Research interest
One of the current challenges in structural biology is to study structure and function of 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 protein crystallography and complementary biophysical techniques.
Ubiquitin and SUMO conjugation
Ubiquitin conjugation processes have emerged as a critical signaling systems that are essential for cell stability, by controlling degradation of short-lived proteins, for DNA repair and targeting to specific areas in the cell through endocytosis. Because of the importance for regulating cell cycle, chromatin regulation, apoptis 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 comples spatial arrangement of ubiquitin, E2, E3 proteins and the target simultaneously in a precise but flexible manner. Although the overall mechanism has been defined, many specificity determing factors are unclear.
We study E2/E3 complexes involved in conjugation of ubiquitin, such as the Rad6/Rad18 complex and Polycomb proteins. The Ring-Ring dimer of the Bmi1/Ring1b complex is active in modifying Histone 2a. We are interested how this ubiquitin ligase activity is regulated in the larger Prc1 complex.
Sumo is a small ubiquitin-related modifier that alters the function of many proteins. Similar to ubiquitin it uses an E1/E2/E3 series of enzymes for modifying its targets. The molecular mechanisms and consequences of this modification are still poorly defined. We solved the original crystal structure of the SUMO E2, Ubc9 (Tong et al., 1977). In collaboration with the group of Frauke Melchior (Göttingen) we studied SUMO modification of the ubiquitin-conjugating enzyme E2-25K and Ubc9 and showed how this affects function.
DNA mismatch repair
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 (Lamers et al., 2000) and how this asymetric ATPase couples DNA mismatch recognition to initiation of repair.
Nicotinic Acetylcholine Receptor homolog AChBP
The Acetylcholine Binding Protein from Lymnea stagnalis is a glial protein with high homology to ligand-binding domain of nicotinic acetylcholine receptors (Smit et al. 2001), identified by Guus Smit at the Free University of Amsterdam. We have solved its crystal structure (Brejc et al., 2001) to provide the first high resolution data on this pharmaceutically important class of ion channels, that also includes GABAA receptors and 5HT3 serotin receptors. The crystal structures of AChBP homologs confirm most of the biochemical data on nicotinic receptor ligand binding, and presented the ligand-binding site at the interface between two monomers, while ligand affinity studies showed details of nAChR ligand binding. We use AchBP as a model system to study ligand binding in cys-loop receptors and to identify novel compounds as potential lead inhibitors of the ion channels.
Key publications
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. doi: 10.1038/sj.emboj. 7601711
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.
Ulens, C., Hogg, R.C., Celie, P.H., 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.
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.
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 alpha-Conotoxin PnIA variant, Nat.Struct.Mol. Biol 12, 582-588.
Pichler, A., Knipscheer, P., Oberhofer, E., van Dijk, W.J., Korner, R., Velgaard Olsen, 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.Struc.Mol.Biol. 12, 264-269.
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.
Brejc, K., van Dijk, W.J., Klaassen, R.V., Schuurmans, M., van der Oost, J., Smit, A.B., Sixma, T.K. (2001) The crystal structure of AChBP, homolog of the N-terminal domain of the nicotinic acetylcholine receptor. Nature, 411, 269-276.
Lamers, M.H., Perrakis, A., Enzlin, J.H., Winterwerp, H.H.K., de Wind, N., Sixma, T.K. (2000) The crystal structure of DNA mismatch repair protein MutS binding to a G:T mismatch. Nature, 407, 711-717.
Biographic sketch
Titia K. Sixma reived her PhD trainig in the group of Wim Hol at Groningen University determining crystal structures of a bacterial protein toxin homolog of cholera toxin, LT (1992). She worked as a post-doctoral fellow in the group of Paul Sigler at Yale University. Since 1994 she has worked as a group leader in the Netherlands Cancer Institute in Amsterdam, using structural biology as a tool to study the regulation of signaling processes within the cell. The group mainly focusses on the process of DNA mismatch repair, the analysis of the proteins involved in ubiquitin conjugation and studies of AChBP, a homolog of the nicotinic acetylcholine receptor ligand-binding domain. Since 2004 she has a part-time appointment as professor of protein structure and function at the Erasmus University in Rotterdam.
Co-workers
Pim van Dijk Technician
Herrie Winterwerp Technician
Marcello Clerici Postdoc
Alexander Fish Postdoc
Rick Hibbert Postdoc
Prakash Rucktooa Postdoc
Mariano Stornaiuolo Postdoc
Azusa Seto Postdoc
Mark Vargas PhD student
Alex Faesen PhD student
Franceska Mattiroli PhD student
Judith Smit PhD student
Flora Groothuizen PhD student
Danny Sahtoe PhD student