Sub theme 3.13
Molecular and cellular basis of genome instability in cancer

Goals of research: general outline
Scientific achievements
Future plans: special goals and approach
Running projects
Associated staff

Goals of research: general outline

Our aim is to provide understanding of structural aspects of errors in cell biology that lead to the development of cancer. Our tool is protein crystallography, the best technique for studying structure at atomic resolution. We use a combination of biochemical and biophysical techniques to help interpret our structural conclusions. In collaboration with cell biologists these data lead to better understanding of carcinogenesis. Our studies help to understand molecular mechanisms and provide a basis for drug design.

Scientific achievements

Nicotinic receptor homolog AChBP

Pentameric ligand-gated ion channels are among the pharmaceutically most important drug targets and represent the best-studied family of neurotransmitters. A homolog to the ligand binding domain of the Nicotinic Acetylcholine Receptor, AChBP (Brejc et al., Nature 2001) provided the first high resolution data for this class of ion channels and structures of the complex of AChBP with nicotine and carbamoyl choline have revealed the details of ligand binding to these receptors (Celie et al., Neuron 2004). Since nAChRs are critical in nicotine addiction, details of the nicotine binding are important for development of compounds that can combat smoking. Our analyses of conotoxins-AChBP complexes provided the first receptor-complex structures for these attractive lead-compounds (Celie et al., NSMB 2005) and generated important insight in nicotinic receptor subtype specificity (Ulens et al, PNAS 2006, Dutertre et al, EMBO J 2007).

Ubiquitin and SUMO conjugation

Ubiquitin and SUMO conjugation provide essential signaling steps in many cellular pathways leading to relocation or degradation of the target proteins. Because of their prevalence in many signaling pathways they present interesting drug targets. We study mechanisms of regulation of the ubiquitin and SUMO pathways. In close collaboration with the groups of Frauke Melchior and Andrea Pichler, we studied the modulation of ubiquitin and SUMO E2 enzymes by the SUMO protein itself. We showed how covalent modification by SUMO modulates E2 enzymatic properties (Pichler et al., NSMB 2005, Knipscheer et al., Mol Cell 2008) and we could show how the non-covalent backside binding is important for SUMO chain formation (Knipscheer et al., EMBO J 2007). Our interest has now shifted to structural and mechanistic questions concerning E3 ligases (Buchwald et al EMBO J 2006, Notenboom et al., NAR 2007) and DUB enzymes (Nijman et al., Cell 2005, van Leuken et al., Cell cycle 2008).

Future plans: special goals and approach


We will use our knowledge of AChBPs to study novel compounds with the potential for interactions with specific subtypes of nicotinic acetylcholine receptors. By a combination of X-ray crystallography and biophysical analysis we explore the ligand binding sites of these proteins. We also use our understanding of AChBP structure to find ways to express water-soluble forms of the cys-loop receptor extracellular domains in a variety of different approaches.


Ubiquitin and Sumo conjugation

We focus on the enzymes involved in ubiquitination and deubiquitination of proteins involved in DNA transactions, such as DNA repair enzymes and chromatin regulators. Here we aim at understanding mechanism and atomic detail that will provide information for future drug screens. We also study small molecule interactions with the HAUSP ubiquitin specific protease.

Most recent publications
  1. Knipscheer P, Flotho A, Klug H, Olsen JV, van Dijk WJ, Fish A, Johnson ES, Mann M, Sixma TK, Pichler A. (2008) Ubc9 sumoylation regulates SUMO target discrimination Mol Cell. 31(3):371-382.
  2. Notenboom V, Hibbert RG, van Rossum-Fikkert SE, Olsen JV, Mann M, Sixma TK. (2007) Functional characterization of Rad18 domains for Rad6, ubiquitin, DNA binding and PCNA modification. Nucleic Acids Res 35(17):5819-30
  3. Knipscheer, P., van Dijk, W.J., Olsen, J.V., Mann M., and Sixma T.K. (2007) Noncovalent interaction between Ubc9 and SUMO promotes SUMO chain formation EMBO J 26(11):2797-2807.
  4. Buchwald, G., van der Stoop, P., Weichenrieder, O., Perrakis, A., van Lohuizen M., Sixma T.K. (2006) Structure and E3-ligase activity of the Ring-Ring complex of polycomb proteins Bmi1 and Ring1b, EMBO J. 25, 2465-2474.
  5. Ulens, C., Hogg, R.C., Celie, P., Bertrand, D., Tsetlin, V., Smit, A.B., Sixma, T.K. (2006) Structural determinants of selective a-conotoxin binding to a nicotinic acetylcholine receptor homolog AChBP Proc. Natl. Acad. Sci. USA, 103, 3615-3620.
  6. Lebbink, J.H.G*, Georgijevic, D*., Natrajan, G*., Fish, A., Winterwerp, H.H.K., Sixma#, T.K., de Wind, N. (2006) Dual roles of MutS glutamate 38 in DNA mismatch discrimination and in the authorization of repair. EMBO J.  25, 409-419
  7. Nijman S.M., Luna-Vargas M.P.A., Velds A., Brummelkamp T.R., Dirac A.M.G., Sixma T.K., Bernards, R. A.  (2005) Genomic and Functional Inventory of Deubiquitinating Enzymes, Cell  123, 773-786.
  8. Celie, P.H*., Kasheverov, I.E*., Mordvintsev, D.Y., Hogg, R.C., van Nierop, P., van Elk, R., van Rossum-Fikkert, S.E., Zhmak, M.N., Bertrand, D., Tsetlin, V., Sixma#, T.K., Smit, A.B. (2005) Crystal structure of AChBP in complex with an a-Conotoxin PnIA variant, Nat. Struct. Mol. Biol. 12, 582-588.
  9. Pichler, A*., Knipscheer, P*., Oberhofer, E., van Dijk, W.J., Korner, R., Velgaard Olson, J., Jentsch, S. , Melchior, F., Sixma, T.K., (2005) SUMO inhibits the ubiquitin E2 enzyme E2-25K via modification of a helical acceptor site, ,  Nat. Struct. Mol. Biol. 12, 264-269.
  10. Celie, P.H.N, van Rossum-Fikkert, S.E. van Dijk, W.J., Brejc, K., Smit, A.B., Sixma, T.K. (2004) Nicotine and carbamylcholine binding to nicotinic receptors studied by AChBP structures, Neuron, 41, 907-914.