Osmoregulatory transport of compatible Solutes in Bacteria: Structure, function and regulation of the TRAP Transporter TeaABCD from Halomonas elogata
Final Report Abstract
The halophilic bacterium Halomonas elongata takes up and accumulates the compatible solute ectoine via the osmoregulated TRAP transporter TeaABCD. TeaABC belongs to the tripartite ATP-independent periplasmic transporters (TRAP-T), a family of secondary transporters functioning in conjunction with periplasmic substrate binding proteins. Our studies on the substrate binding protein TeaA present the first detailed analysis of the molecular mechanisms underlying substrate recognition of the substrate binding protein of an osmoregulated TRAP transporter. We were able to demonstrate by isothermal titration calorimetry measurements that TeaA is a high-affinity ectoine binding protein (K d = 0.19 µ M) that also has a significant affinity to hydroxyectoine (K d = 3.8 µM). Furthermore, we present the structure of TeaA in complex with ectoine at a resolution of 1.55 Å and hydroxyectoine at a resolution of 1.80 Å, which revealed a solvent-mediated specific binding of ectoine to TeaA. A fourth orf (teaD) is located adjacent to the teaABC locus that encodes a putative universal stress protein (USP). By RT-PCR experiments we proved a cotranscription of teaD along with teaABC. Deletion of teaD resulted in an enhanced uptake for ectoine by the transporter TeaABC and hence a negative activity regulation of TeaABC by TeaD. A transcriptional regulation via DNA binding could be excluded. ATP binding to native TeaD was shown by HPLC, and the crystal structure of TeaD was solved in complex with ATP to a resolution of 1.9 Å by molecular replacement. TeaD forms a dimer-dimer complex with one ATP molecule bound to each monomer. We proved an ATP-dependent oligomerization of TeaD, which might have a functional role in the regulatory mechanism of TeaD. The genome of H. elongata was sequenced and annotated. The membrane proteome was quantitatively analyzed by mass spectrometry employing an isotope labeling procedure with the intention to find proteins that might play a role in ectoine efflux. Four putative stretch-sensitive channels were found with a potential role in ectoine release, which are currently under further investigation.
Publications
- (2008) 1.55 A Structure of the Ectoine Binding Protein TeaA of the Osmoregulated TRAP-Transporter TeaABC from Halomonas elongata. Biochemistry 47: 9475-9485
Kuhlmann, S.I., Terwisscha van Scheltinga, A.C., Bienert, R., Kunte, H.J., and Ziegler, C.
- (2010) A Blueprint of Ectoine Metabolism from the Genome of the Industrial Producer Halomonas elongata DSM 2581T. Environ Microbiol.
Schwibbert, K., Marin Sanguino, A., Bagyan, I., Heidrich, G., Lentzen, G., Seitz, H., Rampp, M., Schuster, S. C., Klenk, H. P., Pfeiffer, F., Oesterhelt, D., Kunte, H. J.
(See online at https://doi.org/10.1111/j.1462-2920.2010.02336.x) - (2010) Structure and Function of the Universal Stress Protein TeaD and Its Role in Regulating the Ectoine Transporter TeaABC of Halomonas elongata DSM 2581T. Biochemistry 49: 2194-2204
Schweikhard, E.S., Kuhlmann, S.I., Kunte, H.-J., Grammann, K., and Ziegler, C.M.
