Membrane transporters, channels and enzymes

Project Group of Structural Biology Department

< go back to other projects

Membrane transporters, channels and enzymes

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 1A) 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 1B). 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 1C-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 1. X-ray structures of secondary-active transporters

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 2). 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).