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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. 2).

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 transduction.

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.

Cancer Targets

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.



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