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The Skaggs Institute for Chemical Biology
Scientific Report 1999-2000


The Immune Response and the Purine Biosynthesis Pathway as Targets for Therapeutic Intervention


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.

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 because the immune system must somehow discriminate between self and foreign antigens.

We elucidated 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.

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 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 tissues.

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 signaling.

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.

Catalytic Antibodies

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.

Publications

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. 293:351, 1999.

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, 1999.

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.

 

 







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