Structural analysis of the mechanism of ubiquitin conjugation, a generalized cellular addressing system, that controls protein and DNA stability

Ubiquitin conjugation processes are emerging as a general addressing system that is essential for cell stability, by controlling degradation of short-lived proteins, DNA repair and targeting to specific areas in the cell through endocytosis. Because of its importance for regulating cell cycle (e.g. P53, MDM2, E2Fs, p27, cyclin D), apoptosis (e.g. cCbl, IAPs) and DNA repair (e.g. Rad6, Rad18, Brca1) deregulation of ubiquitin-dependent processes often leads to cancer. The process of conjugation by ubiquitin(-like) proteins involves covalent linking of one or more ubiquitins to a target protein by an E1/E2/E3 cascade of enzymes. Correct ubiquitination requires the complex spatial arrangement of ubiquitin, E2, E3 proteins and the target simultaneously in a precise but flexible manner. Although the overall mechanism has been defined, the atomic details have been lacking and the specificity determining factors are unclear. In this project we will analyze the steps along the pathway by X-ray crystallographic studies complemented by biochemical and biophysical analysis. We will focus on a number of parallel ubiquitination pathways in cell-cycle control, DNA repair and nuclear targeting. Starting from the E3-ligases, themselves often multi-protein complexes, we will gradually include E2s, ubiquitin and target proteins. By making judicious use of chemically modified ubiquitin and mutations in the E2, E3 and targets we will create stable intermediates and determine their structures. By biochemical and biophysical analysis of these complexes both before and after structure solution we will evaluate different stages in the mechanism. This project will provide better understanding of the critical ubiquitin addressing system. By approaching the system in a generalized manner, studying several pathways in parallel we will both improve our progress, creating better opportunities from a practical viewpoint, but also create opportunities for understanding the regulatory details of this ubiquitous system. These studies will give insight in the chemistry of the transfer reaction and the specificity of the various steps in the pathway. This will provide a basis for targeted structure-based drug design.