Protein-Kristallisationssystem
Final Report Abstract
We are performing structural studies of proteins related to the protozoal infections and developing isoform specific inhibitors for the selected targets using a combination of a wide range of experimental and computational methods. In these methods structural studies of protein-inhibitor complexes play a key-role. The modeling of specific active-site based and allosteric inhibitors that can serve as potential drugs is based on the fine-tuned conformational control by the unique residues of the individual protein isoforms. Structures of protein-inhibitor complexes will allow revealing the binding properties of the inhibitors and the inhibitor binding sites, which creates rational basis for increasing the binding affinity and isoform-specificity. Development of highly specific allosteric inhibitors often requires structural reconstruction of complete enzymatic cycles using kinetic crystallography methods. In all our lines of research we are making ongoing efforts to crystallize and determine the structures of single proteins and their complexes with other proteins, nucleic acids and small-molecule inhibitors in various kinetic states to obtain this information. The current lines of research in the Research Unit for Structural Analysis include (i) structural studies of nonmuscle myosins and tropomyosins in complex with small-molecule inhibitors performed by the group of Prof. Dr. D.J. Manstein; (ii) Structural studies of myosin-18A and myosin-18B in their interaction with specific binding partners will be performed by Dr. M.H. Taft and Prof. Dr. D.J. Manstein; (iii) the work on allosteric inhibition of UDP-glucose pyrophosphorylase (UGP) from Leishmania major and the extension of this project line to structural and mechanistic studies of UGP from Trypanosoma brucei, allosteric regulation mechanisms of UDP-sugar pyrophosphorylase (USP) from Leishmania major and structural and mechanistic studies of USP from Trypanosoma cruzi (Dr. R. Fedorov); (iv) investigations on proteins and protein-complexes involved in apoptosis as well as dynamin and dynamin containing complexes are carried out by Dr. S. Eschenburg and Dr. T.F. Reubold; (v) Structural studies of protein-complexes involved in bacterial DNA replication performed by Andrea Bogutzki and Prof. Dr. U. Curth. These lines of research provide new structural data essential for understanding the infection biology of protozoan parasites and for developing new ways to develop anti-parasitic treatments. In 2013-2015 the Institute for Biophysical Chemistry has submitted 15 structures resulting from experiments involving the use of the system. This includes 5 structures of protein complexes involved in the glycosylation pathway of protozoan parasites, 4 structures of innate immune sensor complexes, and 2 structures of human myosin motors.
Publications
- Catalytic mechanism and allosteric regulation of UDP-glucose pyrophosphorylase from Leishmania major. (2013) ACS Catalysis. 3:2976-2985
J. Führing, J.T. Cramer, F.H. Routier, A-C Lamerz, P. Baruch, R. Gerardy-Schahn and R. Fedorov
(See online at https://doi.org/10.1021/cs4007777) - Expression, purification, crystallization and preliminary X-ray crystallographic analysis of human myosin 1c in complex with calmodulin (2013). Acta Cryst. Sect F Struct Biol Cryst Commun 69, 1020- 1022
S. Münnich, D. J. Manstein
(See online at https://doi.org/10.1107/S1744309113020988) - Octamerization is essential for enzymatic function of human UDP-glucose pyrophosphorylase. (2013) Glycobiology. 23(4):426-37
Führing J, Damerow S, Fedorov R, Schneider J, Münster-Kühnel AK, Gerardy-Schahn R
(See online at https://doi.org/10.1093/glycob/cws217) - Crystal structure of human myosin 1c-the motor in GLUT4 exocytosis: implications for Ca 2+ regulation and 14-3-3 binding. (2014) J Mol Biol. 426(10):2070-81
S. Münnich, Taft MH, Manstein DJ
(See online at https://doi.org/10.1016/j.jmb.2014.03.004) - Crystal structure of the rigor-like human non-muscle myosin-2 motor domain. (2014). FEBS Lett 588, 4754-4760
S. Münnich, S. Pathan-Chhatbar, D. J. Manstein
(See online at https://dx.doi.org/10.1016/j.febslet.2014.11.007) - Expression, purification and crystallization of a dye-decolourizing peroxidase from Dictyostelium discoideum. (2014) Acta Cryst. Sect F Struct Biol Cryst 70:252-255
A. Rai, R. Fedorov & D.J. Manstein
(See online at https://doi.org/10.1107/S2053230X14000545) - schenburg, Crystal structure of the leucine-rich repeat domain of the NOD-like receptor NLRP1: implications for binding of muramyl dipeptide. (2014). FEBS Lett 588, 3327-3332
T. F. Reubold, G. Hahne, S. Wohlgemuth, S.
(See online at https://doi.org/10.1016/j.febslet.2014.07.017) - A Quaternary Mechanism Enables the Complex Biological Functions of Octameric Human UDP-glucose Pyrophosphorylase, a Key Enzyme in Cell Metabolism. (2015) Sci. Rep. 5, 9618
Führing JI, Cramer JT, Schneider J, Baruch P, Gerardy-Schahn R, and Fedorov R
(See online at https://doi.org/10.1038/srep09618) - Crystal structure of the dynamin tetramer. (2015) Nature. 525(7569):404-8
Reubold TF, Faelber K, Plattner N, Posor Y, Ketel K, Curth U, Schlegel J, Anand R, Manstein DJ, Noé F, Haucke V, Daumke O, Eschenburg S
(See online at https://doi.org/10.1038/nature14880) - The activation mechanism of 2’-5’-oligoadenylate synthetase gives new insights into OAS/cGAS triggers of innate immunity. (2015) Structure. 23(5):851-862
Lohöfener J, Steinke N, Kay-Fedorov P, Baruch P, Nikulin A, Tishchenko S, Manstein DJ, and Fedorov R
(See online at https://doi.org/10.1016/j.str.2015.03.012)
