Our group is interested in structural aspects of protein degradation. We are currently looking at proteases and at other enzymes that are involved in the regulation of protein degradation. The proteins that we attempt to crystallise are either chosen because of their biological relevance, or because they are not recognisably similar to any proteins of known structure and thus provide the best opportunities to discover new mechanistic principles in proteolysis.
Protein degradation in eukaryotes: ubiquitin system
Our work on the ubqiuitin system relies partially on proteins that we clone and purify ourselves and partially on collaborations with both MPI-CBG (Drs. Zachariae and Drechsel) and IIMCB (Prof. Kuznicki). We have established purification protocols for ubiquitin-activating enzyme (E1), for a ubiquitin conjugating enzyme (E2), for CHIP, a chaperone associated, non-RING ubiquitin ligase (E3) and for several class II ubiquitin hydrolases. We have also purified TRIC (CCT), the chaperone that is required for the activity of APC, a ubiquitin ligase and the clock of the cell cycle.
Protein degradation in prokaryotes
A lot of our work on bacterial proteases has concentrated on the staphopain-staphostatin system. Staphopains are the major secreted cysteine proteases of Staph. aureus and candidate virulence factors of this serious human pathogen that has received a lot of attention due to the emergence of multi-drug resistant strains. Staphostatins are the recently discovered endogenous inhibitors of staphopains.

We have recently:

• solved the 1.25 Å crystal structure of ~110 residue staphostatin B. The structure contains a major surprise: The inhibitor folds into an eight-stranded _-barrel and resembles lipocalins rather than cystatins, the standard inhibitors of cysteine proteases. This work will appear shortly in Protein Science.
• solved the 1.8 Å structure of the inhibitor in complex with its target protease. The structure has added to the surprise from the structure of the free inhibitor, since the inhibitor binds in forward direction to the target protease. It avoids cleavage by protease through a rotation of the potentially ?scissile? amide bond of inhibitor, so that both nucleophilic attack and stabilisation of the oxyanion intermediate are geometrically impossible. To our knowledge, this mechanism is without precedence in cysteine protease inhibition. This work has just appeared on-line in JBC.

In addition, we are working on several other families of bacterial proteases of unknown fold. In one case, we have recently solved a structure to 1.4 Å resolution by MAD and found that the protein has a fold that was never seen before in a proteolytic enzyme. In addition, the structure shows a mechanistically novel strategy to maintain the enzyme in a latent state. Mutagenesis and biochemical work confirms the structural predictions. This information will be updated as soon as our manuscript is accepted.