Structure-based mechanistic analysis of the proteasome

The ubiquitin proteasome pathway is one of the major protein degradation pathways in the cell. This pathway is essential for protein quality control and the regulation of the cell cycle. Proteins subjected to the ubiquitin pathway are cleaved into short peptides by the 26S proteasome. The 26S proteasome consists of the 19S regulatory particle and the 20S core proteasome. Ubiquitinated substrates are recognized through their ubiquitin moiety by the 19S regulatory particle, unfolded and translocated into the 20S core proteasome. Within the 20S proteasome hydrolysis of the protein peptide bonds takes place. The aforementioned significance of proteasome for cell function and the associated fact that the proteasome is a drug target for the treatment of cancer makes it an attractive object for structural analyses. A detailed understanding of the proteasome structure is essential to dissect the function and its modulation by proteins and small molecule effectors. In this thesis the sample quality of purified human 20S proteasomes was improved by biochemical optimization and by the introduction of a chromatography free purification protocol. The newly developed purification protocol was shown to be transferable to the purification of human 26S proteasomes, 20S proteasomes from Saccharomyces cerevisiae, Drosophila melanogaster and Thermoplasma acidophilum. Crystals were grown and structures were solved for all purified 20S proteasomes. The established post-crystallization treatment resulted in human and fruit fly 20S proteasomes crystals diffracting to 2.0 Å on average. The best resolution of 1.8 Å was collected for the human 20S proteasome. The robust purification and crystallization protocol leading to unprecedented resolutions obtained for the human 20S proteasome enabled to reanalyze inhibition mechanism of known 20S proteasome inhibitors. Furthermore, screening efforts using small molecules revealed the diazomethylketone C-3455 as new modulator of the 20S proteasome. Most of the previously described reaction mechanisms could be validated. The largest discrepancy to published reaction mechanisms was found for epoxyketone inhibitors. Instead of the proposed formation of a 1,4-morpholine ring, the newly solved structures suggest the formation of a 1,4-oxazepane ring. The observed reaction mechanism was validated by structural analyses, electronic structure calculations of the reaction pathways and kinetic analyses. Based on these results a new reaction mechanism was proposed for epoxyketone inhibition enabling the design of new 20S proteasome inhibitors. Electron microscopy studies of the 26S proteasome and X-ray crystallographic studies of the fruit fly 20S proteasome revealed for the first time allosteric pathways of the pro- teasome. Inhibition of the proteolytic active sites in the 26S holocomplex influences the dynamics of the 19S lid movements. For the fruit fly 20S proteasome a structural rearrangement in the β5-subunit was observed upon inhibition of the chymotryptic-like site or upon binding of a short peptide activator to the α-rings. In summary, the newly established purification protocol led to 20S/26S proteasome sam- ples of superior quality. This made it possible to analyze structural features of the pro- teasome at resolutions not described before. The established high throughput pipeline for crystallographic analyses of human 20S proteasomes can be used for screening small molecules in future