Structural analysis of Proteins Involved in Apoptosis and Inflammation

Proteins Involved in Apoptosis and Inflammation

Programmed cell death (apoptosis) and inflammation are two closely related cellular processes which are involved in development, immune response and many other important processes in multicellular organisms.

One group of proteins, called caspases (cysteine-dependent aspartate specific proteases) is of particular interest to us because of their central position in many apoptotic and inflammatory pathways and their high specificity. Caspases are involved in cancer, auto-inflammatory and many other diseases.

Our goals are to study the activation pathways of caspases and their catalytic mechanism with biochemical and biophysical methods. In addition, we are interested in the analysis of several adaptor proteins that are either involved in triggering or propagation of the apoptotic and inflammatory signals. These adaptor proteins possess modular domain architectures, comprising protein-protein interaction domains from the death domain, leucine-rich repeat and B30.2 superfamilies.

Besides general biochemical and biophysical methods three-dimensional structure determination using X-ray crystallography is our major tool.
Depicted are the crystal structures of caspase-3 and pyrin B30.2 domain.

Sphingosine-1-phosphate lyase (collaboration with G. Capitani, PSI Villigen)

Sphingosine-1-phosphate (S1P) is an important sphingolipid that is involved in the regulation of cell proliferation, motility and invasiveness. S1P also plays a fundamental role in lymphocyte trafficking. Sphingosine-1-phosphate lyase (SPL) catalyzes the irreversible degradation of S1P and thus takes part in controlling the concentration of this important sphingolipid. We are characterizing SPL by means of biochemical and biophysical methods.

Protein crystallization

The preparation of diffracting crystals is still the major rate-limiting factor in protein crystallography. Therefore innovative methods for the crystallization of proteins will have a large impact on the field of macromolecular crystallography in general. We are interested in two different approaches to increase the speed and the success of crystallization trials.

Crystallization screens

Crystallization screens require a relatively large amount of protein (more than 10 mg), because 1 µl is the minimal volume that can be handled manually. The application of the high-throughput nanodrop crystallization technology (drops of 100 to 200 nl) allows to explore more crystallization conditions with the same amount of pure protein. This is especially interesting for proteins that are difficult to express and to purify. Moreover, we have developped a high-throughput capillary crystallization technique (Crystalharp, in collaboration with Swissci) that complements the nanodrop technology for crystal screening and optimization.

Crystallization chaperones

Target proteins in complex with a ligand or another protein often exhibit superior biophysical properties to those of target proteins alone. Therefore, the crystallization of Fab-complexes is a well known methodology in the field of membrane protein crystallization. However, monoclonal antibodies are time-consuming to obtain and their handling is laborious.
As an alternative method we are using Designed Ankyrin Repeat Proteins (DARPin) as crystallization tools. DARPin that selectively recognize a given target protein are generated using the ribosome-display technology (collaboration with the group of Prof. A. Plückthun, UniZH).
Besides DARPins, we are also work with scFv as crystallization tools using the ETH-2 Gold phage display library (group of Prof. D. Neri, ETHZ). Binders are selected using the phage display technology.

Structural analysis of membrane proteins

Membrane proteins constitute a large group of physiologically and medically important proteins that are challenging to handle and are therefore substantially underrepresented in structure databanks.

We are working on different classes of membrane proteins that are responsible for the controlled transport of nutrients or drugs across the lipid bilayer, such as ABC transporters and representatives of the RND superfamily.

The preparation of diffraction quality crystals remains the major bottleneck in the structural analysis of membranes proteins by X-ray crystallography. For that reason we use crystallization chaperones (DARPin or scFv) as auxiliary proteins that bind to and increase the crystallization probability of a target molecule of interest. Such chaperones may reduce conformational heterogeneity and extend surfaces predisposed to forming crystal contacts. DARPins that influence the functional properties of the targeted proteins such as inhibitors are particularly interesting to be used for co-crystallization as the complex structure often reveals interesting mechanistic details.
Depicted is the crystal structure of a complex between AcrB and a DARPin.

For further information please consult the Institute of Biochemistry homepage