Principal Investigator
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P1 Werner Kühlbrandt
Professor and Director
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Structural Biology
Max Planck Institute of Biophysics
Max-von-Laue-Str. 3
60438 Frankfurt am Main, Germany
Phone +49 (0)69 63 03 30 00
Fax +49 (0)69 63 03 30 02
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P1 Structure and function of archaeal transporters
We use electron and X-ray crystallography to study the structure and molecular mechanisms of secondary transporters in the membrane. Apart from bacterial transporters, we focus on those from hyperthermophilic archaea, which are more stable and more closely related to their medically relevant human homologues. We have cloned and expressed MjNhaP1, the M. jannaschii homologue of the human Na+/H+ antiporter NHE1, in E. coli with a C-terminal His6 tag. Cryo-EM and image processing of 2D crystals grown at pH 4 yielded projection maps at 7Å resolution (Fig. 1a), showing a dimer of similar shape and dimensions as NhaA (Williams, 2000). Incubation at pH 8 on the EM grid resulted in well-defined conformational changes, which are evident in the 7Å projection map (Fig. 1c) but seen more clearly in a Fourier difference map (Vinothkumar et al., 2005) (Fig. 1b). These changes are functionally relevant as MjNhaP1 is inactive at pH 7.5 and above, but active at pH 6, in contrast to NhaA, which is fully active at pH above 8 but inactive below pH 7.
Very recently, we obtained an 7Å 3D map of MjNhaP1 at pH 4 by electron crystallography, which surprisingly shows 13 instead of 12 membrane-spanning helices (Fig. 2; Goswami, Yildiz, Kühlbrandt; unpublished). A fitted homology model of MjNhaP1 indicates an extensive inverted helix repeat, comprising 10 of the 13 helices. Comparison to NhaA at pH4 (Williams, 2000; see also project P3 Michel) indicates that the helix arrangement at the monomer-monomer interface of the two transporters is quite different, but the structure of the 6-helix bundle at either end of the dimer is conserved. This helix bundle undergoes clear substrate-induced conformational changes both in MjNhaP1 (Fig. 1) and NhaA (Appel et al, 2009), which indicate that the site of ion translocation is located in its centre. Projection difference maps show that helix pIV in this bundle moves by ~7 Å in active NhaA. Work towards 3D maps of MjNhaP1 and NhaA in different conformations and on the structures of other membrane transport systems is in progress.
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- Fig. 1: Projection maps at 8 Å resolution of MjNhaP1 dimer at pH4 (a), pH 8 (c), and difference map of both (b), with positive contours in red and negative contours in blue showing overlayed onto the pH 4 projection map (grey).
- Fig. 2: Three-dimensional map MjNhaP1 at 7 Å resolution with fitted homology model based on the NhaA structure (Hunte et al, 2005; see Project P3 Michel). Top view (left) and side view (right). Helices are numbered as in NhaA. One putative monomer is shown in colour, the other one in grey.
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Publications
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Schulze, S., Köster, S., Geldmacher, U., Terwisscha van Scheltinga, A.C. and Kühlbrandt, W. (2010) Structural basis of Na+-independent and cooperative substrate/product antiport in CaiT. Nature, in press.
Daum, B., Nicastro, D., Austin II, J., McIntosh, J.R. and Kühlbrandt, W. (2010) Arrangement of photosystem-II and ATP synthase in chloroplast membranes of spinach and pea. Plant Cell, in press.
Gipson, P., Mills, D., Wouts, R., Grininger, M., Vonck, J. and Kühlbrandt, W. (2010) Direct structural insight into the substrate-shuttling mechanism of yeast fatty acid synthase by electron cryomicroscopy. Proc Natl Acad Sci USA, doi: 10.1073/pnas.0913547107.
Barros, T., Royant, A., Standfuss, J., Dreuw, A. and Kühlbrandt, W. (2009) Crystal structure of plant light-harvesting complex shows the active, energy-transmitting state. EMBO J 28, 298-306.
Strauss, M., Hofhaus, G., Schröder, R.R. and Kühlbrandt, W. (2008) Dimer ribbons of ATP synthase shape the inner mitochondrial membrane. EMBO J 27, 1154-1160.