Research Profile Dr. Christine Ziegler

Dr. Christine Ziegler

Dr. rer. nat. (Physics) University of Kassel, 1996
Postdoc (Proton therapy of brain tumors),
Centre de Proton-Thérapie d'Orsay, France, 1997-1999
At the institute since 2000

Structure and function of transporters involved in environmental stress response

Research in our group is dedicated to membrane proteins involved in cellular stress response. Cells are constantly exposed to diverse forms of environmental stress, including changes in osmolarity, temperature, water content, pH, oxidation, nutritional starvation, and chemical compounds. Under severe stress conditions nucleic acids, proteins, and membranes can be seriously damaged leading to growth inhibition or cell death. To survive these stresses, cells acquire a variety of stress-adaptation mechanisms. A complex network of regulatory systems ensures a coordinated and effective response cascade of alterations in gene expression and protein activity. Transporters, which are capable of sensing stress stimuli, transducing them into signals relevant to cells, and modulating their activity profile accordingly, are the focus of our current work. We investigate stress response mechanisms by a broad methodology ranging from molecular biology and microbiology to structural biology.

Osmotic stress response in bacteria

Cells counteract hyperosmotic stress by accumulating small, osmotically active solutes, the so-called compatible solutes (Fig. 1), and releasing them under hypo-osmotic conditions. Compatible solutes like glycine betaine, or ectoine (Fig. 1) increase the internal osmolality to sustain an appropriate cell turgor. They also function as protectants for cellular proteins against denaturation under stress conditions. Accumulation is achieved preferably by uptake and cells are equipped with a diversity of compatible solute uptake systems. These encompass primary active transporters, as well as secondary carriers including tripartite ATP-independent (TRAP) transporters.

Fig. 1: Accumulation of compatible solutes by synthesis and/or highly specific uptake systems.

Structure and function of the osmoregulated glycine betaine transporter BetP from C. glutamicum

In our main project, we investigate the molecular mechanism of osmoregulated Na⁺-coupled glycine betaine transport mediated by BetP, which senses osmotic stress in Corynebacterium glutamicum and subsequently regulates its activity. For this purpose, we have solved the structure of BetP by X-ray crystallography to 3.35Å resolution (Fig. 2a) and determined a 3D map to 8Å resolution by electron crystallography of 2D crystals (Fig. 2b).

: Movie1 and Fig. 2: Xray structure of BetP to 3.35 Å resolution determined by x-ray crystallography and 3D map of BetP to 8 Å determined by cryo-electron crystallography.

Our studies revealed that BetP is an asymmetric trimer in different transporter conformations and that conformational asymmetry has an important functional impact on transport and regulation upon osmotic stress. Conformational changes in the osmosensing C-terminal domain result in activation via an elaborate interaction network between the different monomers.

To investigate important functional aspects of BetP, we perform extensive mutagenesis studies by ¹⁴[C]glycine betaine transport and voltage patch clamp measurements. There is strong evidence that down-regulation of BetP also involves a mechanosensitive channel, which works as a specific glycine betaine exporter. A structural investigation of this exporter by single-particle analysis and X-ray crystallography is in progress.

: Fig. 3a:Crystals of the periplasmic ectoine binding protein TeaA.

Structure and function of TRAP-transporters involved in osmoregulation in H. elongta

We have studied the osmoregulated ectoine TRAP transporter TeaABC from Halomonas elongata by X-ray crystallography (Fig. 3a) and ITC measurements.We could demonstrate that osmolyte transport and regulation in TeaABC differs significantly from the mechanisms observed in BetP.

: Fig. 3b: X-ray structure of the 1.8Å apo-structure

: Fig. 3c: X-ray structure of the 1.55Å ectoine-bound structure of TeaA.

We solved the structure of the substrate binding protein TeaA in three different conformations (Fig. 3b and c), which in combination with most recent mutagenesis studies will allow us to describe the mechanism of ectoine binding in different environmental conditions

We solved the structure of the universal stress protein TeaD, and identified its role as a negative regulator in a sophisticated recycling circuit to balance the internal ectoine pool (Fig. 4a). In fact, we could show that TeaD is deactivated by an ATP dependent oligomerization (Fig. 4b).

: Fig. 4a: Ectoine export, import and biosynthesis are balanced in H. elongata via TeaD.

: Fig. 4b: ATP-binding in TeaD triggers a deactivating tetramerization.

Future Projects

In future, structure determination of different conformations of BetP in the catalytic and regulatory cycle is currently our highest priority. We are expanding our research to structural and functional studies of eukaryotic glycine betaine transporters, which possess an intriguing topology flipping indicating an evolutionary relation to neurotransmitter transporters of the NSS family. In addition, we are working on eukaryotic osmolyte transporters of the NSS family.

To understand the role of TRAP-transporter regulation by universal stress proteins we have started functional and structural studies on another member of the TRAP transporter family from Halomonas elongata.

Understanding the general mechanisms of stress response is an important theme of our work. We will exploit the unique ability of pathogenic GRAM-positive bacteria to adapt to various stress types in order to understand how physical stress stimuli are perceived and transduced into productive cellular signals, and how transporters are regulated by protein-protein interaction in stress response.

Collaborations

Prof. Reinhard Krämer, Department of Biochemistry at the University of Cologne, Germany

Prof. Daniel Müller, Biotechnical Center (Biotec) of the Technical University of Dresden, Germany

Prof. Erhard Bremer, Department of Microbiology at the University of Marburg, Germany

PD Dr. Hans-Jörg Kunte, BAM Berlin, Germany

Dr. Stephen Kempson, Medical Scholl of Illinois, USA

Grant Support

CEF: Cluster of Excellence Frankfurt Macromolecular Complexes

SFB: Transport and Communication across Biological Membranes

Publications

Krämer, R.and Ziegler, C.: Regulative interactions of the osmosensing C-terminal domain in the trimeric glycine betaine transporter BetP from Corynebacterium glutamicum. Biol. Chem. 390 (8) 685-691 (2009).

Appel, M., Hizlan, D., Vinothkumar, K.R., Ziegler, C., and Kühlbrandt, W.: Conformations of NhaA, the Na+/H+ exchanger from Escherichia coli, in the pH-activated and ion-translocating states. J. Mol. Biol. 388 (3) 659-672 (2009).

Ressl, S., Terwisscha van Scheltinga, A.C., Vonrhein, C., Ott, V. and Ziegler, C.: Molecular basis of transport and regulation in the Na(+)/betaine symporter BetP. Nature 458 (7234) 47-52 (2009).

Kuhlmann, S.I., Terwisscha van Scheltinga, A.C., Bienert, R., Kunte, H.J., and Ziegler, C.: 1.55 A structure of the ectoine binding protein TeaA of the osmoregulated TRAP-transporter TeaABC from Halomonas elongata. Biochemistry 47 (36) 9475-9485 (2008).

Kedrov, A., Appel, M., Baumann, H., Ziegler, C. and Müller, D.J.: Examining the Dynamic Energy landscape of an antiporter upon inhibitor binding. J. Mol. Biol. 375 1258-1266 (2008).

Tsai, C.J., Ejsing, C.S., Shevchenko, A. and Ziegler, C.: The role of lipids in two-dimensional crystallization of BetP, a glycine-betaine transporter from Corynebacterium glutamicum. J. Struc. Biol. 160 275-286 (2007).

Kedrov, A., Ziegler, C. and Müller, D.J.: Differentiating ligand and inhibitor interactions of a single antiporter. J. Mol. Biol. 362 (5) 925-932 (2006).

Kedrov, A., Janovjak, H., Ziegler, C., Kühlbrandt, W. and Müller, D.J.: Observing folding pathways and kinetics of a single sodium-proton antiporter from Escherichia coli. J. Mol. Biol. 355 (1) 2-8 (2006)

Tsai, C.-J. and Ziegler, C.: Structure determination of secondary transport proteins by electron crystallography: Two-dimensional crystallization of the betaine uptake system BetP. J. Mol. Microbiol. Biotechnol. 10 197-207 (2005).

Kedrov, A., Krieg, M., Ziegler, C., Kühlbrandt, W. and Müller, D.J.: Locating ligand binding and activation of a single antiporter. EMBO Rep. 6 (7) 668-674 (2005).

Kedrov, A., Ziegler, C., Janovjak, H., Kühlbrandt, W. and Müller, D.J.: Controlled unfolding and refolding of a single sodium-proton antiporter using atomic force microscopy. J. Mol. Biol. 340 (5)1143-1152 (2004).

Ziegler, C., Morbach, S., Schiller, D., Krämer, R., Tziatzios, C., Schubert, D., and Kühlbrandt, W.: (2004). Projection structure and oligomeric state of the osmoregulated sodium/glycine betaine symporter BetP of Corynebacterium glutamicum. J. Mol. Biol. 337(5) 1137-47 (2004).

Ziegler, C., Bonnefont-Rousselot, D., Delacroix, S., Habrand, J.L. and Mazal A.:Effectiveness of protons and argon ions in initiating lipid peroxidation in low-density lipoproteins. Radiat. Res. 150 (4) 483-487 (1998).

Ziegler, C. and Wessels, J.M.: Investigation of lipid peroxidation in liposomes induced by heavy ion irradiation. Radiat. Environ. Biophys. 37(2) 95-100 (1998).

Kinder, R., Ziegler, C., Wessels, J.M.: Gamma-irradiation and UV-C light-induced lipid peroxidation: a Fourier transform-infrared absorption spectroscopic study. Int. J. Radiat. Biol. 71 (5) 561-571 (1997).

Schön, W., Ziegler, C., Gärtner, H. and Kraft, G.: Heavy ion induced membrane damage: hemolysis of erythrocytes and changes in erythrocyte membrane fluidity. Radiat. Environ. Biophys. 33 (3) 233-241 (1994)

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