Ion transfer mechanisms in Mrp-type antiporters from high resolution cryoEM and molecular dynamics simulations

Multiple resistance and pH adaptation (Mrp) cation/proton antiporters are essential for growth of a variety of halophilic and alkaliphilic bacteria under stress conditions. Mrp-type antiporters are closely related to the membrane domain of respiratory complex I. We determined the structure of the Mrp antiporter from Bacillus pseudofirmus by electron cryo-microscopy at 2.2 Å resolution. The structure resolves more than 99% of the sidechains of the seven membrane subunits MrpA to MrpG plus 360 water molecules, including ~70 in putative ion translocation pathways. Molecular dynamics simulations based on the high-resolution structure revealed details of the antiport mechanism. We find that switching the position of a histidine residue between three hydrated pathways in the MrpA subunit is critical for proton transfer that drives gated trans-membrane sodium translocation. Several lines of evidence indicate that the same histidine-switch mechanism operates in respiratory complex I.


Coordination of internal water molecules by conserved polar and titratable residues
In MrpA, conserved polar and protonatable residues (Fig. 2, Supplementary Fig. 3, Supplementary   Fig. 6, Supplementary Table 4) form a contiguous network extending from the putative proton entry site at Lys408 MrpA /Glu409 MrpA to His248 MrpA in the A conformation. Water molecules W72 -W78 are arranged around the strictly conserved Lys408 MrpA . The ligation of the water molecules indicates that Gln309 MrpA and Tyr447 MrpA and several serine and threonine residues play a crucial role, e.g. water molecule W72 is within hydrogen bonding distance of the highly conserved Ser308 MrpA and Tyr447 MrpA and moderately conserved Gln309 MrpA and Thr444 MrpA .
From His248 MrpA a hydrophilic connection to the cytosolic side is formed by strictly conserved Tyr101 MrpA , Ser244 MrpA , Lys299 MrpA and highly conserved residues Thr241 MrpA and Asp297 MrpA (Fig. 2). Tyr101 MrpA and Thr241 MrpA are within hydrogen bonding distance of W70 and W71. Ser244 MrpA binds water W69. Note that residues corresponding to Ser244 MrpA also bind a water molecule in respiratory complex I 1,2 .
His248 in the B conformation is associated with a network of polar residues that connects to the strictly conserved Glu140 MrpA /Lys223 MrpA pair of MrpA. Water W68 is coordinated by the strictly conserved residues Ser146 MrpA , Thr170 MrpA and non-conserved Ser147 MrpA . The adjacent water molecules W66 and W67 are mainly bound by Glu140 MrpA and the highly conserved residues Ser143 MrpA and Thr222 MrpA . Interestingly, the indole NH moiety of the strictly conserved Trp139 MrpA coordinates both water molecules.
At the interface of MrpA and MrpD, water W65 is bound toTyr136 MrpA and the strictly conserved Lys392 MrpD . In MrpD, we modelled six water molecules (W59 -W64) between Lys392 MrpD and the highly conserved His332 MrpD . The water molecules W59 -W64 are further coordinated by Gln307 MrpD , highly conserved His303 MrpD and several polar residues of which Tyr329 MrpD is highly conserved. Following the hydrated region towards the core of MrpD, Lys250 MrpD , His333 MrpD and strictly conserved Lys337 MrpD form an arrangement that is highly similar to the His349 MrpA /Lys254 MrpA /Lys353 MrpA triad in MrpA described above. Interestingly, the antiporter from In contrast to MrpA, two water molecules (W56 and W57) are located in the center of MrpD, coordinated by the strictly conserved residues Lys250 MrpD and Tyr233 MrpD and highly conserved Thr249 MrpD . A pathway from the center of MrpD to the cytosol is expected but not obvious in the structure. We have proposed that in antiporter-like complex I subunits, conserved residues at the end of TMH10 mark the entrance of a proton channel that can be closed by a conserved phenylalanine residue in TMH11 2 . In Mrp, the corresponding residues at the putative channel entry, Asp295 MrpD and Lys297 MrpD , are also conserved and water molecule W58 is bound in close proximity.
However, no other water molecules were found between W58 and water molecules in the central axis, which might indicate that the pathway is blocked by the strictly conserved Phe341 in TMH11 ( Supplementary Fig. 3) as recently described for complex I 2 . An overlay of ND2, ND4 and MrpD shows that the position of this residue agrees well with the "closed" conformation of the ND4 subunit in complex I ( Supplementary Fig. 4).
A hydrated path runs from the center of MrpD to the neighboring MrpC subunit. Tyr233 MrpD bridges between the W56/W57 pair and a cluster of five water molecules (W51 -W55). This cluster is coordinated by Gln166 MrpD and the highly conserved Tyr162 MrpD . The latter residue engages in a hydrogen bond with the strictly conserved Ser143 MrpD . Water molecule W50 is bound between Lys219 MrpD and Glu137 MrpD , the strictly conserved Lys/Glu pair of MrpD, and is further ligated by Ser170 MrpD . We modelled Lys219 MrpD in two conformations, only one of which allows for a hydrogen bond to the water molecule.
In the neighboring MrpC subunit, TMH2 and 3 are at the center of a highly hydrated area. A cluster of water molecules W32 -W37 at the interface of MrpC and MrpD is coordinated by Glu137 MrpD and by moderately conserved Ser36 MrpC , His40 MrpC (see below), Ser80 MrpC , Thr84 MrpC . Towards the Cterminal domain of MrpA, a large water cluster (W21 -W31) is bound by polar residues of MrpC, His37 MrpC and His40 MrpC , as well as Thr690 MrpA and strictly conserved Gln683 MrpA and Glu687 MrpA . A polar residue at position 690 of MrpA is strictly conserved. Residues His37 MrpC and His40 MrpC in TMH2 were recently described as being critical for sodium binding 3 . We note that neither residue is strictly conserved, but histidine or asparagine are the only residues allowed at position 40. A string of water molecules W16 -W20 connects TMH18 of MrpA with the highly conserved Asp38 MrpF . Residue Ser75 MrpF was modelled in two conformations one of which binds water W16. Two clusters of waters (W6 -W15) are located at the interface of MrpF and MrpG. The larger cluster is arranged around Asp38 MrpF and further binding interactions exist with the adjacent Thr39 MrpF , Ser68 MrpF and Thr40 MrpG , Thr44 MrpG and Thr79 MrpG . Polar residues at positions 40, 44 and 79 of MrpG are highly conserved. The smaller cluster is coordinated by residues from both subunits but none of them is conserved.
A previous study suggested that sodium entry from the cytoplasm occurs at the interface between MrpG and MrpE 3 . We find water molecules W1 -W5 distributed around the proposed sodium entry site and several of the hydrogen-bonding residues including highly conserved His37 MrpG , Thr116 MrpE , and strictly conserved Thr113 MrpE , His131 MrpE . A sodium exit 3 or proton entry 4 site was proposed near the highly conserved residues Asp776 MrpA and Glu780 MrpA in TMH21 of MrpA. Water molecules W45 and W46 are close to the critical residues and are further coordinated by strictly conserved residues Thr777 MrpA and Thr75 MrpC . Water molecules W38 -W44 are found in a cavity that may form a pathway to the cytoplasm. They are coordinated by the strictly conserved residues Asp678 MrpA , Asn766 MrpA , and Asp121 MrpB . Asn766 MrpA was modelled in two different conformations oriented towards different waters in the hydrated path.

Protein hydration and residue-water hydrogen bonding analysis
Our additional analyses reveal that a change in protonation state of an amino acid changes the hydrogen bonding patterns with the surrounding water molecules. As expected, the charged state of a residue forms a larger number of hydrogen bonds with water molecules, whereas fewer hydrogen bonds are observed with charge neutral states ( Supplementary Fig. 10). This is also observed by plotting the radial distribution function of water molecules around the residue, where its charged state shows a clear sharp peak relative to its neutral state ( Supplementary Fig. 10). We find that the internal hydration of the protein stabilizes rapidly (see also 5 ) in both cases when the protein is simulated with structural waters or without them ( Fig. 4a and Supplementary Fig. 9e). Our data also reveal that the internal hydration of the protein resembles the structural water content more when simulations are performed in the P state ( Fig. 4a and Supplementary Fig. 9, panels a-d, see methods).
In the S state simulations, additional water molecules diffuse into the protein interior because of the charged states of amino acid residues. Even though we observe close agreement between the structural waters and the water occupancy map from simulations, some non-overlapping areas are observed ( Fig. 4a and Supplementary Fig. 9). These are likely due to the differences in time scales of simulations and experiments (see also 6 ).
In the simulated time scales, most of the structural water molecules were found to be replaced by bulk water molecules ( Supplementary Fig. 9f). However, the water occupancy in internal regions of the protein remain in agreement with the structural water content, when visualized both at 20% (Fig. 4a) and 50% ( Supplementary Fig. 9, panels a-d) occupancy level. Scrutinizing hydrogen bonding between water molecules and selected titratable residues, as well as their lifetimes allowed us to estimate residence times of water molecules in their vicinity (Supplementary Table 8). This also led to the classification of amino acid residues (see Supplementary Table 8). Titratable residues that face bulk or near-bulk like situations do not show a difference in hydrogen bonding lifetimes when the protonation state changes. Interestingly, a putative sodium binding site acidic residue (Glu780 MrpA ) consistently show nanosecond long residence times of water molecules either in its charge neutral or deprotonated state. This is because in its anionic state, it strongly binds a sodium ion, which replaces the role of its protonated sidechain. In addition, we find that the buried residues with conserved structural water next to them show relatively longer hydrogen bonding residence times of water molecules (Supplementary Table 8).  Hydrogen bonds and radial distribution function of water molecules around selected residues in different protonation states. The histograms display the occupancy of hydrogen bond between a residue and water molecules in different protonation states (PA1, PB1, SA1 and SB1, refers to data from setups PA1, PB1, SA1 and SB1, respectively, see also Supplementary Table 6). The hydrogen bonding criteria used are Donor-Acceptor distance 3.5 Å and Donor-Hydrogen-Acceptor angle 30 degrees. The radial distribution function g(r) is calculated based on distance between sidechain nitrogen/oxygen atom and oxygen of water molecules. Overall, the trend is that the neutralization of amino acid sidechain destabilizes hydrogen bonding with water molecules.