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A two-domain elevator mechanism for sodium/proton antiport

Abstract

Sodium/proton (Na+/H+) antiporters, located at the plasma membrane in every cell, are vital for cell homeostasis1. In humans, their dysfunction has been linked to diseases, such as hypertension, heart failure and epilepsy, and they are well-established drug targets2. The best understood model system for Na+/H+ antiport is NhaA from Escherichia coli1,3, for which both electron microscopy and crystal structures are available4,5,6. NhaA is made up of two distinct domains: a core domain and a dimerization domain. In the NhaA crystal structure a cavity is located between the two domains, providing access to the ion-binding site from the inward-facing surface of the protein1,4. Like many Na+/H+ antiporters, the activity of NhaA is regulated by pH, only becoming active above pH 6.5, at which point a conformational change is thought to occur7. The only reported NhaA crystal structure so far is of the low pH inactivated form4. Here we describe the active-state structure of a Na+/H+ antiporter, NapA from Thermus thermophilus, at 3 Å resolution, solved from crystals grown at pH 7.8. In the NapA structure, the core and dimerization domains are in different positions to those seen in NhaA, and a negatively charged cavity has now opened to the outside. The extracellular cavity allows access to a strictly conserved aspartate residue thought to coordinate ion binding1,8,9 directly, a role supported here by molecular dynamics simulations. To alternate access to this ion-binding site, however, requires a surprisingly large rotation of the core domain, some 20° against the dimerization interface. We conclude that despite their fast transport rates of up to 1,500 ions per second3, Na+/H+ antiporters operate by a two-domain rocking bundle model, revealing themes relevant to secondary-active transporters in general.

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Figure 1: NapA Na+/H+ transport activity is electrogenic.
Figure 2: Outward-facing NapA structure.
Figure 3: Structure of NapA dimer and proposed Na+/(Li+)-binding site.
Figure 4: Alternating access model of sodium-proton antiport.

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Protein Data Bank

Data deposits

The coordinates and the structure factors for NapA have been deposited in the Protein Data Bank under accession code 4BWZ.

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Acknowledgements

We are grateful to D. Slotboom for critical reading of the manuscript and N.-J. Hu for assistance in data collection. Data were collected at the European Synchrotron Radiation Facility and Diamond Light Source, with excellent assistance from beamline scientists. This work was funded by grants from the Medical Research Council (MRC grant G0900990 to A.D.C. and D.D.), the Swedish Research Council (to C.v.B. and D.D.) and the BBSRC (BB/G02325/1 to S.I.). The authors are grateful for the use of the Membrane Protein Laboratory funded by the Wellcome Trust (grant 062164/Z/00/Z) at the Diamond Light Source Limited and The Centre for Biomembrane Research (CBR), supported by the Swedish Foundation for Strategic Research. Computer simulations were partially run on XSEDE resources (grant TG-MCB120151 to O.B.). C.L. was a recipient of a BBSRC-funded PhD scholarship, H.J.K. a Human Frontiers Science Program (HFSP) postdoctoral fellowship, and D.D. acknowledges the support from The Royal Society through the University Research Fellow (URF) scheme.

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Authors

Contributions

A.D.C. and D.D. designed the project. Cloning, expression screening, protein purification and crystallization were carried out by C.L. and D.D. with assistance from H.J.K., S.N., S.I. and A.D.C. Data collection and structural determination were carried out by C.L., D.D. and A.D.C. Experiments for functional analysis were designed by C.v.B. and D.D. and carried out by C.v.B., C.L., P.U. and D.D. Molecular dynamics simulations were carried out by D.L.D. and O.B. A.D.C. and D.D. wrote the manuscript with contributions from C.L., H.J.K., C.v.B. and O.B.

Corresponding authors

Correspondence to Alexander D. Cameron or David Drew.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-8, Supplementary Tables 1-2 and additional references. (PDF 9005 kb)

Alternating access model of Na+/H+ antiport.

Video morphing between the outward-facing NapA crystal structure and a model created by superposing the NapA Core and Dimerisation domains separately onto the NhaA structure. The residues mentioned in the text are shown in a stick representation. Colouring as in Figure 3. (MOV 5509 kb)

Alternating access model of Na+/H+ antiport.

Video morphing between the outward-facing NapA crystal structure and a model created by superposing the NapA Core and Dimerisation domains separately onto the NhaA structure. The residues mentioned in the text are shown in a stick representation. Colouring as in Figure 3. (MOV 8207 kb)

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Lee, C., Kang, H., von Ballmoos, C. et al. A two-domain elevator mechanism for sodium/proton antiport. Nature 501, 573–577 (2013). https://doi.org/10.1038/nature12484

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