Membrane transporters, channels and enzymes (Ö. Yildiz, W. Kühlbrandt)

Membrane transporters, channels and enzymes
(Özkan Yildiz, Werner Kühlbrandt)

The first structure of a secondary-active transporter, the E. coli sodium/proton antiporter NhaA was determined at 6 Å resolution by electron crystallography in this Department (Williams, Nature 2000; Williams et al., EMBO J 1999). Since then, numerous x-ray structures of secondary-active transporters have been published by us and others. With recent developments, even comparably small membrane trans­porters and ion channels are now within reach of single-particle cryoEM.

Crystallography of sodium-proton antiporters

The electroneutral Na+/H+ antiporter NhaP from the hyperthermophilic archaeon Pyro­coccus abyssi (PaNhaP) is a functional homologue of the medically important human antiporter NHE1. We determined the structure of PaNhaP by x-ray crystallography at pH 8 (Figure 16A) and pH 4. The substrate ion was resolved for the first time in such a transporter by soaking crystals with thallium, an anomalous x-ray scatterer that mimics Na+ in the binding site (Figure 16B). Transport measurements show that thallium is a substrate for PaNhaP. The substrate ion is coordinated by three acidic sidechains, a water molecule, a serine and a main-chain carbonyl in the unwound stretch of TM helix 5 (Wöhlert et al., eLife 2014).

We also determined the structure of the related Na+/H+ antiporter NhaP1 from Methano­coccus jan­naschii (MjNhaP1) in two complementary states. The 3.5 Å x-ray structure of the inward-open state in the presence of sodium at pH 8, where the transporter is highly active, was solved by molecular replacement with PaNhaP. The 6 Å structure of the com­ple­mentary outward-open state was obtained by electron crystallography of 2D crys­tals at pH 4, where the transporter is inactive (Paulino et al., eLife 2014). Com­parison revealed a 7° tilt of the 6‑helix bundle that includes the ion-binding site. Pro­gressive sub­strate-induced conformational changes in MjNhaP1 were investigated by dif­ference maps obtained from 2D crystals incubated with a range of physiological pH and Na+ conditions (Paulino and Kühlbrandt, eLife 2014). Changes in Na+ concentration caused a marked confor­ma­­tional change that was largely pH-independent, con­sis­tent with substrate com­petition for a common ion-binding site, as confirmed by electro­physio­logical studies in collaboration with Klaus Fendler in the Bamberg department (Calinescu et al., J Biol Chem 2014). Projection difference maps indicated helix movements that convert MjNhaP1from the proton-bound, outward-open state to the Na+-bound, inward-open state. Oscillation between the two states results in rapid antiport.

The citrate transporter SeCitS We solved the x-ray structure of the sodium/citrate symporter SeCitS from the human patho­gen Salmonella enterica, which takes up citrate as a nutrient. Unexpected­ly, we found that the structure of bacterial citrate transporters closely resembles that of the sodium/proton anti­por­ters, without detectable sequence homology. The 2.5 Å resolution x-ray structure of SeCitS con­tains two dimers. In each dimer, the two protomers assume different, inside-open or outside-open con­for­mations that do not result from crystal contacts. The structure thus shows three functional states of the active protomer in one asymmetric unit (Figure 16C-E), which is unprecedented. Together with com­prehensive func­tional studies of SeCitS reconstituted into liposomes, the structures explain the trans­port mechanism as a six-step process, with a rigid-body 31° rotation of a helix bundle around an axis in the membrane plane, perpendicular to the long dimer axis. The rotation of the helix bundle translocates the bound substrates vertically by 16 Å across the membrane. We expect similar transport mechanisms apply to a wide range of secondary transporters (Wöhlert et al., eLife 2015).

Figure 16. X-ray structures of secondary-active transporters (A) Structure of the electroneutral Na+/H+ antiporter PaNhaP from P. abyssi. (B) Substrate ion coordination in the archaeal sodium/proton antiporter PaNhaP. The acidic side chains of Glu73, Asp159, a water molecule held by Asp130, the hydroxyl group of Ser155 and the main-chain carbonyl of Thr129 coordinate the substrate ion (grey sphere). The anomalous density for the Tl+ ion in the substrate-binding site between helix H3, H6 and the unwound stretch of H5 is shown in magenta at 4σ. Red spheres are water molecules. From (Wöhlert et al., eLife 2014). (C) The two different dimers of the citrate transporter SeCitS from S. enterica in the asymmetric unit. (D) Inward-open and outward-open protomers in the asymmetrical SeCitS dimer. (E) Details of the substrate-binding site. In the outward-facing protomer (left), citrate is closely coordinated by sidechains of two helix hairpins and helix H13. In the inward-facing protomer B (centre), citrate is hydrated and attached weakly to the glycine-rich loop of helix H12. In protomer B’ (right), only the Na1 site is occupied. Two citrate molecules are resolved, outlining a likely trajectory for citrate release. From (Wöhlert et al., eLife 2015).

Figure 17. Morph of conformational changes in one protomer of the SeCitS dimer, showing the transition from the outward-facing to the inward-facing state. Arg402, Arg428 and Tyr348, which coordinate citrate in the outward-facing conformation, are drawn as stick models, while the Na+ ions are represented as grey spheres. Na+ ions bind to their respective sites in the helix bundle, followed by citrate binding between helix bundle and dimer contact domain. Subsequently the substrates are translocated by a rotation of the bundle. Citrate release is independent from the release of either Na+ ion. Due to the empty Na2 binding site in protomer B’ we assume that this ion is released immediately after the citrate. After substrate release the empty transporter changes its conformation back to the outward-facing state to repeat the cycle.

CryoEM of transporters and ion channels
The human TRP channel Polycystin-2 (PC2) responds to changes in cytosolic calcium con­centration. PC2 mutations trigger autosomal dominant polycystic kidney disease. PC2 is found predominately in the endoplasmic reticulum (ER), in the primary cilium and, depen­ding on cell type, in the plasma membrane. We determined cryoEM structures of two full-length PC2 channels from the plasma membrane or the endoplasmic reticulum at 4.2 Å resolution (Figure 8E). The structures show two distinct open states in complex with lipids and Ca2+. A single Ca2+ is bound in the selectivity filter, while multiple Ca2+ ions along the translocation pathway suggest an electrostatic knock-off mechanism to increase Ca2+-selectivity outside the ER (Wilkes et al., NSMB 2016).

PilQ, a bacterial protein export/DNA uptake system
Bacteria use flagella or pili for locomotion. While flagellar motors are well-characterized, much less is known about the machinery that generates bacterial pili. The type 4 pilus (T4P) secretion complex from Thermus thermophilus is a 1100 kDa assembly of several different membrane and membrane-associated proteins. The T4P complex is active both in the extru­sion of pili and in the uptake of DNA during bacterial transformation. In collabo­ration with Beate Averhoff (Frankfurt University) we are determining the structure of the T4P complex by cryoEM. Subtomogram averages of T4P in entire Thermus thermophilus cells show the complex in the closed and open state, with a pilus in the process of being extruded (Gold et al., eLife 2015).

Figure 18. Side view (A)and top view (B) of Polycycstin-2 ion channel tetramer. Colours indicate local map resolution: blue, 3.5 Å; cyan, 4 Å; green 4.5 Å; yellow, red and orange 5.0, 5.5 and 6.0 Å. From Wilkes et al, Nat Struct Mol Biol 2016. (C,D) Open and closed state of the type IV pilus machinery of Thermus thermophilus. From Gold et al, eLife 2015.


Max Planck Institute of Biophysics

Prof. Dr. Werner Kühlbrandt, Director
Department of Structural Biology
Secretary: Monika Hobrack

Phone: +49 (0) 69 6303-3001
Fax: +49 (0) 69 6303-3002
E-mail: monika.hobrack(at)