News and Publications
The Skaggs Institute for Chemical Biology
Scientific Report 1999-2000
The Immune Response and the Purine Biosynthesis Pathway as Targets for Therapeutic
I.A. Wilson, R.A. Forcada Lowrie, S.E. Greasley, P.A. Horton, J.G. Luz, M.
Rudolph, R.S. Stefanko, D. Wolan, M. Yu, X. Zhu, V.M. Reyes, D.B. Williams
We use x-ray crystallography to investigate the structure and function of
proteins that are potential targets for therapeutic drugs or that harness the
immune system for generation of novel catalysts. Knowledge gained from the crystal
structures of these proteins will enable the design of potential reagents for
cancer therapy, for the treatment of diseases involving immune recognition or
hematopoiesis, and for use in biology, biotechnology and medicine.
The T-cell receptor (TCR) is a heterodimeric glycoprotein expressed on the
surface of T lymphocytes. The central event in immune responses mediated by T
cells is TCR recognition of peptide antigens from foreign pathogens in the context
of the MHC. Interactions between TCRs and MHCs are finely modulated because the
immune system must somehow discriminate between self and foreign antigens.
the crystal structures of 2C, a murine TCR, in complexes with agonist and superagonist
ligands, which are represented by the MHC molecule H-2Kb bound to
dEV8 and SIYR peptides, respectively (Fig. 1). The superagonist complex showed
that only relatively subtle changes in the structure of the interface are associated
with remarkably large increases in biological potency. We have now collected
x-ray diffraction data to 2.4 Å on a crystalline complex consisting of
2C and an alloligand, H-2Kbm3, bound to the dEV8 peptide. This information
markedly extends the resolution of the available data on 2C bound to antigen-presenting
molecules and should provide insights into how 2C responds to both agonist and
antagonist ligands. Structural studies of such class I MHC-restricted TCRs for
which antagonistic and agonistic MHC-peptide complexes are available may reveal
the structural basis of positive and negative selection of T cells.
We are also investigating the interaction between the coreceptor CD8 and
the TCR-MHC-peptide complex. CD8 on cytotoxic T lymphocytes interacts with both
the TCR and the MHC, thereby producing a ternary complex that is essential for
intracellular signaling. The soluble, glycosylated extracellular domains of all
3 proteins have been expressed and purified in insect cells, and microcrystals
of the ternary complex have been grown. These studies will help determine the
role of CD8 in signal transduction during the immune response.
Some MHC molecules are strongly associated with autoimmune diseases. A particularly
interesting example is the association of insulin-dependent diabetes mellitus
with the absence of a peptide aspartate residue (ß57) in I-Ag7 in
mice and in HLA-DQ alleles in humans. In nonobese diabetic mice, spontaneous
diseases occur because I-Ag7 is the sole class II MHC allele present.
The crystal structure of I-Ag7 in the presence of a proposed autoreactive
peptide (GAD65, an autoantigen found in the ß cells in pancreatic islets)
reveals a substantially altered peptide-binding pocket that differs from the
pocket in other class II MHC alleles (Fig. 2).
The absence of the MHC aspartate residue can be compensated for by the presence
of a carboxyl residue in the peptide itself. Thus, this MHC allele selects a
different subset of peptides for binding to its peptide-binding groove. These
results also indicate that I-Ag7 is as stable as some of the class
II MHC alleles, such as I-Ad, and that it still binds peptide in the
classical manner. All the studies on TCRs and MHCs were done in collaboration
with L. Teyton, The Scripps Research Institute.
The receptor for erythropoietin is a member of the class 1 cytokine receptor
superfamily. Binding of cytokine hormones and oligomerization of their receptors
induce the intracellular response, which promotes the biological function of
cell activation or proliferation. Erythropoietin, a 34-kD glycoprotein, is the
primary hormone that regulates the differentiation and proliferation of immature
erythroid cells. Erythropoietin functions through binding to its receptor on
the surface of committed progenitor cells in bone marrow and other hematopoietic
We determined the crystal structures of the extracellular domain of the receptor
in complex with both agonist and antagonist peptides. On the basis of the structural
and chemical knowledge derived from the structure of the complex containing the
agonist, we are pursuing a multidisciplinary drug discovery effort to detect
novel erythropoietin agonists and antagonists. This work is being done in collaboration
with L. Jolliffe, R.W. Johnson Pharmaceutical Research Institute, and D. Boger,
the Skaggs Institute.
The exciting finding that antagonist peptides dimerize the receptor but do
not signal caused us to reevaluate the nature and role of dimerization in signal
transduction and cell proliferation. The recently determined crystal structure
of the extracellular domain of the erythropoietin receptor in the receptor's
unliganded form unexpectedly revealed a dimer and provided evidence for preformed
dimers of the receptor on the surface before ligand activation. These results
explain how a relatively low density of receptor on the cell surface can signal
even when the binding constant of the second site is in the micromolar range.
These findings, obtained in collaborative studies with S. Michnick, University
of Montreal, represent a new model for the role of dimerization in cytokine receptor
Enzymatic Cancer Targets
Glycinamide ribonucleotide (GAR) transformylase and ATIC (aminoimidazole
carboxamide ribonucleotide [AICAR] transformylase inosine monophosphate cyclohydrolase
[IMPCH]) are folate-dependent enzymes involved in de novo purine biosynthesis
and are potential targets for anticancer drugs. In collaboration with G.P. Beardsley,
Yale University, S.J. Benkovic, Pennsylvania State University, and D. Boger,
the Skaggs Institute, we are using structure-based drug design to develop novel
compounds specific for these enzymes, thereby reducing potential cross-reactivities
with other folate-dependent enzymes in the cell. We determined several crystal
structures of GAR transformylase both in the presence and absence of bound substrate, ß-GAR,
and cofactor-derived inhibitors. The details provided by these structures suggested
improvements for the next generation of GAR transformylase inhibitors.
ATIC (also known as PurH) is a 64-kD bifunctional enzyme with both AICAR
transformylase and IMPCH activities. AICAR transformylase catalyzes the transformylation
of AICAR and 10-formyl-tetrahydrofolate to produce formyl-AICAR and tetrahydrofolate,
whereas IMPCH is responsible for the conversion of formyl-AICAR to inosine monophosphate.
Thus, ATIC catalyzes the penultimate and final steps in the purine biosynthesis
pathway. ATIC is therefore a prime target for rational drug design for antineoplastic
intervention. However, it has not been investigated as intensively as the other
folate-requiring enzymes because no crystal structure is available to aid in
the design of ATIC inhibitors.
Sequence comparisons of various AICAR transformylases with other folate-using
enzymes indicated no obvious homology with any known folate- or AICAR-binding
domains. Recently, we determined the crystal structure of avian ATIC to 1.75-Å resolution
(Fig. 3). In addition, complexes of both human and avian ATIC are being determined
to provide valuable information about the possible mechanism of action of both
the AICAR transformylase and IMPCH and to advance the design of inhibitors.
ATIC has been implicated as the target of nonsteroidal anti-inflammatory
drugs and of the anti-inflammatory response induced by low doses of methotrexate-polyglutamate.
The discovery of inhibitors of ATIC may therefore lead to the treatment of both
cancer and diseases associated with the inflammatory response.
Much of our efforts on catalytic antibodies focuses on antibodies that catalyze
reactions for which no natural enzymes are known or reactions that are highly
disfavored. An exciting new structure this year was that of a Diels-Alderase
antibody, 1E9, that is highly related to both a progesterone antibody that we
worked on several years ago and another Diels-Alderase catalytic antibody developed
by R. Stevens and P. Schultz, The Scripps Research Institute and the Skaggs Institute.
These 3 antibodies came from the same primordial germline; hence, we can follow
the evolution of catalytic activity for the 2 Diels-Alderase antibodies.
Only 2 somatic mutations in 1E9 appear to be responsible for a huge increase
in 1E9 activity compared with the activity of the other Diels-Alderase. An unexpected
mutation in a highly conserved framework residue, Trp47Leu, allowed reconfiguration
of the binding pocket such that the transition-state analog fit almost perfectly.
The close correlation of the structure of the transition-state analog with the
true transition-state molecule thus allowed the antibody complementarity to the
bound transition-state molecule to be fully harnessed into catalytic activity.
Such revealing analyses are only possible because of the increasing number of
catalytic antibody structures being deposited in the Protein Data Bank. Hence,
the use of catalytic antibodies to study the evolution of catalysis can now become
more fruitful as the number of abzyme structures continues to increase. The research
on 1E9 was done in collaboration with D. Hilvert, ETH, Zürich, Switzerland.
Degano, M., Garcia, K.C., Apostolopoulos, V., Rudolph, M.G., Teyton, L.,
Wilson, I.A. A functional hot spot for antigen recognition in a superagonist
TCR/MHC complex. Immunity 12:251, 2000.
Greasley, S.E., Yamashita, M.M., Cai, H., Benkovic, S.J., Boger, D.L.,
Wilson, I.A. New insights into inhibitor design from the crystal structure
and NMR studies of Escherichia coli GAR transformylase in complex with ß-GAR
and 10-formyl-5,8,10-trideazafolic acid. Biochemistry 38:16783, 1999.
Middleton, S.A., Barbone, F.P., Johnson, D.L., Thurmond, R.L., You, Y.,
McMahon, F.J., Jin, R., Livnah, O., Tullai, J., Farrell, F.X., Goldsmith, M.A.,
Wilson, I.A., Jolliffe, L.K. Shared and unique determinants of the erythropoietin
(EPO) receptor are important for binding EPO and EPO mimetic peptide. J. Biol.
Chem. 274:14163, 1999.
Rudd, P.M., Wormald, M.R., Stanfield, R.L., Huang, M., Mattsson, N., Speir,
J.A., DiGennaro, J.A., Fetrow, J.S., Dwek, R.A., Wilson, I.A. Roles for glycosylation
of cell surface receptors involved in cellular immune recognition. J. Mol. Biol.
Wilson, I.A. Perspectives: Protein structure. Class-conscious TCR?
Science 286:1867, 1999.
Wilson, I.A., Jolliffe, L.K. The structure, organization, activation
and plasticity of the erythropoietin receptor. Curr. Opin. Struct. Biol. 9:696,
Xu, J., Deng, Q., Chen, J., Houk, K.N., Bartek, J., Hilvert, D., Wilson,
I.A. Evolution of shape complementarity and catalytic efficiency from a primordial
antibody template. Science 286:2345, 1999.