News and Publications
The Skaggs Institute For Chemical Biology
Scientific Report 1997-1998
X-ray Crystallography of Therapeutically Important Macromolecules
I.A. Wilson, K.C. Garcia, M. Degano, S.E. Greasley, T. Horton, M. Huang, O.
Livnah, V.M. Reyes, R.S. Stefanko, Y. Su, D.B. Williams, M.M. Yamashita
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. The 3-dimensional structures
of TCRs alone and of TCRs in complex with different peptide-MHC ligands can provide
insights on the structural basis of peptide-MHC specificity.
Using single-crystal x-ray diffraction, we determined the structure of a
murine TCR (2C) to 2.5 Å and the structure of a complex formed by this
TCR with Kb-dEV8, a syngeneic MHC with a self-peptide, to 3.2 Å.
The TCR is oriented diagonally across the surface of the peptide-MHC molecule,
with the α chain of the TCR over the peptide N-terminal residues and the ß chain
covering the peptide C-terminus (Fig. 1).
Three of the complementarity-determining regions (CDRs) of the TCR undergo
conformational changes during binding, providing a means of accommodating a variety
of different peptide antigens. The CDR2's of the α and ß chains of
the TCR are located on top of the MHC α-helices and interact with highly
conserved residues. These findings suggest a generalized orientation for TCR-MHC
interaction mediated primarily by the variable domain of the TCR α chain.
We recently refined at 3.2-Å resolution the 3-dimensional structure
of the syngeneic complex consisting of 2C and Kb-dEV8. The dEV8 peptide
is a weak agonist that may be involved in positive selection of 2C in the thymus.
The interface between 2C and the peptide-MHC molecule has poor surface complementarity,
with large, unfilled spaces between the peptide and the TCR. This poor fit explains
how 2C can interact with a large set of peptide antigens bound to Kb,
thus rationalizing at the atomic level its broad specificity. All CDRs are involved
in contacts with the peptide-MHC molecule; the CDR2's of both the α and
the ß chains of the TCR contact exclusively the MHC part of the molecule,
thus providing a scaffold for fine sampling of the peptide residues by the other
CDRs. CDRs 1 and 3 of both TCR chains contact both peptide and MHC and undergo
segmental and conformational changes upon binding of the antigen.
Hence, these structural rearrangements provide an induced-fit mechanism that
can accommodate a variety of different peptide ligands. The short CDR3 of the ß chain
has surprisingly few contacts with the peptide antigen. The CDR1 and CDR2 contact
residues of the variable domain of the α chain appear to be highly conserved
across different TCRs, suggesting a dominant role of the α chain in determining
the orientation of the TCR-MHC complex.
We are currently refining the structure of a complex that consists of the
same MHC and TCR, but with a foreign (synthetic) peptide antigen. Comparison
of the structures of these 2 complexes can explain at the atomic level how the
immune system discriminates between self and foreign antigens and how the additional
kinetic stabilization that appears to promote signaling in the T cell is achieved.
This project is a collaboration with L. Teyton, Department of Immunology, The
Scripps Research Institute.
TCRs specific for class II--restricted MHC molecules have also been engineered
as either variable domain or complete exodomain heterodimer constructs. Comparative
structural analysis will provide valuable insights into how class II--restricted
TCRs discriminate between mutually exclusive major T-cell epitopes at the midpoint
and at the carboxy end of insulin peptides.
Erythropoietin, a 34-kD glycoprotein molecule, 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. Recombinant
human erythropoietin is widely used in the treatment of anemia, and sales of
the drug generate more than $2 billion per year. However, because of its large
molecular size, erythropoietin must be administered either intravenously or subcutaneously.
We have determined the crystal structures of the extracellular domain of
the receptor for erythropoietin (EBP) in complex with several different peptides,
including EMP1, an agonist, and EMP33, an antagonist. Although the same regions
of EBP are used to bind the peptides in both complexes, the dimerization modes
of the 2 complexes differ. In the EBP-EMP1 complex, 2 EMP1 molecules are required
to dimerize the EBP molecules in an almost perfect 2-fold symmetric configuration.
However, this symmetry is not maintained in the EBP-EMP33 complex, which has
an asymmetric EBP dimer. Indeed, it is surprising that the antagonist peptide
still dimerizes the receptor at all. The only difference between EMP1 and EMP33
is that a tyrosine in EMP1 is replaced with a 3,5-dibromotyrosine in EMP33 (Fig.
Thus, dimerization is not a sufficient prerequisite for signal transduction
and subsequent cell division and proliferation. These pairs of structures suggest
that both proximity and orientation of the receptors are important for signal
On the basis of the structural and chemical knowledge derived from these
crystal structures of EBP-EMP1 and EBP-EMP33, we are pursuing a multidisciplinary
drug discovery effort to detect novel erythropoietin agonists and antagonists.
In collaboration with L. Jolliffe, R.W. Johnson Pharmaceutical Research Institute,
and D. Boger, Department of Chemistry, The Scripps Research Institute, we are
exploring different possible pharmacophores and are generating libraries of small
molecules. We will pursue crystallization of EBP with any lead compounds. Information
from such structural analyses can be critical for the optimization and redesign
of new compounds.
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. Work is in progress on a number of folate-derived
and nonfolate inhibitors synthesized in Dr. Boger's laboratory and cocrystallized
with wild-type Escherichia coli GAR transformylase (Fig. 3).
These x-ray structures will guide future development toward novel compounds
specific for GAR transformylase, thereby reducing nonspecific interaction with
other folate-dependent enzymes in the cell.
Recently, a mutant form of E coli GAR transformylase lacking the ability
to form dimers was developed by S. Benkovic, Pennsylvania State University, and,
in collaborative studies with P. Jenning, University of California, San Diego,
has been extensively characterized. This mutant and other structures determined
in our laboratory have defined a series of events along the reaction pathway
that involve the ordering of both folate and pH (or substrate)-dependent loops
critical for catalytic activity.
To explore the catalytic mechanism of GAR transformylase, we are refining
2 active-site mutants of the enzyme in complex with the Burroughs-Wellcome inhibitor
BW147U89. It appears that the mutations affect interactions with other key active-site
residues. These combined structural and biochemical studies have led to a model
in which the enzyme has distinct conformational states that are dependent on
pH, substrate, and cofactor binding. The ability to exploit these various conformational
forms should enhance the design of potent inhibitors of the enzyme.
Garcia, K.C., Degano, M., Pease, L.R., Huang, M., Peterson, P.A., Teyton,
L., Wilson, I.A. Structural basis of plasticity in T-cell receptor recognition
of a self peptide-MHC antigen. Science 279:1166, 1998.
Garcia, K.C., Tallquist, M.D., Pease, L.R., Brunmark, A., Scott, C.A.,
Degano, M., Stura, E.A., Peterson, P.A., Wilson, I.A., Teyton, L. αß T-cell
receptor interactions with syngeneic and allogeneic ligands: Affinity measurements
and crystallization. Proc. Natl. Acad. Sci. U.S.A. 94:13838, 1997.
Johnson, D.L., Farrell, F.X., Barbone, F.P., McMahon, F.J., Tullai, J.,
Hoey, K., Livnah, O., Wrighton, N.C., Middleton, S.A., Loughney, D.A., Stura,
E.A., Dower, W.J., Mulcahy, L.S., Wilson, I.A., Jolliffe, L.K. Identification
of a 13 amino acid peptide mimetic of erythropoietin and description of amino
acids critical for the mimetic activity of EMP1. Biochemistry 37:3699, 1998.
Wilson, I.A., Garcia, K.C. T-cell receptor structure and TCR complexes.
Curr. Opin. Struct. Biol. 7:839, 1997.