Contributed by Robert M. Stroud, October 4, 2013 (sent for review August 14, 2013)
Transmembrane efflux pumps belonging to the resistance–nodulation–cell division (RND) superfamily are found in all kingdoms of life, and transport substrates out of cells, powered by an electrochemical proton gradient. Here we report two X-ray crystal structures of a Zn(II) efflux pump, ZneA, that capture different intermediate states along the transport cycle. The structures show how passage of substrates through ZneA is regulated by a series of conformational changes in the efflux pump. By comparing the structures of ZneA with other RND efflux pumps, we present a coherent mechanistic model for RND-mediated substrate efflux, which ensures efficient transport of substrates out of the cell.
Efflux pumps belonging to the ubiquitous resistance–nodulation–cell division (RND) superfamily transport substrates out of cells by coupling proton conduction across the membrane to a conformationally driven pumping cycle. The heavy metal-resistant bacteria Cupriavidus metallidurans CH34 relies notably on as many as 12 heavy metal efflux pumps of the RND superfamily. Here we show that C. metallidurans CH34 ZneA is a proton driven efflux pump specific for Zn(II), and that transport of substrates through the transmembrane domain may be electrogenic. We report two X-ray crystal structures of ZneA in intermediate transport conformations, at 3.0 and 3.7 Å resolution. The trimeric ZneA structures capture protomer conformations that differ in the spatial arrangement and Zn(II) occupancies at a proximal and a distal substrate binding site. Structural comparison shows that transport of substrates through a tunnel that links the two binding sites, toward an exit portal, is mediated by the conformation of a short 14-aa loop. Taken together, the ZneA structures presented here provide mechanistic insights into the conformational changes required for substrate efflux by RND superfamily transporters.
↵1J.E.P. and E.N.E. contributed equally to this work.
Author contributions: J.E.P., E.N.E., F.D.A., G.V., and R.M.S. designed research; J.E.P., E.N.E., E.G.K., J.D.O., F.D.A., M.B.T., K.-M.D., Y.R.-C., R.A.R., and G.V. performed research; J.E.P., E.N.E., G.V., and R.M.S. analyzed data; and J.E.P., E.N.E., E.G., G.V., and R.M.S. wrote the paper.
The authors declare no conflict of interest.
Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4K0E and 4K0J).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1318705110/-/DCSupplemental.
