Maintaining intracellular homeostasis is crucial for the survival of all organisms. Alkaliphilic and halophilic bacteria live in challenging conditions, where pH and ion concentration in the cytosol must be carefully controlled. For this task, many bacteria harness Mrp-type antiporters (multiple resistance and pH adaptation cation/proton antiporters) ^1-3. In recent years, the seven-subunit Mrp-type antiporter complexes have gained significant interest due to their evolutionary relationship with a superfamily of energy converting enzyme complexes including respiratory complex I. Mitochondrial complex I has a central role in aerobic energy metabolism and its dysfunction is associated with severe human diseases ⁴.

We report a 2.2 Å resolution structure of the Mrp antiporter complex from Bacillus pseudofirmus determined by single-particle electron cryo-microscopy. The antiporter complex is a dimer of seven-subunit protomers. A variance in the angle between the two protomers proved to be a limiting factor for the achievable resolution, but this obstacle was overcome by symmetry expansion and focused 3D refinement of one protomer. The structure includes 1957 residues (with 99 % of the sidechains resolved), three phospholipids and 360 water molecules. For several residues, the map indicated alternative sidechain orientations and we observed even more extensive main-chain rearrangements in a short sequence stretch interrupting a transmembrane helix of the MrpA subunit. 

The large number of resolved water molecules enabled us to identify putative pathways for protons and sodium ions – a central question with far-reaching implications also for the complex I superfamily. To further study the water and ion dynamics in the antiporter, we performed molecular dynamics (MD) simulations. The Mrp antiporter protomer was embedded in a POPG:POPE model membrane in a solvent box, yielding a model system of roughly 450,000 atoms that was studied by atomistic MD simulations totaling about 19.5 µs. The hydration observed from simulation data matched the internal water clusters of the structural model well, corroborating the proposed proton and sodium pathways.

 In the N-terminal domain of subunit MrpA (corresponding to ND5 in mitochondrial complex  I), we recognized conformational changes that led us to propose a histidine switch mechanism. Structural data revealed that the strictly conserved His248 sits in the center of a Y-shaped arrangement of pathways connecting to the periplasm, the central hydrophilic axis and the cytoplasm. The histidine forms alternating contacts to Thr306, Ser146 and to a protein-bound water molecule. Strikingly, simulations showed that the position of the histidine residue depends on its protonation state. These data strongly suggest that the histidine is central for gated proton translocation which is coupled to sodium transport.

 To study the potential sodium pathways in the Mrp antiporter, we combined an analysis of internal cavities in the protein with MD simulations. We propose two possible uptake pathways for sodium, both converging at Glu687 in MrpA. A critical role of this highly conserved residue is in agreement with data from previous site-directed mutagenesis studies ⁵. Our simulations also provide clear evidence for a second sodium binding site in the exit pathway to the periplasmic side. We consider two different connecting pathways between the sodium-loading site at Glu687 and the sodium-loading site in the exit pathway, but both routes require the passage of hydrophobic barriers. Our simulations provide clues to a possible coupling mechanism, as the binding of sodium to different acidic residues is dependent on their protonation state.

 In conclusion, we propose that switching of a histidine residue between two different positions permits the gated transfer of protons to different pathways in subunit MrpA. The histidine switch mechanism has intriguing implications for our understanding of respiratory complex I and related enzyme complexes (see also ⁶). The recognition of sodium loading sites opens new avenues for studying the sodium transfer in the Mrp antiporter and in related redox-driven sodium pumps. Our paper demonstrates the importance of combining structural and biochemical research with computer simulations. While the coupling mechanism is not yet fully understood, our results provide novel concepts applicable to a wide range of research topics.

1.          Ito, M., Morino, M. & Krulwich, T. A. Mrp Antiporters Have Important Roles in Diverse Bacteria and Archaea. Front Microbiol 8, 2325, doi:10.3389/fmicb.2017.02325 (2017).

2.          Steiner, J. & Sazanov, L. Structure and mechanism of the Mrp complex, an ancient cation/proton antiporter. Elife 9, doi:10.7554/eLife.59407 (2020).

3.          Li, B. et al. Structure of the Dietzia Mrp complex reveals molecular mechanism of this giant bacterial sodium proton pump. Proc Natl Acad Sci U S A 117, 31166-31176, doi:10.1073/pnas.2006276117 (2020).

4.          Parey, K., Wirth, C., Vonck, J. & Zickermann, V. Respiratory complex I — structure, mechanism and evolution. Current Opinion in Structural Biology 63, 1-9, doi:10.1016/j.sbi.2020.01.004(2020).

5.          Kajiyama, Y., Otagiri, M., Sekiguchi, J., Kudo, T. & Kosono, S. The MrpA, MrpB and MrpD subunits of the Mrp antiporter complex in Bacillus subtilis contain membrane-embedded and essential acidic residues. Microbiology 155, 2137-2147, doi:10.1099/mic.0.025205-0 (2009).

6.          Djurabekova, A., Haapanen, O. & Sharma, V. Proton motive function of the terminal antiporter-like subunit in respiratory complex I. Biochim Biophys Acta Bioenerg 1861, 148185, doi:10.1016/j.bbabio.2020.148185 (2020).