News and Publications
The Skaggs Institute for Chemical Biology
Scientific Report 1998-1999
X-ray Crystallography of Therapeutically Important Macromolecules
I.A. Wilson, K.C. Garcia, M. Degano, R.A. Forcada Lowrie, S.E. Greasley, P.A.
Horton, M. Huang, J.G. Luz, V.M. Reyes, R.S. Stefanko, Y. Su, D.B. Williams,
M.M. Yamashita, M. Yu
Our research program involves determining the x-ray crystallographic structures
of proteins that are potential targets for therapeutic drugs. These studies may
be of use in cancer therapy, in diseases involving immune recognition, and in
disorders involving hematopoiesis.
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 in order
to discriminate between self and foreign antigens.
We determined the structure of 2C, a murine TCR, to 2.5 Å; the structure
of a self-complex formed by this TCR with Kb/dEV8, a syngeneic MHC
with a self-peptide, to 3.2 Å; and the structure of a foreign complex formed
by 2C with the highly potent Kb/SIYR to 3.0 Å. In collaboration
with L. Teyton, The Scripps Research Institute, we are studying the structures
of other TCR-peptide-MHC complexes to understand the molecular details of this
complex system. Currently, we are trying to obtain x-ray quality crystals for
a complex consisting of the TCR B3 and MHC H-2Kb, bound to both antagonist
and agonist peptides. Structural studies of MHC class Irestricted 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 also used a bacterial expression system to produce soluble MHC class IIrestricted
helper TCRs that recognize insulin peptides when the peptides are presented by
the IAd class II MHC restricting element of the TCRs. These TCRs have
a unique ability to discriminate a 2 amino acid difference between the human
insulin A1-13 peptide and several isoforms of insulin. Data to 2.6-Å resolution
have been collected on tetragonal crystals of the scFv TCR fragment. Structural
studies will address whether differences exist in the abilities of helper TCRs
and cytotoxic TCRs to recognize the receptors' specific class II and class I
restricting elements, respectively.
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 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 tissues.
We determined the crystal structures of the extracellular domain of the erythropoietin
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
with 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 provides evidence for the occurrence
of preformed dimers of the receptor before ligand activation (Fig. 1).
Glycinamide ribonucleotide (GAR) transformylase and aminoimidazole carboxamide
ribonucleotide transformylase are folate-dependent enzymes involved in the de
novo biosynthesis of purine. These enzymes are potential targets for anticancer
and antiinflammatory drugs. In collaborative studies with Dr. Boger and with
S. Benkovic, Pennsylvania State University, and P. Beardsley, Yale University,
we are using structure-based drug design to develop novel compounds specific
for these enzymes, thereby reducing nonspecific interactions with other folate-dependent
enzymes in the cell. We have determined several crystal structures at high resolution
(2.41.6 Å) of wild-type and mutant forms of GAR transformylase, in
both the presence and the absence of bound substrate, ß-GAR, and cofactor-derived
inhibitors. In particular, we recently determined the structure of a complex
with a compound that has time-dependent inhibition; the 1.6-Å electron
density map clearly revealed the existence of a multisubstrate adduct, formed
between the compound and ß-GAR, which was the rationale behind the inhibitor
design (Fig. 2). The details provided by these structures suggested improvements
for the next generation of inhibitor design and yielded valuable information
about the possible mechanism of the formyl transfer reaction that will further
aid the design of novel anticancer agents.
Garcia, K.C., Degano, M., Speir, J.A., Wilson, I.A. Emerging principles
for T cell receptor recognition of antigen in cellular immunity. Rev. Immunogenet.
Garcia, K.C., Teyton, L., Wilson, I.A. Structural basis of T cell recognition.
Annu. Rev. Immunol. 17:369, 1999.
Grant, E.P., Degano, M., Rosat, J.P., Stenger, S., Modlin, R.L., Wilson,
I.A., Porcelli, S.A., Brenner, M.B. Molecular recognition of lipid antigens
by T cell receptors. J. Exp. Med. 189:195, 1999.
Livnah, O., Johnson, D.L., Stura, E.A., Farrell, F.X., Barbone, F.P., You,
Y., Liu, K.D., Goldsmith, M.A., He, W., Krause, C.D., Pestka, S., Jolliffe, L.K.,
Wilson, I.A. An antagonist peptide-EPO receptor complex suggests that receptor
dimerization is not sufficient for activation. Nat. Struct. Biol. 5:993, 1998.
Livnah, O., Stura, E.A., Middleton, S.A., Johnson, D.L., Jolliffe, L.K.,
Wilson, I.A. Crystallographic evidence for preformed dimers of erythropoietin
receptor before ligand activation. Science 283:987, 1999.
Manning, T.C., Schlueter, C.J., Brondnicki, T.C., Parke, E.A., Speir, J.A.,
Garcia, K.C., Teyton, L., Wilson, I.A., Kranz, D.M. Alanine scanning mutagenesis
of an αß T cell receptor: Mapping the energy of antigen recognition.
Immunity 8:413, 1998.
Remy, I., Wilson, I.A., Michnick, S.W. Erythropoietin receptor activation
by a ligand-induced conformation change. Science 283:990, 1999.
Speir, J.A., Garcia, K.C., Brünmark, A., Degano, M., Peterson, P.A.,
Teyton, L., Wilson, I.A. Structural basis of 2C TCR allorecognition of H-2Ld peptide
complexes. Immunity 8:553, 1998.
Su, Y., Yamashita, M.M., Greasley, S.E., Mullen, C.A., Shim, J.H., Jennings,
P.A., Benkovic, S.J., Wilson, I.A. A pH-dependent stabilization of an active
site loop observed from low and high pH crystal structures of mutant monomeric
glycinamide ribonucleotide transformylase at 1.8 to 1.9 Å. J. Mol. Biol.