printing_only_background

Principal Investigator

linie

P01  Werner Kühlbrandt
Professor and Director

linie
 

Structural Biology
Max Planck Institute of Biophysics
Max-von-Laue-Str. 3
60438 Frankfurt am Main, Germany

Phone +49 (0)69 6303-3000
Fax +49 (0)69 6303-3002

> send email
> homepage

> publications

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which surprisingly shows 13 instead of 12 membrane-spanning helices (Fig. 2; Goswami, Yildiz, Kühlbrandt; unpublished).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.
 

linie
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.
linie

Publications

linie

Wöhlert D, Grötzinger MJ, Kühlbrandt W, Yildiz Ö (2015) Mechanism of Na+-dependent citrate transport from the structure of an asymmetrical CitS dimer. eLife 4, e09375.

 

Kühlbrandt W, Davies K (2015) Rotary ATP synthase: A new twist to an ancient machine. Trends Biochem Sci 41, 106-116. 

 

Allegretti M, Klusch N, Mills DJ, Vonck J, Kühlbrandt W, Davies KM (2015) Horizontal membrane-intrinsic α-helices in the stator a-subunit of an F-type ATP synthase. Nature 521, 237-40. 

 

Gold VAM, Salzer R, Averhoff B, Kühlbrandt W (2015) Structure of a type IV pilus machinery in the open and closed state. eLife 4, e07380. 


Müller M, Bamann C, Bamberg E, Kühlbrandt W (2015) Light-induced helix movements in channelrhodopsin-2. J Mol Biol 427, 341-349.

Calinescu O, Paulino C, Kühlbrandt W, Fendler K (2014) Keeping it simple, transport mechanism and pH regulation in Na+/H+ exchangers. J Biol Chem 289, 13168-13176. 

Paulino C, Wöhlert D, Kapotova E, Yildiz Ö, Kühlbrandt W (2014) Structure and transport mechanism of the sodium/proton antiporter MjNhaP1. eLife 3, e03583.

Paulino C, Kühlbrandt W (2014) pH- and sodium-induced changes in a sodium/proton antiporter. eLife 3, e01412.

Wöhlert D, Kühlbrandt W, Yildiz Ö (2014) Structure and substrate ion binding in the sodium/proton antiporter PaNhaP. eLife 3, e03579.

Kalayil S, Schulze S, Kühlbrandt W (2013) Arginine oscillation explains Na+ independence in the substrate/product antiporter CaiT. PNAS 110, 17296-17301.

Kühlbrandt W (2014) Science Perspective: The resolution revolution. Science 343, 1443.

Gold V, Ieva R, Walter A, Pfanner N, van der Laan M, Kühlbrandt W (2014) Visualizing active membrane protein complexes by electron cryo-tomography. Nat Commun 5, 4129.

Köster S, Van Pee K, Hudel M, Leustik M, Rhinow D, Kühlbrandt W, Chakraborty T, Yildiz Ö (2014) Crystal structure of listeriolysin O reveals molecular details of oligomerization and pore formation. Nat Commun 5, 3690.

Allegretti M, Mills DJ, McMullan G, Kühlbrandt W, Vonck J (2014) Atomic model of the F420-reducing [NiFe] hydrogenase by electron cryo-microscopy using a direct electron detector. eLife 3, e01963.

Daum B, Walter A, Horst A, Osiewacz HD, Kühlbrandt W (2013) Age-dependent dissociation of ATP synthase dimers and loss of inner membrane cristae in mitochondria. PNAS 110, 15301-15306.

Davies KM, Anselmi C, Wittig I, Faraldo-Gómez JD, Kühlbrandt W (2012) Structure of the yeast F1Fo-ATP synthase dimer and its role in shaping the mitochondrial cristae. PNAS 109, 13602-13607. 

top

linie

Collaborations

linie

Dötsch/Bernhard (P02), Tampé (P16), Schleiff (P17), Pos (P18), Hänelt (P22),
Hummer (P25)