One of the current challenges in structural biology is to analyse the large and often transient complexes that are important in cellular systems. We study structure and function of complexes in ubiquitin conjugation, DNA mismatch repair and chromatin remodeling using cryo-EM, protein crystallography and complementary biophysical techniques.
Ubiquitin conjugation processes are critical signaling systems for most cellular processes. They attach one or more ubiquitins to target proteins and change their cellular fate, by promoting novel interactions, controlling degradation of short-lived proteins or inducing a relocalization. Because of the importance for regulating cell cycle, chromatin regulation, apoptosis and DNA repair deregulation of ubiquitin-dependent processes often leads to cancer. The process of conjugation by ubiquitin(-like) proteins involves covalent linking of one or more 76-amino-acid 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. As these are reversible processes the role of deubiquitinating enzymes (DUBs) in the process is equally important to maintain the balance in the system.
We are interested in the regulation of the process of ubiquitin conjugation and deconjugation. We study the specificity of E2/E3 complexes and DUBs for specific targets such as H2A and PCNA or ubiquitin itself. We are interested how these ubiquitin ligases choose their target and how their actions are regulated.
The activity of DUBs is regulated in the cell is carefully controlled. We study regulation of intrinsic catalytic activity of a number of different DUBs by partners, cofactors or additional domains outside the catalytic subunit. Knowledge of this type of regulation can be of interest for drug development targeting these signaling proteins.
One of the most prevalent forms of human hereditary cancer, HNPCC, is caused by mutations in the genes encoding the DNA mismatch repair proteins. These proteins are the human homologs of E. coli MutS and MutL, which execute the first two steps in repair of misincorporated base pairs during DNA duplication besides playing a role in the prevention of recombination of homeologous sequences. We study the crystal structure of MutS complexed to mismatched DNA and how this asymmetric ATPase couples DNA mismatch recognition to initiation of repair.
In the past we have studied the molluscan Acetylcholine Binding Protein (AchBP) as a mimic for ligand binding in nicotinic acetylcholine receptors (nAChR). This glial protein has conserved all the ligand binding properties and hence has served very well as a model system for studying ion channel function for the cys-loop receptors that include the GABAa and 5HT3 serotonin receptors, particularly with respect to their ligand binding properties.
We have used AChBP as a model system to study many aspects of ligand binding in Cys-loop receptors. These studies revealed ligand binding properties for a variety of ligands, the role of the modulating complementary subunit, the flexibility of the binding site and options for changing ligand properties. We analyzed anti-smoking compounds and used this model system for identification of novel ligands.