THE SOLUTION STRUCTURE OF ABL SH3, AND ITS RELATIONSHIP TO SH2 IN THESH(32) CONSTRUCT

Background: The Src homology domains, SH3 and SH2, of Ab1 protein tyro sine kinase regulate enzymatic activity in vivo. Ab1 SH3 suppresses ki nase activity, whereas Ab1 SH2 is required for the transforming activi ty of the activated form of Ab1. We expect that the solution structure s of Ab1 SH3, Ab1 SH2 and Ab1 SH(32) (a dual domain comprising SH3 and SH2 subdomains) will contribute to a structural basis for understandi ng the mechanism of the Ab1 'regulatory apparatus'. Results: We presen t the solution structure of the free Ab1 SH3 domain and a structural c haracterization of the Ab1 regulatory apparatus, the SH(32) dual domai n. The solution structure of Ab1 SH3 was determined using multidimensi onal double resonance NMR spectroscopy. It consists of two antiparalle l beta sheets packed orthogonally, an arrangement first shown in spect rin SH3. Compared with the crystal structure of the Ab1 SH3 complexed with a natural ligand, there is no significant difference in overall f olding pattern. The structure of the Ab1 SH(32) dual domain was charac terized by NMR spectroscopy using the H-1 and N-15 resonance assignmen t of Ab1 SH3 and Ab1 SH2. On the basis of the high degree of similarit y in chemical shifts' and hydrogen/deuterium exchange pattern for the individual domains of SH3 and SH2 compared with those of the SH(32) du al domain, a structural model of the Ab1 SH(32) regulatory apparatus i s suggested. This model is in good agreement with the ligand-binding c haracteristics of Ab1 SH3, SH2 and SH(32). The binding constants for i solated SH3 and SH2 domains when binding to natural ligands, measured by intrinsic fluorescence quenching, do not differ significantly from the constants of these domains within SH(32). Conclusion: The solution structures of free Ab1 SH3 and Ab1 SH2, and the structural model of A b1 SH(32), provide information about the overall topology of these mod ular domains. The structural model of Ab1 SH(32), a monomer, consists of the SH3 and SH2 domains connected by a flexible linker. Sites of li gand binding for the two subdomains are independent.