.) This application's objective is the structural explanation of the specificity of the transforming growth factor beta (TGFbeta) binding to the TGFbeta receptors (TbetaR). TGFbeta is bound by exoplasmic domains of the type II and type I TbetaR. Both types are transmembrane serine/threonine kinase receptors. Depending on the cellular context, TGFbeta inhibits or stimulates cellular division in animals and humans. In breast carcinomas, hepatomas, gastric, colon and skin tumors, as well as B and T lymphomas, inactivating mutations in both alleles of the Tbeta genes occur and correlate with loss of sensitivity to TGFbeta allowing unrestrained cell growth and tumor progression. Very little is known about thesecondary and/or tertiary structures of the ligand binding domains of TbetaRs (exTbetaR).
Specific aims of this application are: 1. Production of 15N/13C labeled exTbetaR type II in milligram quantities 2. Sequence specific assignments of 1H, 15N, and 13C nuclei of the exTbetaR type II 3. Solution structure of the exTbetaR type II 4. Experimental identification of the TGFbeta1 binding sites on the surface of the exTR type II 5. Production of 15N13C labeled exTbetaR type I in milligram quantities 6. Sequence specific assignments of 1H, 15N, and 13C nuclei of the exTbetaR type I 7. Solution structure of the exTbetaR type I The exTbetaRs are expressed as fusion proteins in an E. coli recombinant system. Affinity chromatography is used to purify fusion proteins containing exTbetaRs, which are then purified by affinity and size exclusion chromatography. The highly optimized expression system is used for enrichment of the purified protein in 15N and 13C isotopes. The structure determination o the exTbetaRs is carried out in solution using NMR methods. multidimensional heteronuclear NMR techniques are used for resonance assignments and extraction of the conformational constraints. These constraints will be used to calculate the 3D structure with distance geometry and energy minimization methods. The structures of the ligand binding domains of receptors will form the basis for studies of ligand-receptor interactions. Identification of the critical sites and changes occurring in them upon binding of the model ligand will follow. This research will allow future design of agonists and antagonists of cell proliferation and differentiation during animal development, physiology, and tumor progression. There is an ever increasing number of known sequences of receptors in the TGFbeta superfamily of proteins as derived from large scale sequencing efforts. The long range goal of this research is to discover the principles that determine locations of binding sites and specificities of new receptors, for which the direct experimental approach to the ligand-receptor complex is not feasible.
| Chim, Nicholas; Gall, Walter E; Xiao, Jing et al. (2004) Solution structure of the ubiquitin-binding domain in Swa2p from Saccharomyces cerevisiae. Proteins 54:784-93 |
