ATP Hydrolysis Mechanism in a Maltose Transporter Explored by QM

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The ATP Hydrolysis Mechanism in a Maltose Transporter Explored by QM/MM Metadynamics Simulation Wei-Lin Hsu, Tadaomi Furuta, and Minoru Sakurai J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b07332 • Publication Date (Web): 07 Oct 2016 Downloaded from http://pubs.acs.org on October 8, 2016

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The Journal of Physical Chemistry

The ATP Hydrolysis Mechanism in a Maltose Transporter Explored by QM/MM Metadynamics Simulation

Wei-Lin Hsu, † Tadaomi Furuta, † and Minoru Sakurai*†

†Center for Biological Resources and Informatics, Tokyo Institute of Technology, 4259-B-62,

Nagatsuta-cho, Midori-ku, Yokohama, 226-8501, Japan

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ABSTRACT The translocation of substrates across the cell membrane by ABC transporters depends on the energy provided by ATP hydrolysis within the nucleotide-binding domains (NBDs). However, the detailed mechanism remains unclear. In this study, we focused on maltose transporter NBDs (MalK2) and performed a QM/MM well-tempered metadynamics simulation to address this issue. We explored the free energy profile along an assigned collective variable. As a result, it was determined that the activation free energy is approximately 10.5 kcal/mol, and the reaction released approximately 3.8 kcal/mol free energy, indicating the reaction of interest is a one-step exothermic reaction. The dissociation of the ATP γ-phosphate seems to be the rate-limiting step, which supports the so-called dissociative model. Moreover, the Glu159 located in the Walker B motif acts as a base to abstract the proton from the lytic water but is not the catalytic base, which corresponds to an atypical general base catalysis (GBC) model. We also observed two interesting proton transfers: the transfer from the His192 ε-position nitrogen to the dissociated inorganic phosphate, Pi and the other transfer from the Lys42 side chain to ADP β-phosphate. These proton transfers would stabilize the post-hydrolysis state. Our study provides a significant insight into the ATP hydrolysis mechanism in MalK2 from a dynamical viewpoint, and this insight would be applicable to other ABC transporters. 2


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INTRODUCTION ATP-binding cassette (ABC) transporters translocate substrates across the cell membrane, typically against a concentration gradient. This type of proteins can be classified into two subtypes based on the direction of substrate transport. The importers, presenting only in prokaryotes, deliver captured molecules from the extracellular side to the intracellular side. On the other hand, the exporters recruit their substrates directly from the cytoplasm or from the inner leaflet of the lipid bilayer and then expel them out of the cell.1–3 These transporters are constructed from some common structural subunits: the highly conserved two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs) that are more variable. The energy source of their functional motions is the free energy released from the hydrolysis of ATP molecules bound to the NBDs. There are many key issues that have been debated for a long time concerning the mechanism of ATP hydrolysis. The first of these is the idea of “associative” versus “dissociative”.4,5 In the associative model, the bond formation between the terminal phosphorus atom and the lytic water occurs prior to the cleavage of the terminal phosphoanhydride bond. Conversely, in the dissociative model, the breaking in ATP bond precedes the nucleophilic attack of the lytic water on the γ-phosphate. It has been thought for a long time that it is difficult to distinguish experimentally between these

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two models.6 However, recent fluorescence experiments by Herdendorf et al. successfully demonstrated that the ATP hydrolysis in T4 Rad 50 proceeds via a dissociative-like mechanism.7 Despite that, the experimental investigation for this issue is still limited. Instead, computational methods would provide another way to evaluate the mechanism quantitatively or at least semi-quantitatively at atomic level. In addition, they would allow us to consider that the above mechanisms represent only the two limiting cases, and there are various levels of concerted reactions, in which bond formation and breaking occur simultaneously with a single transition state.4,8 The second issue concerns the identity of the final acceptor of the lytic water proton. In aqueous solution, the ATP γ-phosphate group itself acts as a catalytic base to abstract the proton from the lytic water; thus we refer to this mechanism as the substrate-assisted catalysis (SAC) model.9–12 On the other hand, ATP hydrolysis seems to be catalyzed by amino acids with a carboxylate group (Glu, Asp) in many enzymes, such as the F1-ATPase and kinesin. In some circumstances, the catalytic base can also be histidine (His), e.g. in hemolysin B (HlyB). In either case, the mechanism by which the amino acid surrounding the ATP γ-phosphate acts as a catalytic base is the called general base catalysis (GBC) model.12–17 The last key issue would be how many water molecules are needed for the ATP

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hydrolysis reaction, and this question is usually referred to as the one water (1W) model versus two water (2W) model debate. This debate was recently summarized by Prasad et al., although they did not conclude with a final answer.18 In some cases, e.g. kinesin, multiple water molecules seem to be obligatorily involved for the ATP hydrolysis reaction.16 Our previous study performed molecular dynamics simulations on MalK2, the subunits that mainly contain the nucleotide-binding domains (NBDs) of the maltose transporter from E. coli. During the MD simulations of MalK2, we also observed that some water molecules remained at the closed ATP binding pockets (ABPs). Those water molecules remaining at the ABPs formed several hydrogen bonds with ATP and its neighboring residues, and their arrangements were in good agreement with the possible pre-hydrolysis position stated in many other previous studies.19,20 Therefore, we believe that MalK2 would be an appropriate target to elucidate the catalytic mechanism of enzyme-assisted ATP hydrolysis. Recently, Huang et al. used the QM/MM nudged elastic band (NEB) method to investigate the ATP hydrolysis reaction in MalK2.14 In their study, the potential of mean force (PMF) along the reaction pathway was obtained with an activation free energy of 19.2 kcal/mol in agreement with experiments. According to their results, the reaction

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can be summarized as a dissociative and one water (1W) model. Moreover, the glutamate residue in the Walker B motif acted as a general base to abstract the proton from the lytic water; however, the rate-limiting step was the bond cleavage between the γ- and β-phosphates rather than the deprotonation of the lytic water. As a result, their model followed an atypical GBC pathway, and thus for the first time well explained the experimental results for the deuterium kinetic isotope effect (KIE).12 Currently, it is of great importance to provide theoretical support to this new model using other types of free energy calculations. In this study, we used the QM/MM metadynamics method to investigate ATP hydrolysis in MalK2. This enhanced sampling method enables us to directly observe the bond cleavage and formation, even with a high activation energy barrier,21,22 and, moreover, to rebuild the free energy surface along a given reaction coordinates. The present metadynamics results revealed that one water molecule is enough to trigger the ATP hydrolysis reaction, and that this reaction follows a more dissociative-like model in MalK2. In addition, several interesting proton transfers were also observed. Our results strongly support the model that Huang et al. have proposed,14 and the details will be discussed in the following sections.

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METHODS Structure Preparation for the QM/MM Calculation.

First, the closed structure of

MalK2 was truncated from that of the structure of the outward-facing state of the full transporter (PDB ID: 3RLF).23 The truncated structure contained 738 residues in total from Ala2 to Gly370 for each MalK monomer, along with AMP-PNP, an Mg2+ ion, and crystal water. Each AMP-PNP molecule was replaced by an ATP molecule. The closed ATP-Mg2+ bound MalK2 structure obtained was exactly the same as the one we had used in our previous work.24 Next, we predicted the possible positions of water molecules using the three-dimensional reference interaction site model (3D-RISM) and its related placevent method.25 Before running the placevent, we first performed the 1D-RISM calculation to obtain the 1D susceptibility function using the chosen solvent mixture. The concentrations of water, Na+, and the Cl- ions were set to 55.5 M, 0.005 M, and 0.005 M, respectively. The temperature was set to 310 K. After acquiring the susceptibility function for the solvent mixture, we ran the 3D-RISM with the default parameters. Finally, we performed the placevent calculation to predict the positions of water molecules for the closed MalK2 structure. Notably, crystal water in the closed MalK2 structure was deleted first to perform the placevent calculation. The predicted water

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positions were then introduced into our MalK2 structure file. However, we deleted those predicted water molecules if they were located within 3 Å of the crystal water. As a result, one water molecule was confirmed to be located at one ATP binding pocket (ABP), hereafter denoted as ABP1 and is illustrated in Figure S1 of the supporting information. This structure was then solvated with TIP3P water using the AMBER 14 program26,27 and the resultant structure was used for the following QM/MM study. QM/MM Modeling.

We first defined the QM region at the ABP1 site (shown in

Figure 1-(a)), in which Mg2+-ATP and the amino acid residues responsible for the ATP hydrolysis were included; to be more specific, Gln82 in the Q-loop, Glu159 in the Walker B motif, His192 in the H-loop on MalK_A, and Asn163 in the D-loop of MalK_B were included. Additionally, all of the residues within 5 Å of the Mg2+ ion and the ATP γ-phosphate, except for glycine and proline, were also included. The corresponding residues were Ser38, Lys42, and Ser43 in the Walker A motif; Asp158 in the Walker B motif of MalK_A; and Ser135 and Gln138 in the signature motif of the MalK_B subunit. The predicted water molecule that remained at the ABP1 site, as well as the two water molecules that coordinated with the Mg2+ ion, were also included in the QM region. As a result, our QM region included ATP, an Mg2+ ion with two coordinating water molecules, one lytic water, and the ten aforementioned amino acid

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residues of Ser38, Lys42, Ser43, Gln82, Asp158, Glu159, and His192 on one side of MalK2 (MalK_A) and Ser135, Gln138, and Asn163 on the other side of MalK2 (MalK_B). The other part of the system was defined as the MM region, which was described with the ff99SBildn force field.28 Notably, we used the Integrated Molecular Orbital Molecular Mechanics (IMOMM) method to define the interface: namely, the QM region only contained the side chain of each residue, and their main chains were taken as the interface of the QM and MM region.29 After the determination of the QM and MM regions, we performed the geometry optimization of the whole system through the QM/MM interface in AMBER 14, where the QM region was optimized by using the Gaussian 09 revision D.01,30 and the MM region was optimized using the Sander module of AMBER 14. The optimization calculation for the QM region was performed at the B3LYP/6-31G(d, p) level.31 The long-range interaction in the MM region was calculated using the smooth particle mesh Ewald (SPME) method.32 The cutoff of the non-bonding interaction radius was set to 12 Å. Finally, the Limited-memory Broyden-Fletcher-Goldfarb-Shanno quasi-Newton algorithm (LBFGS)33 was used for the energy minimization calculation, and then the energy minimization convergence criterion was set to 1 × 10 kcal/(mol Å). QM/MM Metadynamics.

The QM/MM metadynamics calculations were carried

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out using the CP2K 2.6.2 program.34 The QUICKSTEP routines was used for computing the energy based on the Kohn-Sham density functional theory (DFT).35 Becke-Lee-Yang-Parr (BLYP)36–38 was chosen for the exchange-correlation functional and the dispersion correlation39 was also included. The plane-wave cutoff for the QM region was set to 300 Ry, and we used the wavelet poison solver to calculate the electrostatic potential of the QM region.40,41 The force field of MM region was the same as that stated in the previous section, i.e., ff99SBildn. We used the following distance constraints to maintain the geometries of all of the the water molecules except for those in the QM region: O-H and H-H distances were fixed to 0.957 Å and 1.513 Å, respectively. The electrostatic potential was calculated by the SPME method, which is also consistent with the previous section. The electrostatic interaction correlation from the MM region to the QM region was calculated by the Gaussian Expansion of the QM/MM Electrostatic Potential (GEEP) method42, and the electrostatic potential of the MM region was set to 15 Gaussian functions. The temperature was set to 310 K, and the Canonical Sampling through Velocity Rescaling (CSVR) method43 was used to maintain the temperature, and the time step was 0.5 fs. After the 5 ps equilibration, the velocity rescaling was changed to use the Nosé-Hoover method.44,45 Finally the QM/MM metadynamics simulation was performed to investigate the ATP hydrolysis mechanism

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in MalK2. In this study, we adopted well-tempered metadynamics,46,47 where the distance of O3Β-Owater minus Pγ-Owater (dist1 – dist2 in Figure 1-(b)) was chosen as the only collective variable (CV). A parameter with the dimension of a temperature, ∆, was introduced in this well-tempered metadynamics, and was set to 3000 K. The Gaussian height was set to 0.003 hartree, and the width was set to be 10% of the assigned CV. The repulsive Gaussian potential was placed every 20 fs. The metadynamics calculation was terminated soon after the reversed hydrolysis reaction began to be observed. The details of the well-tempered metadynamics are summarized in the supporting information. Finally, we reweighted the metadynamics trajectory to recover the unbiased distribution by the method proposed by Bonomi et al.48 Potential Energy Surface (PES) and Charge Distribution Calculations.

To obtain

the potential energy surface (PES) along the proton transfer pathways predicted from the metadynamics simulation, a series of single point energy (SPE) calculations were carried out. In the case of proton transfer from His192, all of the atoms were fixed except for the His192 HE2 atom and then the distance between the His192 HE2 and ATP O3B (from 0.84 Å to 2.12 Å, 17 points) was taken to be the reaction coordinate. As for the proton transfer from Lys42, all of the atoms other than the Lys42 HZ1 atom were

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fixed, and the distance between Lys42 HZ1 and ATP O1B (from 092 Å to 1.88 Å, 13 points) was taken as the reaction coordinate. The definition of atom names used in this article are illustrated in Figure S2 of the supporting information. In addition, from the metadynamics trajectory obtained, we picked up three structures, including the reactant state (RS), the transition state (TS), and the product state (PS). For each of these states, the Hirshfeld charge distribution calculations were carried out to understand the quantitative description of the molecular charge distribution.49 Both the PES and charge distribution calculations were applied to the same QM/MM system (Fig. 1) as described in the previous sections. Then, for better accuracy, Becke, 3-parameter, Lee-Yang-Parr (B3LYP)31 was chosen for the exchange-correlation functional instead of BLYP hybrid functional. All of the calculations were also performed using the CP2K 2.6.2 program.

RESULTS The potential of mean force (PMF) obtained by reweighting the metadynamics trajectory is shown in Figure 2. It indicates a single energy barrier between the two minima. The calculated activation energy (∆ ) is approximately 10.5 kcal/mol, which is lower than the experimental value, 16.3~18.3 kcal/mol.14,50,51 The possible origins of

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such numerical errors will be discussed in the next section. Despite the errors, the estimated activation energy in the protein is clearly lower than the experimental value (27.9 kcal/mol) for the hydrolysis of free ATP or GTP in water.52,53 Therefore, the present simulation reproduces well the fact that the ATP hydrolysis reaction is catalyzed by MalK2. The calculated reaction free energy (∆ ) is around -3.8 kcal/mol, which is smaller than the hydrolysis energy (-7.3 kcal/mol) of free ATP in water. This difference for ∆ may be due to the fact that in the present simulation, the Pi release to the bulk water was not taken into account. With the time evolution of CV (shown in Figure S3 in supporting information), the ATP bond breaking occurred at approximately 12 ps after the production run had been started, and the backward reaction from ADP-Pi to ATP was observed at approximately 65 ps. Therefore we terminated the production run at 67 ps. The reaction process animations can be viewed in the attached files, si_001.avi and si_002.avi. The time evolution of the several atom-atom distances responsible for the ATP hydrolysis was plotted in Figure S4 of the supporting information. At approximately 12 ps, the significant cross point between the distances Pγ-O3B and Pγ-Owater was observed (Figure S3-(a)), indicating the formation of the TS, and was consistent with the animation. The

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other cross point was observed at approximately 65 ps, which indicated the passing of the TS in the backward reaction. As a result, the initial 12 ps period can be considered as the RS sampling period, while the subsequent 12 ps~65 ps period can be considered as the PS sampling period. The other distance data illustrated in Figure S3 will be discussed in detail in the following sections. The RS, TS, and PS structures from our metadynamics simulation are shown in Figure 3-(a), -(b), and -(c), respectively. Our results indicated that Pγ-O3B bond breakage occurred prior to the formation of the bond Pγ-Owater, which supports the dissociative model over the associative model.14 To confirm this, we superimposed the ABP1 site of our TS structure with the 3PUV crystal structure23 that was ligated with ADP-VO4, an ATP analog that can mimic the transition state of ATP hydrolysis (Figure 4). The RMSD value between the two structures is only 0.45 Å with respect to the Cα atoms, which proved that our TS is quite similar to the crystal structure, indicating that the reaction pathway obtained here is reliable. Despite their similarity, the 3PUV structure suggests that the V-O1 and V-O2 bonds are covalent bonds and that their distances are 1.98 Å and 2.14 Å, respectively. This ATP-analog formed a trigonal bipyramid-shaped structure; as a result, Oldham et al. proposed an associative ATP hydrolysis reaction in MalK2.23 On the contrary, the captured TS structure in this study

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shows longer distances for O3B-Pγ and for Pγ-Owater, which are 2.29 Å and 2.50 Å, respectively, indicating that they are non-covalent bonds. As a result, a triangle-shaped structure is observed in the TS; thus, this reaction is more likely to follow a dissociative pathway. In addition to bond breakage and formation, we also observed a change in the side chain orientation of Ser135. From the RS to the TS, this side chain pointed toward the O1G atom of the ATP γ-phosphate, forming a stable hydrogen bond (Figure 3-(a) and -(b)); however, as the PS was reached, the orientation of this side chain changed to form a hydrogen bond with the O3B atom (Figure 3-(c)). Several proton transfers were also observed as the PS was reached. The proton removed from the lytic water was transferred to Glu159 in the Walker B motif, and this residue is considered as the best candidate to receive such a proton according to the general base catalysis (GBC) model. Moreover, we further observed two other interesting proton transfers at the PS. The first one pertains to the proton of the His192 ε nitrogen (HE2), which transferred to the O3G of the γ-phosphate. This proton transfer has also been reported by Huang et al.14 and is consistent with several previous experimental studies, which suggested that the mutation of His192 would lead to a lower ATPase activity.54 The other one is the proton transfer from the Lys42 side chain

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(HZ1) in the Walker A motif to the O1B atom of the ATP. Interestingly, the HZ1 was not firmly fixed at one place, but went back and forth between the ATP and Lys42 (attached file si_003.avi). This phenomenon was unable to be seen during the RS sampling period.

DISCUSSION The Reliability of Metadynamcs Simulation.

The calculated PMF (Figure 2)

from our metadynamics simulation indicated that the ATP hydrolysis in MalK2 is a one-step exothermic reaction, where there is only one energy barrier between the RS and the PS. The calculated activation energy was approximately 10.5 kcal/mol. As described in the previous section, this is lower than the corresponding experimental values 16.3~18.3 kcal/mol.14,50,51 To explore the origin of this error, we searched similar computational studies on ATP hydrolysis using almost the same QM/MM metadynamics methods with the BLYP functional. Glaves et al. investigated the hydrolysis reaction of triphosphate in aqueous solution.55 Their estimation of the activation energy (29 kcal/mol) showed a good agreement with the experimental value (27.9 kcal/mol).52,53 This result is better than those (32.5~35.1 kcal/mol) from other QM/MM MD simulations10,18,56,57 instead of using the metadynamics method. Therefore, it can safely

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be said that the QM/MM metadynamics method itself is useful to address the issue of ATP hydrolysis. Unfortunately, when the method is applied to the ATP hydrolysis in protein, the activation barrier tends to be underestimated. As similar to the present study, a study of the ATP hydrolysis in kinesin by McGrath et al. also provided a lowered activation energy value (11 kcal/mol) compared with the corresponding experimental value (17.5 kcal/mol).16 They stated that it would be necessary to use more general collective variables, more exact density functionals, longer simulation time, and multiple runs for the same CV starting from different configurations to converge the free energy estimations. However, such sophisticated calculation are cost-prohibitive and beyond the current computational capacities. In addition to the above factors, it is known that the free energy surface obtained from a metadynamics simulation depends on the timing to terminate the simulation. There are mainly two criteria for metadynamics convergence. If one is interested in reconstructing the free energy surface of a given reaction, the simulation should be stopped when the motion of the given CVs become diffusive in the region of interest.47 In fact, we were not able to continue the simulation until such a condition was satisfied due to the extremely large size of the system. Instead, we attempted to use another

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criterion: namely, we stopped the production run soon after the reversed hydrolysis reaction began to occur. As a result, the present simulation gave the reasonable PMF for the ATP hydrolysis (Figure 2), although the activation barrier was underestimated. However, if one has special attention to find the closest saddle point, one should stop the simulation as soon as the system exits from the minimum.47 Based on this criterion, we reconstructed the PMF of 13 ps production run, and the result is shown in Figure S5 in supporting information. As a result, we could obtain two possible saddle points, and thus the activation energy was estimated to be 14.8~15.3 kcal/mol, which is much more consistent with the experimental value, 16.3~18.3 kcal/mol.14,50,51 Taken together, the numerical results of QM/MM metadyamics depend on various factors, and thus the validation of this methodology is not fully discussed in a single paper. Despite the quantitative discrepancy of the activation energy value, the hydrolysis pathway observed from the present metadynamics simulation enables one to characterize clearly mechanistic features of the reaction as described below. The Non-associative Model Is the Favored Mechanism for the ATP Hydrolysis in MalK2.

The TS structure obtained in this study showed a good agreement with the

crystal structure (3PUV) of the ADP-VO4–bound protein. Based on the 3PUV structure, Oldham et al. mentioned that the ADP-VO4 formed a trigonal bipyramid-shaped TS

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structure; thus the ATP hydrolysis reaction in MalK2 may go through an associative model.23 On the contrary, our simulation suggested that the bond formation of Pγ-Owater occurs after the bond breaking of Pγ-O3B and that the TS structure is thus more like a triangle-shaped structure. In fact, several recent theoretical studies have also suggested that the ATP hydrolysis likely proceeds in accordance with a non-associative model regardless of whether it occurs in pure water or in protein.10,14,16 Glu159 Acts as a General Base Instead of a Catalytic Base to Abstract the Proton from the Lytic Water. Our results indeed support the results reported by Huang et al., who stated that the ATP hydrolysis in MalK2 does not follow a normal GBC model. The value of the OE2-Hwater distance (Figure S3-(a), red line) indicated that the proton (Hwater) of the lytic water sometimes transferred to the Glu159 OE2 atom and then went back to the lytic water during the RS sampling period. However, after the reaction went across the TS, Glu159 received the lytic water hydrogen almost at the same time as the Pγ-Owater bond formed. This result indicates that the rate-limiting step should be the Pγ-O3B bond breakage instead of the abstraction of hydrogen from the lytic water to Glu159. This result was very consistent with the experimental results of deuterium kinetic isotope effects (KIE), which indicated that the kinetics of the ATP hydrolysis in ABC proteins remain unchanged regardless of whether it occurs in H2O or D2O.12

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Proton Transfer from His192 to the γ-Phosphate.

The doubly protonated His192

in the H-loop motif is believed to recognize as well as to fix the ATP γ-phosphate.2,58 Several studies also suggested that this highly conserved histidine somehow plays an important role in the ATP hydrolysis reaction, because the mutation from histidine to alanine would make the ATPase activity drop dramatically.11 Zhou et al. even noted that in HlyB, the H-loop histidine is the general base that abstracts the lytic proton rather than the glutamate in the Walker B motif.12 According to our results, Glu159 is the general base. However, it was also found that during the RS sampling period, His192 formed a stable hydrogen bond with the γ-phosphate and sometimes the HE2 would transfer to the ATP O3G atom (Figure S3-(b)). This hydrogen bond might be able to stabilize the transition state and thus lower the activation energy. After the formation of ADP and Pi, the proton (HE2) of His192 would transfer to the Pi O3G atom instead of staying at its original place. During the PS sampling period, the HE2 sometimes went back to the His192, but mostly, this proton preferred to stay at the γ-phosphate. For a better understanding of the H192 proton transfer, we obtained the PES of each state as described in the method section, and the results are shown in Figure 5. We found out that before the TS, the structure was more stable when the HE2 stayed at the His192; however after the formation of ADP and Pi, the proton would prefer to stay at the Pi.

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Accordingly, this proton transfer must be important for stabilizing the PS. Proton Transfer from Lys42 to the β -Phosphate.

In addition to the proton

transfers as mentioned above, there was one more interesting proton transfer that we found; that is the transfer of the side chain HZ1 of Lys42 in the Walker A motif to the ADP O1B during the PS sampling period. This observation was fascinating because the lysine side chain usually has an extremely high pKa value, approximately 10.53. Under most physiological circumstances, the deprotonaion of a lysine side chain seems to be unfavorable. However, during the PS sampling period, we observed the phenomenon that the HZ1 went back and forth between the ADP O1B and the Lys42 side chain. Therefore, we performed pKa calculations by use of PROPKA59,60 on the PDB2PQR server (http://nbcr-222.ucsd.edu/pdb2pqr_2.0.0/).61,62 Interestingly, the pKa value of the Lys42 side chain in the PS was evaluated to be 5.57, which strongly supports the observation of Lys42 side chain deprotonation. This lysine residue is considered important for the capture of the ATP, because its mutation drastically lowers the nucleotide binding capability.54,63 However, previous studies suggested that the mutation from lysine to methionine in the Walker A motif of the ABCG2 transporter does not affect the ATP binding capacity but eliminates the binding ability of ADP-VO4.64,65 Until now the real function of this highly conserved lysine in the Walker A motif has

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remained elusive. From the RS structure (Figure 3-(a) left panels), we can see that the Lys42 side chain is located between the γ-phosphate O3G atom and the β-phosphate O1B atom, forming two stable hydrogen bonds, O3G…HZ2 and O1B…HZ1 at a distance 1.56 Å and 1.91 Å, respectively. On the other hand, in the PS (Figure 3-(c) left panels), although the hydrogen bond between O3G…HZ2 continued to exist, the distance increased to 1.85 Å, while the O1B was protonated, and the O1B…HZ1 distance decreased to 1.06 Å. Moreover, according to the Hirshfeld charge distribution for each state shown in Figure 3 (right panels), the His192 proton transfer that we mentioned in the previous section caused a decrease of the negative charge on O3G. Both the distance changes and the protonation of O3G affected the electrostatic interaction balance: the attraction with the O3G negative charge was weakened, while that with the O1B charge was enhanced as soon as the O3B-Pγ bond broke. In Figure 3, the right panel, the charge distribution of O1B changed from -0.837 (RS) to -0.929 (TS), and then decreased to -0.750 (PS). As a result, the Lys42 side chain was easily deprotonated and the HZ1 was able to go back and forth between the O1B and the Lys42 side chain. We believe that this proton transfer is able to decrease the negative charge distribution over the β-phosphate, and stabilize the product state. In fact, we also calculated the Hirshfeld charge distribution

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for the structure in which the Lys42 HZ1 did not transfer its proton to the ADP O1B atom (Figure S6 in supporting information). We found that the total negative charge distributions of the O1B, O2B, and O3B atoms were indeed higher when the HZ1 atom was not transferred. In addition to this Lys42 proton transfer, the side chain of Ser135 underwent a large conformational change on going from the TS to the PS as shown in Figure 3-(b) and -(c) might also contribute to the electrostatic stabilization of the PS by pointing its hydroxyl group toward the β-phosphate in the PS. For a better understanding of the above Lys42 proton transfer, we also obtained the PES for each state, and the results are shown in Figure 6. In the RS, the HZ1 was obviously stable when it stayed at the Lys42 side chain. In the TS, the energy barrier decreased significantly, but the proton was still more stable remaining at the Lys42 side chain than at the ADP. However, after the ATP hydrolysis was completed, the energy barrier was lowered to a mere 2~3 kcal/mol, making it possible for the proton to go back and forth between the two sides. As a result, it is likely that the proton transfer from the Lys42 side chain would be able to decrease the β-phosphate negative charge distribution, stabilize the post-hydrolyzed state, and make the Pi more difficult to go back to form the Pγ-O3B bond. This observation may also be able to explain why the mutation from lysine to

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methionine in the Walker A motif of ABCG2 transporter would eliminate the ability to form the vanadate-induced transition state intermediate, because the methionine is not able to share a proton to alleviate the electrostatic interaction imbalance caused by the elongated V-O bonds.

CONCLUSION Our present theoretical study successfully provided a detailed picture for the ATP hydrolysis mechanism in MalK2 that is consistent with the previous study.14 According to the RISM calculations, one water molecule was observed to remain at the closed ABP site, forming several hydrogen bonds with its surrounding residues, including Asn163 on the D-loop and Glu159 in the Walker B motif. The oxygen atom of the water molecule directly pointed to the γ-phosphate, and thus was arranged on the straight line connecting O3B and Pγ. According to our metadynamics results, this water molecule was confirmed to act as the lytic water, and was the only water molecule that was able to trigger the ATP hydrolysis reaction, which supports the one water (1W) model. A triangle-shaped structure was captured at the transition state, where the Pγ-O3B bond broke prior to the deprotonation of the lytic water concomitant with the Pγ-Owater bond formation. Therefore, the ATP hydrolysis in MalK2 would follow a dissociative model,

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or at least a non-associative model. Moreover, the deprotonation of the lytic water occurred as soon as the Pγ-Owater bond formed, and the proton was abstracted by Glu159 in the Walker B motif. Although the Glu159 is the best candidate for the general base in the GBC model, the rate-determining step is more likely to be the Pγ-O3B bond break instead of the deprotonation of the lytic water. After the ADP Pi has formed, two interesting proton transfers were observed, the His192 HE2 to the O3G of Pi and the Lys42 HZ1 to the O1B of ADP. The His192 proton transfer has already been observed in a previous study and was thought to stabilize the production state.14 The Lys42 proton transfer, together with the Ser135 side chain reorientation, would also be able to electrostatically stabilize the final production state. We found the Lys42 proton transfer to be both surprisingly and highly significant because this observation may be critical to explain why a lysine residue is highly conserved in most of the Walker A motifs. The lysine may be able to not only capture the ATP but also to stabilize the post-hydrolysis state and thus make it more difficult for the Pi to regenerate the Pγ-O3B bond. Our results provided an atomic-level insight into the ATP hydrolysis mechanism in MalK2, and this insight is helpful for understanding the ATP hydrolysis process in other ABC transporters.

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ASSOCIATED CONTENTS Supporting Information. The detailed well-tempered metadynamics calculations and the reweighting method; the figure of closed MalK2 structure and lytic water position; the atom names used in this article; the time evolution of CV; the time evolution of several distances in ATP hydrolysis; the Hirshfeld charge distributions in the PS without Lys42 proton transfer. Animations of the reaction process (si_001.avi, si_002.avi, and si_003.avi). These materials are available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected]; Phone: +81-45-924-5795

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS This work was supported by JSPS KAKENHI JP16H00825 and JP15K00400.

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Figure Captions Figure 1. QM region and CV defined in the QM/MM metadynamics simulation. (a) The QM region includes ATP, a Mg2+ ion with two coordination water, lytic water, and ten residues consisting of Ser38, Lys42, Ser43, Gln82, Asp158, Glu159, His192 on one side of MalK2 and Ser135, Gln138, Asn163 on the other side of MalK2. (b) The CV is defined as the distance of O3B-Owater (dist1) minus Pγ-Owater (dist2), where dist1 and dist2 are indicated by the blue and green arrows, respectively. Figure 2. PMF along the CV for the ATP hydrolysis in MalK2. The estimated activation energy (∆ ) is approximately 10.5 kcal/mol, and the estimated reaction free energy (∆ ) is about -3.8 kcal/mol, which indicates that the ATP hydrolysis reaction is an exothermic reaction. Figure 3. Structures of the RS, TS, and PS. (a) The structure of the reaction state (RS) is illustrated in a ball and stick model. The left panel labels all of the relevant atom-atom distances described in the text, and the left-top figure show the distances between the Mg2+ ion, the coordination oxygen atoms of ATP and the two coordinating water molecules. The right panel labels the Hirshfeld charges on the O1B, O2B, O3B, O3G, and Pγ atoms. The structures of the TS and PS are illustrated in (b) and (c), respectively, in the same manner as (a).

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Figure 4. Superimposed QM region of the TS structure and the 3PUV crystal structure.23 Both of the structures are illustrated in stick models, and the TS structure is colored only in yellow. The Cα RMSD value is 0.45 Å. The distances of O3B-Pγ, Pγ-Owater, V-O1, and V-O2 are 2.29 Å, 2.50 Å, 1.98 Å, and 2.14 Å, respectively. Figure 5. PES for the His192 proton transfer. (a), (b) and (c) correspond to the results for the RS, TS, and PS states. The reaction coordinate (x-axis) was taken to be the distance between His192 HE2 and ATP O3G and is illustrated in (d). Therefore, the left-hand side of the x-axis can be considered as the ATP side, while the right-hand side is specified as the His192 side. Figure 6. Potential energy surface (PES) for the Lys42 proton transfer. (a), (b) and (c) correspond to the results for the RS, TS, and PS states. The reaction coordinate (x-axis) was taken to be the distance between Lys42 HZ1 and ATP O1B and is illustrated in (d). Therefore, the left-hand side of the x-axis can be considered as the ATP side, while the right-hand side is specified as the Lys42 side.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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TOC graphic

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ATP Hydrolysis Mechanism in a Maltose Transporter Explored by QM

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