Structure and mechanism of the ubiquitin activating enzyme

点击翻译按钮获取中文摘要

The activation of ubiquitin by the ubiquitin-activating enzyme (E1) represents the initial step in the covalent attachment of ubiquitin molecules to the side chains of lysine residues in target substrates. This modification is found in a multitude of cellular process including the selected degradation of the target protein by the proteasome. The E1-catalyzed activation of ubiquitin is a three-step process in which ubiquitin is adenylated at its C-terminal glycine, followed by the covalent attachment of ubiquitin to an essential cysteine residue. Finally, the enzyme ensures that ubiquitin is transferred in a trans-thioesterification reaction to one of several ubiquitin conjugating (E2) enzymes but not to any of the E2 enzymes of a ubiquitinlike protein modifier such as SUMO or NEDD8. Based on our crystal structure of the complex between the yeast E1 enzyme with ubiquitin bound at the adenylation active site, the first crystal structure of an intact ubiquitin E1, we intend to further characterize this enzyme with following aims in mind: (1) We will study how the enzyme recognizes cognate E2 enzymes and discriminates against E2 enzymes involved in SUMO or NEDD8 transfer. (2) We will characterize how the E1 enzyme is inhibited by small molecules to further probe its catalytic mechanism and as a first step towards the design of highly specific inhibitors. (3) We will determine the crystal structure of a quarternary complex consisting of the E1 enzyme, two bound ubiquitin molecules and an E2 enzyme to determine the conformational changes accompanying trans-thioesterification.