Word - Laboratory of Molecular Biophysics

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7.

E. YVONNE JONES


7.1 Cell surface receptors, their recognition and signalling complexes.


E.Y. Jones, D. I. Stuart, K. Harlos, J. Bravo Sicilia, S. Ikemizu, K. Maenaka, W
-
C
Hon, M. Sami, J. Maloney, B. Browning, A. May, M. Deller, N. Zaccai, T.
Batuwang
ala and T. Maenaka.


Summary



The cells in a multicellular organism, such as a human, need to communicate with each
other to co
-
ordinate their functions. Direct cell
-
cell communication is achieved through
the interactions of specialised receptor molecu
les protruding from the cell surface.
Similarly interactions at a distance employ molecules secreted by one cell to deliver a
signal at a target cell by binding to the appropriate receptor molecules on its surface.
Research in the group aims to harness the

techniques of protein crystallography to
visualise these recognition and signalling events in atomic detail. Such information
should allow us to address fundamental questions about the biological processes that
underpin human health. How are signalling as
semblies arranged? Which features are
necessary for normal signal transduction into the cell? What mechanisms trigger
dysfunctional signalling? If we can understand the ways in which cells communicate with
each other we can aim to manipulate these mechanis
ms in the design of novel clinical
therapies.


One of our most established areas of study is gp130 mediated cytokine/receptor
signalling (in collaboration with Professor JK Heath (Department of Biochemistry,
University of Birmingham). Marc Deller has recen
tly completed a structure
determination of the gp130
-
binding cytokine Oncostatin M (OSM). This study was
crucially dependent on Marc engineering a mutant form of OSM with much improved
solubility that proved amenable to crystallisation. Janet Moloney and W
ai
-
Ching Hon are
now focusing on crystallisation trials of various gp130
-
containing cytokine/receptor
complexes, studies that are critically dependent on tissue culture based protein
expression. Brenda Browning joined us early in 1999 and has proved invalu
able in the
setting up and subsequent day
-
to
-
day running of our new tissue culture facilities. Malkit
Sami and Thil Batuwangala have also joined the group to trail
-
blaze structural studies
into cell surface signalling systems of importance in developmental

biology. Structural
immunology results have arisen from the work of Katsumi Maenaka, Taeko Maenaka and
Nathan Zaccai on the killer cell inhibitory receptors (KIRs) and from Shinji Ikemizu’s
studies (in collaboration with Dr SJ Davis, Nuffield Department o
f Clinical Medicine,
Oxford) on B7
-
1 (manuscript submitted).


This year Andrew May gained his doctorate, as did Chris O’Callaghan (who joined the
group, from the Institute for Molecular Medicine, for part of his thesis studies); both have
taken up Wellcome

Trust funded fellowships to study in Stanford, USA. The active
interplay between the group and the MRC Human Immunology Unit at the Institute for
Molecular Medicine represents one facet of our increasingly interdisciplinary ties within
the biomedical rese
arch community in Oxford. Two results from our published work this
year serve to illustrate this trend particularly well and are summarised in the following
two subsections. These strengthening ties were a major factor in the decision to move the
group acr
oss Oxford to the new Wellcome Trust Centre for Human Genetics building on
the clinical school site in Headington. The move, which was successfully accomplished
in early June, obviously made this year particularly eventful for us. We thank our
colleagues i
n our old home at the Laboratory of Molecular Biophysics for all their help
and patience and look forward to maintaining close links. In particular we remain very
grateful to Louise Johnson for the very strong support and steadfast encouragement she
has pr
ovided over the last eight years, all of which played a crucial role in the
development of this research programme.



(a) Biacore studies on KIR/peptide
-
MHC interactions


K. Maenaka, T. Maenaka, N.R. Zaccai, D.I. Stuart and E.Y. Jones in collaboration with

P. A. van der Merwe (Sir William Dunn School of Pathology, Oxford).


Natural Killer (NK) cells and CD8+ T lymphocytes (T cells) have complementary roles
in the cellular immune response. Whereas T cells kill cells presenting non
-
self peptides
on MHC class
I molecules, NK cells kill cells deficient in MHC class I molecules. In
doing so they make it difficult for intracellular pathogens to evade the immune response
by interfering with the expression of MHC class I molecules. It is thought that NK cells
are st
imulated to kill somatic cells by ligation of one or more activatory receptors but are
held in check if inhibitory receptors are able to bind MHC class I molecules on these
cells. In humans a family of killer cell immunoglobulin
-
like receptors (KIRs) has b
een
identified on NK cells, and a subset of T cells, that bind to MHC class I molecules. Last
year we reported the structure of one such receptor, KIR2DL2. Cell based assays have
indicated that KIR2DL2 binds preferentially to group 2 HLA
-
C alleles (Cw1, Cw
3, Cw7
and Cw8) whilst the related KIR KIR2DL1 binds o group 1 HLA
-
C alleles (Cw2, Cw4,
Cw5, Cw6 and Cw15). However there appears to be some cross
-
reactivity between these
groups. In addition, KIR recognition of peptide
-
MHC class I has been shown to depend

to some extent on the peptide presented on the MHC molecule. These observations
suggest that a single KIR will bind different peptode
-
MHC complexes with different
affinities, Raising the question as to what the affinity and/or kinetic threshold is for
fun
ctional recognition? We decided to extend the existing studies (including some
Biacore data) by providing more precise kinetic measurements of the KIR2DL3/peptide
-
HLA
-
Cw7 interaction, obtaining thermodynamic data, and determining the stoichiometry
of the i
nteraction. We also used affinity measurements to quantitate the effects of the
peptide on the binding affinity and the degree of cross
-
reactivity between KIR2DL1 and
HLA
-
Cw7.


Soluble MHC class I molecules, KIR2DL1 and KIR2DL3 were produced by expression
in E. coli and refolding. Surface plasmon resonance experiments were performed using a
BIAcore 2000 with proteins immobilized either by exploiting biotinylated forms of the
protein to bind via streptavidin covalently coupled to CM5 sensor chips or forms wi
th
oligohistidine tags to bind to Ni
-
NTA sensor chips. The binding of KIR2DL1 and
KIR2DL3 to a series of HLA
-
Cw7
-
peptide complexes was studied. KIR2DL3 bound to
the HLA
-
Cw7 allele presenting the peptide RYRPGTVAL with a 1:1 stoichiometry and
an affinity (K
d ~7

M at 25 °C) within the range of values measured for other cell
-
cell
recognition molecules, including the TCR. Although KIR2DL1 is reported not to
recognize the HLA
-
Cw7 allele in functional assays, it bound RYRPGTVAL/HLA
-
Cw7,
albeit with a 10
-
20
-
fold l
ower affinity. TCR/peptide
-
MHC interactions have been are
characterized has having comparatively slow kinetics and unfavourable entropic changes
suggesting that binding is accompanied by conformational adjustments. In contrast, our
measurements for KIR2DL3

binding to RYRPGTVAL/HLA
-
Cw7 reveal fast kinetics
and a favourable binding entropy, consistent with rigid body association. These results
indicate that KIR/peptide
-
MHC class I interactions have properties typical of other cell
-
cell recognition molecules,
and they highlight the unusual nature of TCR/peptide
-
MHC
recognition.


(b) Structure of MHC class I/Glycopeptide complexes


J. Tormo and E.Y. Jones with A. Glithero and T. Elliott (Molecular Immunology,
Nuffield Department of Clinical Medicine, Oxford)


Ma
jor histocompatibility complex (MHC) class I and II molecules have evolved to
present antigenic peptides at the cell surface, where they can be recognized by circulating
T lymphocytes. In recent years, there has been increasing evidence to show that
glycop
eptides can also be presented by MHC molecules in the same way. In at least some
of these cases the T cells are glycopeptide
-
specific and sensitive to the fine structure of
the sugar, indicating that the sugar may be recognized directly by the T cell recep
tor. In
addition to the N
-

and O
-
linked glycosylation that occur in the endoplasmic reticulum
and Golgi apparatus, O
-

-
linked N
-
aetylglucosamine (O
-
GlcNAc) substitution of serine
and threonine residues is found abundantly on proteins in the cytosol and nucleus of
mammalian cells. Since cytosolic protein is the preferred source of peptides for
presentation by MHC class I
molecules, it is possible that peptides carrying O
-
GlcNAc
residues could enter the class I presentation pathway.


In collaboration with Dr Gemma Arsequell (Unit for Glycoconjugate Chemistry, CID
-
CSIC, Barcelona, Spain) Dr Tim Elliott’s group have shown th
at synthetic glycopeptides
carrying single O
-
GlcNAc residues can bind to the murine class I molecule H
-
2Db and
are immunogenic, giving rise to glycan
-
specific T cells. Two O
-
GlcNAc substituted
peptides were used, both being analogs of the Sendai virus nucl
eoprotein residues 324
-
332 (FAPGNYPAL). This peptide binds to H
-
2Db using the side
-
chains asparagine at
position 5 and leucine at position 9 as anchor residues. The glycopeptides FAPS(O
-
GlcNAc)NYPAL (designated K3G) and FAPGS(O
-
GlcNAc)YPAL (designated K2G
)
thus carry the substitutions on positions 4 and 5, respectively. In the case of K2G this is a
modification of the P5 anchor residue. Several K3G and K2G specific T cell clones were
isolated and shown to require the glycan for recognition. Intriguingly, a
ll the K2G
specific clones exhibited a strong cross
-
reactivity toward K3G whilst K3G
-
specific
clones recognized K2G very poorly or not at all. In order to investigate the recognition of
MHC class I
-
glycopeptide complexes by T cell receptors we determined t
he crystal
structures of the wildtype peptide and the two N
-
acetylglucosamine
-
bearing
glycopeptides complexed with the murine class I allele H
-
2Db. Then using existing MHC
class I/TCR crystal structures as a basis, we modeled the glycopeptide
-
specific TCR
clones raised against both K2G and K3G onto our MHC
-
glycopeptide complexes. This
exercise was surprisingly successful in suggesting a molecular basis for T cell cross
-
reactivity to glycopeptide antigens.


Recombinant, soluble H
-
2Db class I was crystallized

in complex with the various peptides allowing the
corresponding series of three
-
dimensional structures to be determined by molecular replacement and
refined at a resolution of 2.85Å or better (using data collected on station 7.2 at Daresbury or station BM
14
at the ESRF). In both the K2G and K3G complex the glycan is solvent exposed and available for direct
recognition by the T cell receptor. For K3G the electron density indicates the occurrence of two major
conformations for the O
-
GlcNAg residue, each at s
imilar occupancy. In the K2G structure the bulky O
-
GlcNAc ring is too large to be accomodated in the peptide binding groove in the same way as the normal
asparagine anchor residue and as a result the peptide backbone is forced to undergo a major local
rear
rangement. This results in the glycan once again being orientated away from the peptide binding
groove, the central potion of the peptide is positioned further above the peptide binding groove than is
usual and is more mobile than in the wildtype and K3G s
tructures. Electron density (calculated in the
absence of any sugar coordinates) delineates a crown
-
shaped arc indicative of at least three major sugar
conformations.


Several important points are immediately apparent from modeling the juxtaposition of
th
ese MHC class I


glycopeptide complexes with T cell receptors (TCR). In both K2G
and K3G the single saccharide residue can be accommodated in the standard TCR
-
MHC
binding geometry. The glycan appears to be bound in a central cavity formed between the
two
CDR3 loops at the interface between the TCR


and


chains. The size of this cavity
may impose some restrictions on the number of conformations that the glycan could
adopt when it is part of the recognition complex. Notably, the TCRs capable of binding
the more prominent K2G all have a shorter

CDR3

loop, a feature likely to provide a
larger cavity. This difference appears to result in a highly promiscuous TCR that displays
a high degree of cross
-
reactivity toward variant peptides (for example recognizing K3G
in which the glycan occupies a smaller bu
t overlapping volume of space). Such glycan
triggered cross
-
reactivity may be an important contributory mechanism in autoimmune
diseases.


References.

K. Maenaka, T. Juji, T. Nakayama, J.R. Wyer, G.F. Gao, T. Maenaka, N.R. Zaccai, A.
Kikuchi, T. Yabe, K.
Tokunaga, K. Tadokoro, D.I. Stuart, E.Y. Jones and P.A. van der
Merwe. (1999) Killer cell immunoglobulin receptors and T cell receptors bind peptide
-
major histocompatibility complex class I with distinct thermodynamic and kinetic
properties.
J. Biol. Chem.

274

28329
-
28334.

Glithero, J. Tormo, J.S. Haurum, G. Arsequell, G. Valencia, J. Edwards, S. Springer, A.
Townsend, Y
-
L. Pao, M. Wormald, R.A. Dwek, E.Y. Jones and T. Elliott. (1999) Crystal
sttructures of two H
-
2Db/glycopeptide complexes suggest a molecul
ar basis for CTL
cross
-
reactivity.
Immunity

10

63
-
74.