Mechanisms for Two-Step Proton Transfer Reactions in the Outward-Facing Form of MATE Transporter Wataru Nishima,1,2 Wataru Mizukami,1 Yoshiki Tanaka,5 Ryuichiro Ishitani,6 Osamu Nureki,6 and Yuji Sugita1,2,3,4,* 1 Theoretical Molecular Science Laboratory and 2Interdisciplinary Theoretical Science Research Group (iTHES), RIKEN, Wako, Saitama, Japan; 3Quantitative Biology Center and 4Advanced Institute for Computational Science, RIKEN, Kobe, Hyogo, Japan; 5Laboratory of Membrane Molecular Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara, Japan; and 6 Department of Biophysics and Biochemistry, The University of Tokyo, Tokyo, Japan

ABSTRACT Bacterial pathogens or cancer cells can acquire multidrug resistance, which causes serious clinical problems. In cells with multidrug resistance, various drugs or antibiotics are extruded across the cell membrane by multidrug transporters. The multidrug and toxic compound extrusion (MATE) transporter is one of the five families of multidrug transporters. MATE from Pyrococcus furiosus uses Hþ to transport a substrate from the cytoplasm to the outside of a cell. Crystal structures of MATE from P. furiosus provide essential information on the relevant Hþ-binding sites (D41 and D184). Hybrid quantum mechanical/molecular mechanical simulations and continuum electrostatic calculations on the crystal structures predict that D41 is protonated in one structure (Straight) and, both D41 and D184 protonated in another (Bent). All-atom molecular dynamics simulations suggest a dynamic equilibrium between the protonation states of the two aspartic acids and that the protonation state affects hydration in the substrate binding cavity and lipid intrusion in the cleft between the N- and C-lobes. This hypothesis is examined in more detail by quantum mechanical/molecular mechanical calculations on snapshots taken from the molecular dynamics trajectories. We find the possibility of two proton transfer (PT) reactions in Straight: the 1st PT takes place between side-chains D41 and D184 through a transient formation of low-barrier hydrogen bonds and the 2nd through another Hþ from the headgroup of a lipid that intrudes into the cleft resulting in a doubly protonated (both D41 and D184) state. The 1st PT affects the local hydrogen bond network and hydration in the N-lobe cavity, which would impinge on the substrate-binding affinity. The 2nd PT would drive the conformational change from Straight to Bent. This model may be applicable to several prokaryotic Hþ-coupled MATE multidrug transporters with the relevant aspartic acids.

INTRODUCTION Multidrug transporters play major roles in the acquisition of multidrug resistance in bacterial pathogens (1–3) and cancer cells (4,5). They reduce the therapeutic effectiveness of drugs by an efflux mechanism, and cause serious human health problems. In the rocker-switch model (6), the transport protein opens to either the extracellular (outward-facing) or cytoplasmic (inward-facing) sides, whereas the other side is closed to prevent leakage. A conformational change from inward- to outward-facing forms and vice versa, driven by ATP-hydrolysis or Hþ/Naþ motive forces drive the conformational changes (7), and is considered

Submitted October 26, 2015, and accepted for publication January 21, 2016. *Correspondence: [email protected] Editor: Scott Feller. Ó 2016 Biophysical Society

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fundamental to the mechanism. Atomic structures of multidrug transporters in different physiological states in the transport cycle are invaluable for detailed understanding of the drug extrusion mechanism (8), which, in turn, is helpful for the development of inhibitors. More than 50 crystal structures of multidrug transporters are in the Protein Data Bank (PDB: Multidrug and toxic compound extrusion (MATE) is one of five multidrug transporter superfamilies and ubiquitously exists in all three domains of life (Archaea, Bacteria, and Eukarya) (9,10). They use a concentration gradient of either Naþ or Hþ to extrude lipophilic and cationic substrates from the cytoplasm (11,12). Eukaryotic MATE, including that of humans, typically uses Hþ (13–16). There are crystal structures of NorM, a drug-Naþ antiporter, from Vibrio cholera in the outward-facing form with/without cations (17) and with drugs (18). A series of molecular dynamics (MD)

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simulations have been performed on NorM previously (19–22). More recently, multiple atomic structures of MATE from Pyrococcus furiosus (PfMATE) have been determined also in the outward-facing form and the transporter is found to be an Hþ coupled antiporter (23). Both NorM and PfMATE show a twofold rotational symmetry for the first six (N-lobe) and last six (C-lobe) transmembrane (TM) helices. Based on the high-resolution x-ray crystal structures of PfMATE, D41 and D184 are suggested as the functionally relevant Hþ-binding sites (23). Most recently, structures of MATE from Bacillus halodurans (DinF-BH) have been determined in the outward-facing form (14,16). There are two distinct structures of PfMATE and both are in the outward-facing form (23) (Fig. S1 in the Supporting ˚ ), Material). In one form, Straight (PDB: 3VVN, 2.4 A TM1 is almost a straight a-helix, which allows for a hydrophilic cavity in the N-lobe (N-lobe cavity). A Norfloxacinderivative substrate (Br-NRF) is bound in the N-lobe cavity in one of the Straight structures (Straight with drug, PDB: ˚ ). In contrast, in the Bent form (PDB: 3VVO, 3VVP, 2.9 A ˚ 2.5 A), TM1 bends at P26 and G30, concealing the N-lobe cavity together with TM2. The conformational change from Straight to Bent is considered to be essential for pushing substrates toward the extracellular region. These distinct forms of Straight and Bent are obtained under different pH environments (Straight: ~6.0, Bent: 7.0–8.0) and no other protonatable acidic residues are located in the N-lobe cavity, suggesting that, Hþ-affinities at D41 and D184 are different in Straight and Bent. In the structural study, D184 is postulated to be protonated in Straight, and both D41 and D184 protonated in Bent. Thus, the additional protonation of D41 is considered to trigger the structural change from Straight to Bent. D41 and D184 are well-conserved residues in archaeal and bacterial MATE (23) (Fig. S2) and these two residues are experimentally known to be crucial to the substrate transport. We consider that the protonation states of D41 and D184 are essential for understanding the molecular mechanisms underlying Hþ/substrate antiport. In this study, we predicted the protonation states of D41 and D184 for the static crystal structures of PfMATE using hybrid quantum mechanical/molecular mechanical (QM/ MM) simulations and continuum electrostatic theory. The protonation states in membrane proteins can be affected by conformational fluctuations and protein-lipid interactions. We therefore carried out all-atom MD simulations of PfMATE in explicit solvent and a lipid bilayer, assuming several different protonation states and initial lipid positions. The MD simulations suggested the possibility of proton transfers between the protonation sites in the outward-facing form of PfMATE. The potential energy surfaces (PESs) for two proton transfer (PT) reactions by QM/MM calculations on selected MD snapshots support this view.

MATERIALS AND METHODS Continuum electrostatic calculations for prediction of pKa values pKa values of D41 and D184 in the crystal structures were predicted by solving the continuum Poisson equation. We used the multiflex program in the MEAD package (24) considering the correlation of protonated and deprotonated states. We included water molecules whose oxygen atoms ˚ of the Od atoms of the two aspartic acids in the continexist within 3.5 A uum electrostatic calculations.

MD simulations We used NAMD (25) and CHARMM force field (26,27) for MD simulations. Production MD runs were carried out for 100–1000 ns with a NPT ensemble without any restraints. A constant temperature of 310 K and a constant pressure of 1 atm were maintained using Langevin dynamics. Long-range electrostatic forces were calculated using the smooth particle ˚ , whereas Lenmesh Ewald summation method with a grid size of 25 A

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Nishima et al. the side chains of the QM-region aspartic acids (i.e., D41 and/or D184) ˚ away from those Od atoms from our QM/MM calculations. Atoms >16 A were kept fixed during the geometry optimizations of Straight and Bent structures (Figs. S5 and S6). An approach called adiabatic mapping (39) was applied to compute a PES along with a reaction coordinate. The method scans potential energies using a restrained minimization at each point of the ˚ . We restrained coordinate successively. The step size for the scan was 0.1 A reaction coordinates by employing a harmonic potential whose force con˚ 2. The adiabatic mapping optimizations were stant was 4000 kcal/mol$A performed repeatedly until obtaining a smooth and converged potential energy surface, because it is well known that this method is prone to yield a discontinuous potential energy curve for a large system. All quantum chemical calculations were performed at the B3LYP/6-31þG* level of theory. Force fields were the same as those in the MD simulations. Further details on pKa calculations, all-atom MD simulations, and QM/MM simulations are described in the Supporting Material.

RESULTS AND DISCUSSION Protonation states of D41 and D184 in the crystal structures The minimum side-chain distances between D41 and D184 ˚ and 2.71 A ˚ in the two crystal structures, Straight are 2.73 A and Bent, respectively. They suggest that a hydrogen bond (H-bond) exists between the Od atoms. Alanine substitutions of D41 or D184 abolish substrate transport and Hþ antiport, indicating their functional importance (23). The protonation states of D41 and D184 were examined using QM/MM simulations and continuum electrostatics theory (see Supporting Material). We initially optimized the position of Hþ in the two aspartic acids and hydrogen atoms in surrounding residues by simulated annealing. To validate the calculations, the procedure was repeated five times. QM/MM optimized structures indicate that only D41 is protonated in Straight, whereas both D41 and D184 are protonated in Bent (Table S2). The electrostatic potential on the surface of the N-lobe is drastically altered by the protonation changes (Fig. S7). These protonation-state predictions are supported by the continuum electrostatic theory (Table S3). For continuum electrostatic calculations, we selected conformations where D41 and D184 have the largest number of H-bonds (Fig. S8). The result for Bent agrees with the suggestion in the structural study (23). It is easily understood, because the environment around D41 and D184 in Bent is hydrophobic (Fig. 1). Although the protonation state predicted for Straight is opposite to that suggested from the structure (23), the QM/MM optimization suggests the possibility of PT between D184 and D41. The side chain of D184 has a larger number of H-bonds with water molecules, and one of the Od atoms in D41 does not form H-bonds in the energy-minimized structure (Fig. S8 b). All-atom MD simulations with different protonation states We next carried out all-atom MD simulations of PfMATE with explicit solvent and a lipid bilayer (Fig. S3). In all

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FIGURE 1 Close-up views of PfMATE structures in the vicinity of the Hþ-binding sites. (a) The Hþ-binding sites in Straight and Bent, viewed from the extracellular side. Bands indicate slices of the solvent accessible surface area at different levels (blue and orange). (b) Typical interaction network at D41 and D184 in MD simulations (left, SL41; middle, SL184; right, BL41-184). To see this figure in color, go online.

the MD simulations, the overall structure of PfMATE was stable. SL41 and BL41-184, in particular, show the smallest conformational deviations from the starting structures (Fig. S9). In S41 and SL41, the Hþ at D41 participated in forming an H-bond with the D184 side chain. Y37 also interacted with one of the Od atoms in D184 (Fig. 2 and S10). In contrast, in S184 and SL184, water molecules interacted with the side chain of D41 (Figs. 2 and S10) and the distance between D41 and D184 became longer because of frequent water mediation. In S41-184 and SL41-184, D41 and D184 did not form H-bonds with each other and the side chain conformations were flexible. In B41-184, BL41-184, the Od atoms in D41 formed two H-bonds with D184 and T202 side chains (Fig. 1 b, right). The H-bond networks in each of the protonation states were unchanged by the existence of lipids in the cleft (Fig. S11). In SL41 and B41-184, the side chains of D41 and D184 formed almost unique H-bonding structures, suggesting that their protonation states are most stable in Straight and Bent, respectively (Table S4). In all the simulations, Naþ ions did not enter the cavity and it is consistent with the experimental evidence that the substrate transport by PfMATE is driven by Hþ (and not Naþ) (23). What dictates the protonation states of D41 and D184 in Straight and Bent? There is no acidic residue near D41 and D184 in the N-lobe cavity in both forms (Fig. 1 a). A direct or water-mediated H-bond between D41 and D184 is frequently formed in Dp41 (Table S5 and Fig. S11), suggesting the possibility of PT from D41 to D184 or vice versa in Straight. Indeed, pKa calculations on each snapshot in the

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FIGURE 2 H-bonds around the Hþ-binding sites. (a) Close-up view of the entrance of the N-lobe cavity in MD simulations (left, SL41 at 59.2 ns; middle, SL184 at 65.4 ns; right, SL41-184 at 19.0 ns). (b) Time course of nearest heavy atom distances between D41 and Y37 (red), D184 and Y37 (green), and D41 and D184 (blue). To see this figure in color, go online.

S41 simulation trajectory after removing Hþ at D41 (Fig. S12) show that the protonation states of D41 and D184 become exchangeable three times. Accordingly, we propose a hypothesis based on an exchangeable protonation state in D41 and D184 in the outward-facing form. In Straight, the most stable form is protonated D41, but the Hþ attached at D41 is likely to be able to be transferred to D184. Later, both D41 and D184 are protonated and stabilized by forming an H-bond network. Two key questions then arise. How do the protonation states affect the physical properties of the substrate-binding site in the N-lobe and the cleft between the N- and C-lobes? Which factor drives the PT reactions? In other words, how and when do the PT reactions connecting Dp41, Dp184, and Dp41þDp184 take place? To answer these questions, we further analyzed the MD trajectories and carried out hybrid QM/MM calculations on selected snapshots from the MD simulations. The effect of the protonation states on the hydrophobic cleft and the N-lobe cavity Two lipid molecules initially placed at the cleft in SL41, SL184, and SL41-184 stayed in almost the same positions for 100 ns. In S41, S184, S41-184, the hydrophilic headgroups of the lipid molecules intruded into the crossing junction between the N- and C-lobes, although entry for the hydrophobic tails was blocked (Fig. S13). We extended two simulations (S41, S184) up to 1 ms and observed that a lipid molecule started to intrude into the cleft at 400 ns only in S184 (Fig. 3 a). At the end of the simulation (at 1 ms), one lipid tail interacted with the C-lobe and another tail remained out of the cleft over the junction. In contrast, no lipid tail intruded into the cleft in S41. We confirmed the tendency with additional shorter (500 ns) trajectories (Fig. S14). Evidently, protonation affects lipid intrusion. We examined the correlation between lipid intrusion and local conformational changes in S184. First of all, local conformational changes occurred at ~130 ns near D41 (green line in Fig. 3 a) and then a part of TM1 became distorted largely as a consequence of a peptide-plane flip (p-flip) (40) (Fig. 3, a, c, and d). The movement of TM2

made one of the crossing junctions wider (red arrow in Fig. 3 c) at ~370 ns, causing a lipid to intrude though the junction (Fig. 3 a). In contrast, in S41 and SL41, the H-bond between D41 and D184 was more stable, (Fig. 3 a; Table S4), and side-chain rigidity here could prohibit the p-flip motion, and accordingly no lipid intrusion was observed even with the 1 ms MD simulation of Dp41 (S41). It is also interesting to examine the closest oxygen-oxygen (O-O) distance between the lipid phosphate group and the protonation sites (Fig. S15). The peaks of the O-O dis˚ except for tance were distributed between 10 and 15 A SL184 (for 100 ns) and S184 (for 1 ms). In these two simulations, the O-O distance between D41 and the lipid phosphate group was much closer. We repeated SL41 and SL184 three times for statistical reliability and obtained consistent results (Fig. S15). Based on the crystallographic evidence (Fig. S1) and the simulations, it is likely that there are a few lipid molecules in the cleft between the N- and C-lobes for Dp184. More water molecules were observed in the N-lobe cavity in SL184 compared with SL41 and SL41-184 (Fig. 2 a). The hydrophobic nature of the N-lobe cavity in SL41 (and SL41-184) was realized mainly by an H-bond network involving Y37, D41, and D184. In particular, Y37 likely plays a key role to keep a tight interaction between TM1 and TM5, which prevented water intrusion from the extracellular side in SL41 and SL41-184. In contrast to SL184, the flexibility of the side chains breaks the water shield creating a hydrophilic environment. The hydrophilicity of the N-lobe cavity of Dp184 may be functionally important for extrusion of substrate from the N-lobe cavity in the outward facing form. Molecular mechanisms underlying the PT reactions Observations of H-bond formations in MD simulations with different protonation states imply two PT reactions: one connecting Dp41 and Dp184, and another between Dp184, and Dp41þDp184. To examine these possibilities, we carried out QM/MM calculations of several snapshots of MD simulations (Figs. S5 and S6). Fig. 4 a shows two PESs of

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FIGURE 4 The PESs of the 1st PT and optimized structures. (a) Two PESs obtained from two consecutive MD snapshots in S41 at 25.965 ns (red), and 25.970 ns (green). RC is defined using Hþ, Od atoms of D41 and D184 (negative, Dp41; positive, Dp184). Two figures at the bottom are optimized structures for two snapshots (I, before; II, after). (b) Three PESs obtained after removing the configured water molecule from the after structure. (c) Optimized structures at the global minima after removal of the configured water molecule. (I, II, III: removal of water a, b, c in the after structure, respectively). To see this figure in color, go online.

FIGURE 3 Lipid intrusion events and their mechanism appeared in 1 ms simulation of S41 and S184. (a) Time course of the lipid intrusion (red: phosphate, green: center of the lipid molecule). Distance between the center of intruded lipid and the center of 4 TMs (TM1, 2, 7, and 8) is shown. Characteristic distances (red, Ca atoms at D41-D184; green, Ca atoms at L39-W44; violet, nearest side chain heavy atoms at G65-S254) and dihedral angles (red, j(D41) in S184; violet, 4(G42) in S184, j(D41) in S41; violet, 4(G42) in S41). (b) Snapshot at 1 ms viewed from the top. An intruded POPC molecule is highlighted using orange color for carbon atoms. Cyan is used for carbons of other POPCs. (c) Structural change of TM1-TM2 region (left, top view; right, side view). The structure obtained in S184 (green) is compared with the crystal structure (orange). (d) Close-up figure of (c) (red dotted region in Fig. 5 b). D41 and G42 are drawn with atomic resolution. Peptide-plane between D41 and G42 is in yellow. To see this figure in color, go online.

the first PT obtained from two successive MD structures (t ¼ 25.965 ns and 25.970 ns in S41), which caused large pKa jumps observed in Fig. S12. Of importance, the energy barrier for the PT is low (~1 kcal/mol), suggesting that the barrier is negligible at the physiological condition. The PES before the pKa jump shows that Dp184 is slightly unstable, while that after indicates it to be mildly stable. The difference of PESs is likely attributable to the water coordination to D41, which is consistent with hydration features in SL41 and SL184 (Figs. 1 b and S11). We further examined the PESs after the anomalous pKa shift, removing each water

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molecule coordinated to D41 (Fig. 4 b). In one of the three cases of water removal (case I in Fig. 4 c), Dp41 was slightly stabilized by the water coordination to D184 after the QM/MM calculations. The same calculations for the two other cases showing the pKa in S41 confirmed that the 1st PT readily happens at the physiological condition (Figs. S16 and S17). Water intrusion with a specific coordination is a rare event in the Dp41 state, but once it happens, the 1st PT reaction becomes possible. In SL184 and S184 (for 1 ms), we observed that the O-O distance between the side chain of D41 and the phosphate group was much shorter than in the other conditions. In fact, we could observe a water-mediated H-bond between a phosphate and D41 in SL184 (Fig. 5), suggesting the possibility of a 2nd PT from bound H3Oþ to the lipid headgroup in Dp184. We calculated PESs using six snapshots in SL184, where water-mediated H-bonds were observed and after replacing the water with H3Oþ (Fig. S18). In Fig. 5, one of the PESs, the reactant (Phosphate þ H3Oþ, or, protonated Phosphate þ H2O) and product (D41p) structures for the PT are shown. Compared to the 1st PT, the energy barrier is much higher and the energy difference between the reactant and product is larger, suggesting that the 2nd PT reaction is irreversible only occurring in the direction of the doubly protonated state (Dp41þDp184). The QM/MM calculations suggest different molecular mechanisms underlying the two PT reactions. The low energy barrier of the 1st PT is reminiscent of the low barrier H-bond (LBHB) according to the definition of Shutz and Warshel (41). Indeed, the Od-Od distances in the MD

Proton Transfer Mechanisms of MATE

FIGURE 5 The PESs of the 2nd PT and optimized structures at minima. (a) PES obtained from MD snapshot (t ¼ 54.88 ns) in SL184.c. RC is defined using Hþ, O atoms of D41 and bridging water (negative: Dp41 þDp184, positive: protonation of water/phosphate group). (b) Optimized structures corresponding two local minima (I: protonation of phosphate group, II: Dp41þDp184). To see this figure in color, go online.

˚ . The formation of a snapshots were shorter than 2.5 A LBHB is worthy of attention, because it often underlies functionally important PT reactions (42). However, its characteristics (e.g., stabilities and formation mechanism in biological systems) are still controversial (43,44). Our results can be interpreted as follows: a LBHB (having similar pKa) is temporally and locally stabilized by a particular H-bond network of configured water molecules, and facilitates the 1st PT. However, thermal fluctuations easily disturb the coordination of waters and can cause a much larger pKa difference, which means the formation of a normal H-bond and the stabilization of either protonated state. These functional concepts of a LBHB have been presented for other biological systems (45). In PfMATE, hydration of D41 is probably the driving force to shift the dynamic equilibrium toward Dp184. The latter is synergistically associated with hydration of the N-lobe cavity. The 2nd PT is likely mediated by a lipid headgroup, as indicated in other experimental studies (46–48) and simulations (49) on membrane proteins (e.g., cytochrome c oxidase (50), bacteriorhodopsin (51), and the bacterial reaction center (52)). A lipid headgroup, acting as a proton conductor, helps fast and distant lateral diffusion of Hþ along the membrane surface compared to bulk water (53,54). In the six cases (Figs. 5 and S18), the reactants took two different forms after the QM/MM optimization (three as a protonated phosphate group and another three as an H3Oþ attached to the phosphate group). In both cases, a Grotthuss mechanism, by which excess Hþ diffuses through the H-bond network, could underlie the 2nd PT. Hierarchical computational approach to elucidate PT reactions and limitations of this method Our hierarchical computational approach includes continuum electrostatic theory, all-atom MD simulations, and the hybrid QM/MM calculations. MD simulation can take

care of longer timescale events such as proton-lipid interactions and conformational fluctuations, whereas QM/MM calculations can describe PT reactions that require cleavage of covalent bonds. pKa calculations using electrostatics are usually applied to stable structures. In this study, we obtained instantaneous pKa value from each MD snapshot to connect MD simulation and QM/MM calculations efficiently. The consistent results suggest our choice of simulation methods is reasonable. Because of its generality, the framework could be applied to other biological systems. However, there are several limitations to mixed computational approaches. First, it is hard to obtain accurate statistical pictures of biological phenomena. For example, in the last section, we discussed how the PT reaction is possibly a rare event due to the need for the formation of appropriate water coordination. In the current approach, we could not provide an accurate estimate of its rarity due to limitation in sampling. Second, there is a need to perform multiple simulations assuming different models in mixed approaches, which escalate computational cost. Third, kinetics is almost lost in such independent simulations. Statistical approaches, such as in the Markov state model (55), would reveal the kinetic properties of a system, though more assumptions and sufficient conformational sampling of complex biological events are necessary. Role of two-step proton transfers in MATE transporters function We have shown the existence of a dynamic equilibrium between protonation states and their molecular mechanisms in the PfMATE multidrug transporter in two outward-facing forms (Straight and Bent), using high-resolution crystal structures and molecular simulations. Our independent computations consistently support the same conclusion, which is summarized in Fig. 6. Both PTs occur in transient Straight-like structures. Hydration of the N-lobe cavity contributes to the population shift in equilibrium during the 1st PT and an intruded lipid headgroup facilitates the 2nd PT. The larger energy barrier for the 2nd PT suggests that lipid intrusion is a crucial event for driving the transport cycle in one direction—the reverse reaction seldom occurs. After the Dp41þDp184 state is established, the transporter would undergo the conformational change from Straight to Bent. The coupling of different protonation states with phenomenological events such as hydration and lipid intrusion are likely highly relevant to MATE transporter function. For example, hydration of the N-lobe cavity may weaken the binding stability of a substrate, and perhaps drug release occurs before lipid intrusion. Different lipid compositions (with individual pKa values of their headgroups) and pH would also obviously affect drug transport. There is still comparatively little known about the effect of lipids on the multidrug transporter (56,57). Indeed, monoolein stabilizes Straight at relatively high pH (7.0~8.0) as evidenced by crystal formation and

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FIGURE 6 A proposed model of two-step proton transfer (PT) reactions. The first PT changes hydration states of N-lobe cavity as well as recruit a lipid into the cleft. The second PT occurs while intervening the protonated lipid or H3Oþ attached to the headgroup. To see this figure in color, go online.

the atomic structure (23). The high pKa of the monoolein headgroup likely inhibits the 2nd PT, and the hydrophobicity of the intruded monoolein located near D41 can stabilize Dp41. However, further computational analyses based on energetics or simulation trials with drug models are required to clarify feasibility and mechanisms of series events such as drug release and the conformational change from Straight to Bent. There is no available experimental data concerning the Hþ/substrate stoichiometry in PfMATE. An isolated aspartic acid residue can only accommodate a single proton, suggesting that PfMATE binds two protons at most at D41 and D184. In this study, we confirm that one proton is mainly used to maintain the H-bond distance of D41 and D184. Actually, such close distances are always observed in the outward-facing form of crystal structures. If the distance is maintained in the inward-facing form in the same way, the proton attached to one may be used for maintaining the distance and another may be transferred across the membrane, producing an Hþ/substrate stoichiometry of 1. Finally, we mention the similarity between PfMATE and another structure of an Hþ-coupled MATE, namely DinF-BH (14,16). Interestingly, the structures of D40 and D184 and Y36 are quite similar to those of PfMATE (Fig. S19). The O-O distance between D41 and D184 is ˚ ) without the stabilizwithin a weak H-bond distance (3.76 A ing effect of crystal water. However, the aforementioned structural and functional study proposed a different proton transfer mechanism, wherein the Bent conformation is nonexistent and there is a one-step PT reaction involving direct competition between the Hþ and drug. However, as

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the authors pointed out the absence of Bent may be because DinF-BH was crystallized in the absence of monoolein (16). In our model, the Bent structure is realized when the 2nd PT takes place after intrusion of lipid into the cleft. Indeed, the formation of Bent with its hydrophobic surface is a reasonable structure to follow the outward-facing form to promote the occlusion form. Other experimental facts on DinF-BH also support our model—substitutions D184A and D184N abolish transport function and the O-O distance between D41 and D184 is close (14,16). They imply the importance of D184 and stable mediation of Hþ in between D41 and D184. Thus, our model with two PT reactions occurring in two steps, may also apply to DinF-BH. The eukaryotic MATEs, like human MATE, may be different as the Hþ binding site may be located in the C-lobe (16,20). However, definitive conclusions on the general applicability of our mechanism need crystal structures for human MATE and other Hþ-coupled MATE transporters and analyses such as performed here.

SUPPORTING MATERIAL Supporting Materials and Methods, nineteen figures, five tables, and two movies are available at S0006-3495(16)00136-3.

AUTHOR CONTRIBUTIONS W.N., R.I., O.N., and Y.S. designed research. W.N. and W.M. performed research and analyzed data. W.N., W.M., Y.T., R,I., O.N., and Y.S. discussed analyzed results. W.N., W.M., and Y.S. wrote the article.

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16. Radchenko, M., J. Symersky, ., M. Lu. 2015. Structural basis for the blockade of MATE multidrug efflux pumps. Nat. Commun. 6:7995.

We are grateful to Dr. Takaharu Mori for providing information on initial protein orientation. We thank Drs. Suyong Re and Kiyoshi Yagi for reading the manuscript carefully and giving us their valuable comments.

17. He, X., P. Szewczyk, ., G. Chang. 2010. Structure of a cation-bound multidrug and toxic compound extrusion transporter. Nature. 467:991–994.

This research was supported in part by the RIKEN iTHES project, and the Strategic Programs for Innovative Research Field 1 by Ministry of Education, Culture, Sports, Science, and Technology (MEXT) (to Y.S.), a Grantin-Aid for Scientific Research on Innovative Areas ‘‘Novel measurement techniques for visualizing ‘live’ protein molecules at work’’ (No. 26119006) (to Y.S.), a grant from Japan Science and Technology (JST) CREST on ‘‘Structural Life Science and Advanced Core Technologies for Innovative Life Science Research’’ (to Y.S.). We also thank the RIKEN Integrated Cluster of Clusters (RICC) and the HPCI system research project (project ID hp150060) for providing computational resources.

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Mechanisms for Two-Step Proton Transfer Reactions in the Outward-Facing Form of MATE Transporter.

Bacterial pathogens or cancer cells can acquire multidrug resistance, which causes serious clinical problems. In cells with multidrug resistance, vari...
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