogen to catch at.
We have expression systems for members of all of these classes of bacterial
adhesins and crystallisation trials of the proteins in isolation and in complex
with CD55 are underway.
4. Echovirus 11
David Pettigrew with the group of Dr. D. Brown (University of Cambridge)
Picornaviruses and their receptors have been much studied by the crystallographic
community. We have determined the structure of a clinical isolate
(Figure 4) 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.
To date all data have been collected from crystals mounted at RT for reasons
of disease security - we hope to develop contained freezing protocols to allow
collection of complete data sets from single crystals so speeding data collection.
Complexes will be generated by soaking or co-crystallisation of virus with
receptor.
Reference
1. Stuart, A., McKee, T., Williams, P., Stuart, D., Brown, T. & Lea,
S. (2002) The structure of a DAF binding clinical isolate of echovirus 11
at 2.9A resolution and mapping of variant virus sequences suggest location
of a DAF binding site on the virion. J. Virol 76:7694-7704
5. EGF-TM7 family proteins
Saskia Neudek, Rachel Abbott & Petra Lukacik in collaboration with
the groups of Dr. P. Handford (Department of Biochemistry) and Dr. S. Gordon
(Sir William Dunn School of Pathology)
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 identification 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.
We have collected an optimised SAD data set for a Gd derivative of EMR2
to 2.5A - these data allow positioning of the Gd sites but due to the low
solvent content of the crystals (35%) do not allow complete structure solution.
We have Ba, Sr and Ca crystal forms (although the symmetry varies) and
are currently producing SeMet labelled protein - a combination of data from
these crystals should allow phasing of this structure. In the future we hope
to study other isoforms of this molecule and a complex between CD97 and CD55
Reference
1. Hsi-Hsien, L., Stacey, M., Saxby, C., Knott, V., Chaudhry, Y., Evans,
D., Gordon, S., McKnight, A., Handford, P. & Lea, S. (2001) Molecular
dissection of the CD55-CD97 complex provides insights into EGF-like domain
mediated cell-cell interaction, J-Biol. Chem. 276:24160-24169
6. Complement System Components
Jason Billington & Petra Lukacik 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 six complement proteins
in crystallisation trials at present.
7. 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.
