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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.

T-Cell Receptor

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.

Erythropoietin Receptor

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).

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. 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.

Publications

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. 1:75, 1999.

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. 281:485, 1998.

 

 







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