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Research Themes Membrane proteins

Membrane Proteome: Pumping Out Heavy Metal

SBKB [doi:10.1038/sbkb.2014.194]
Featured Article - April 2014
Short description: Crystal structures of a heavy-metal efflux pump reveal intermediate transport states.

Overall structure of ZneA trimer at pH7.5 (PDB 4K0E). Each protomer is represented in a different color; Zn2+ ions are shown as red spheres. Reprinted with permission from PNAS. 1

Efflux pumps actively transport a range of substrates, including toxic agents such as antibiotic or heavy metals, across the membrane and out of the cell. The resistance-modulation-cell division (RND) superfamily of efflux pumps uses the proton gradient to drive conformational changes during its pumping cycle. RND forms a homotrimer in the inner membrane of Gram-negative bacteria; each protomer has 12 transmembrane helices, a substrate-binding porter domain located in the periplasm and a docking domain that couples with an outer membrane factor.

Previous structures of RND multidrug exporters showed each promoter trapped in a different conformation, suggesting a rotating mechanism for transport. In contrast, the structure of heavy-metal exporter CusA showed a three-fold symmetry. Recent work by Stroud, Vandenbussche and colleagues (PSI CSMP) now sheds light on the conformational transitions involved in RND transport of heavy metals and explains the ability of these pumps to transport metal ions located in the periplasmic space.

Working on a RND pump from Cupriavidus metallidurans CH34, the authors initially showed that ZneA is a proton-dependent antiporter specific for Zn2+. They then solved the crystal structures of ZneA at two different conditions, pH 7.5 (PDB 4K0E, 3.7-Å resolution) and pH 5.2 (PDB 4K0J, 3.0-Å resolution). At the lower pH, the ZneA trimer is in an asymmetric conformation, with two protomers featuring a single Zn2+ bound at a site in the porter domain (the “proximal” site). In the higher pH structure, the presence of Zn2+ was observed at the proximal sites of each of the three protomers, as well as at a "distal" site near the exit funnel between the protomers.

In all protomers, at either pH condition, a continuous tunnel, ∼16 Å in length, was observed traversing the porter domain, connecting the proximal and distal Zn2+ binding sites and then extending until reaching a side opening that provides access to the exit funnel. In the protomer lacking Zn2+ at the proximal site, the access loop adopts a different conformation and disrupts the proximal site. By comparing this to the CusA structure, the authors propose that the access loop prevents substrates from flowing back to the proximal site.

The conformational states of ZneA captured here seem to correspond to late steps in the transport cycle, with substrates being transported from a periplasmic location to the outside of the cell (periplasmic efflux). Combined with previous work on other members of this family, the authors present a model for how periplasmic substrates enter, traverse and exit the porter domain, with conformational changes that serve as switches to regulate each step.

Inês Chen

References

  1. J.E. Pak et al. Structures of intermediate transport states of ZneA, a Zn(II)/proton antiporter.
    Proc Natl Acad Sci U S A. 110, 18484-9 (2013). doi:10.1073/pnas.1318705110

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