Catalytic Mechanism of the Maltose Transporter Hydrolyzing ATP

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Catalytic Mechanism of the Maltose Transporter Hydrolyzing ATP Wenting Huang, and Jie-Lou Liao Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.5b00970 • Publication Date (Web): 15 Dec 2015 Downloaded from http://pubs.acs.org on December 19, 2015

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Biochemistry

Catalytic Mechanism of the Maltose Transporter Hydrolyzing ATP Wenting Huang and Jie-Lou Liao* Department of Chemical Physics, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui Province, People’s Republic of China, 230026

1 Environment ACS Paragon Plus

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ABSTRACT: We use quantum mechanics/molecular mechanics (QM/MM) simulations to study ATP hydrolysis catalyzed by the maltose transporter. This protein is a prototypical member of a large family which consists of adenosine triphosphate (ATP) binding cassette (ABC) transporters. The ABC proteins catalyze ATP hydrolysis to perform a variety of biological functions. Despite extensive research efforts, the precise molecular mechanism of ATP hydrolysis catalyzed by the ABC enzymes still remains elusive. In this work, the reaction pathway for ATP hydrolysis in the maltose transporter is evaluated using a QM/MM implementation of the nudged elastic band method without presuming reaction coordinates. The potential of mean force along the reaction pathway is obtained with an activation free energy of 19.2kcal/mol in agreement with experiments. The results demonstrate that the reaction proceeds via a dissociative-like pathway with a trigonal bipyramidal transition state where the cleavage of the γ-phosphate P-O bond occurs and the O-H bond of the lytic water molecule is not yet broken. Our calculations clearly show that the Walker B glutamate as well as the switch histidine stabilizes the transition state via electrostatic interactions rather than serves as a catalytic base. The results are consistent with biochemical and structural experiments, providing novel insight into the molecular mechanism of ATP hydrolysis in the ABC proteins.


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The ATP-binding cassette (ABC) transporter family contains more than 2,000 members1 that utilize the energy from ATP binding and hydrolysis to perform important biological functions including translocation of various substrates across cell membranes, translation of RNA and DNA repair.2-3 The ABC proteins also catalyze ATP hydrolysis, playing a key role in many biological processes. Some ABC proteins are implicated in diseases including juvenile diabetes,4 cystic fibrosis,5 and drug resistance in cancer.6 Because of their biological importance, substantial structural and biochemical data have been achieved for understanding the ABC transporters. However, the precise molecular mechanism of ATP hydrolysis in the ABC enzymes still remains elusive. A common basic architecture of the ABC transporters comprises two highly variable transmembrane domains (TMDs) that form a substrate translocation pathway and two nucleotide binding domains (NBDs) that bind and hydrolyze ATP. The ABC transporters alternate between inward- and outward-facing conformations during a transport/catalytic cycle for substrate translocation as a delicate molecular machine.7-11In the outward-facing conformation, the two NBDs form a closed head-to-tail dimer that can tightly bind and hydrolyze ATP at their interface. The NBDs feature several highly conserved structural motifs. These motifs include the Walker A- and B- motif, the Q-loop, the C- or signature motif, the H-loop or switch region, and the D-loop, contributing residues to form the enzymatic active site in which ATP hydrolysis is catalyzed. The highly conserved nature of the active site in sequence and structure implies that the hydrolysis of ATP shares a common mechanism in the ABC protein family.12-13 There have been two major hypothetical mechanisms regarding the catalytic role of the ABC proteins. One is the general base catalysis (GBC). Based on biochemical and structural studies, the conserved glutamate (E) residue, which is C-terminal to the Walker B motif, was proposed to


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act as a catalytic base.12,14-16In the GBC, the catalytic base abstracts a proton from the lytic water prior to or at the transition state (TS), which is the rate-limiting step.17But some other experimental data contradicted this role of the residue as the catalytic base.17-19 An alternative mechanism, the substrate-assisted catalysis (SAC), was thus proposed, suggesting that the γphosphate group of ATP itself acts as the catalytic base for cleavage of the lytic water molecule before the TS is formed.17 The SAC was proposed based on mutation of the switch histidine to alanine (H662A) in the isolated NBDs of the ABC transporter, Haemolysin B (HlyB). In the SAC, the switch histidine functions together with the Walker B glutamate to stabilize the TS rather than to act as the catalytic base. However, the X-ray structure of the H662A HylB mutant (PDB: 1XEF)17shows that the conserved glutamine residue (Q550) in the Q-loop is displaced away from Mg2+ likely due to the absence of the TMDs, compared to the wild-type full-length maltose transporter (PDB: 3PUY),20 in which Mg2+ is coordinated by the corresponding Q-loop glutamine, Q82, in the 3PUY structure. Furthermore, the side chain of the Walker B glutamate, whose counterpart in the maltose transporter is E159,20 in the H622A HylB mutant is hydrogen bonded (H-bonded) to the substituted A662 rather than pointing toward the catalytic water molecule. As argued by Oldham and Chen,20 the loss of the ATPase activity in the H662A mutant can be explained by the configurational rearrangements of these key residues in the mutant. The SAC seems to be unlikely on basis of experimental and theoretical studies of the different systems.21-23 In a recent QM/MM study based on the above H662A mutant structure, the switch histidine was proposed as the catalytic base to deprotonate the lytic water molecule.24The resulting associative pentacovalent TS structure is the common feature of the reaction in the GBC and the SAC.17 However, the calculations cannot satisfactorily explain the deuterium kinetic isotope effect (KIE) reported for HlyB-NBDs, as also pointed out by the authors.24 In


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addition, the pH-rate profiles for WT and H536Q Rad 50 were obtained by fitting the data to a bell-shaped curve characterized with two pKa values.21 The results show that both WT (7.2 ± 0.1 and 9.1 ± 0.1) and H536Q (7.0 ± 0.1 and 9.4 ± 0.1) have similar pKa values, arguing against H536 acting as a catalytic base.21 The currently dominant view based on high-resolution full-length X-ray studies,12,20, 25-26 is that the Walker B glutamate is the catalytic base as mentioned above. In this GBC, the associative pentacovalent structure is formed at the TS where the proton is abstracted from the lytic water molecule by the Walker B glutamate and the proton abstraction is the rate-limiting step in the reaction. As ATP hydrolysis is the slowest step in the catalytic cycle demonstrated in biochemical experiments,21 the reaction rate should be considerably faster (~6-10 times) in H2O than in D2O in the GBC due to the primary deuterium KIE in which the isotopically labeled atom (H/D) is directly involved in the bond breaking.27-29 However, recent kinetic experiments have showed that the ABC ATPase activities remain essentially unchanged in D2O and H2O, suggesting that there is no catalytic base in the ABC enzymes.21 The GBC, which is inferred from the high-resolution X-ray studies,12,20,25-26 is thus contradictory to the experimental deuterium KIE data, highlighting the urgent need for further research into the detailed molecular mechanism of ATP hydrolysis catalyzed by the ABC proteins. In particular, the reaction path, the TS structure and the exact catalytic roles of the Walker B glutamate and the switch histidine in the active site are currently unclear. In this respect, a hybrid quantum mechanical and molecular mechanical (QM/MM) approach provides currently a most suitable way to resolve the fundamental mechanistic controversies in the reaction.30 To address the above issues in this work, we report a QM/MM study of ATP hydrolysis in the maltose transporter. This protein has served as the prototype to elucidate the fundamental


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mechanism of ABC transporters for several decades.20,31 A QM/MM nudged elastic band (NEB) method32-33 is used for calculating the reaction pathway in the present study. The NEB method requires the reactant state (RS) and product state (PS) structures as two endpoints for the input without predefining reaction coordinates. An NEB path is then constructed to connect the RS and PS endpoints with a number of discrete structures (images). The QM/MM NEB method used here has been successfully extended to study large complex biological systems 32,34-35as well as ATP hydrolysis in aqueous solution.30 In the present work, a reliable reaction pathway is obtained through extensive unbiased QM/MM optimizations of the NEB path. Our calculations show that ATP hydrolysis proceeds through a concerted (dissociative-like) transition state similar to that in solution.30 Our results are in good agreement with available experimental data for the reaction in ABC proteins as discussed below. COMPUTATIONAL DETAILS QM/MM modeling and structural optimizations. The maltose transporter consists of a periplasmic maltose-binding protein (MBP), two integral transmembrane proteins, MalF and MalG (TMDs), and a cytoplasmic nucleotide-binding homodimer, MalK (NBDs) (Supporting Information, SI; Figure S1). Several high-resolution X-ray structures have been determined for the full-length wild-type maltose transporter including complexes with adenosine 5’-(β,γ-imido) triphosphate (AMP-PNP) (PDB code: 3PUY), ADP-VO4 (PDB code:3PUV), and ADP-AlF4 (PDB code:3PUW).20 These complexes resemble different states of ATP hydrolysis in the protein. However, the protein structures are found to be essentially identical to each other, indicating that no major conformational rearrangement occurs in the protein during ATP hydrolysis.12,20 Although the above X-ray complex with ADP-VO4 or ADP-AlF4 was considered to be a TS analog, the exact structure of the TS, which is inherently unstable, is yet unknown.


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Here, the aforementioned QM/MM NEB method is applied for an extensive unbiased search for the TS structure in the following discussion. To provide an initial structure for the RS we used the NBD dimer taken out from the 3PUY complex where MgAMP-PNP was replaced by MgATP. This structure, in which hydrogen atoms were added to the heavy atoms, was then solvated with a 10nm cubic box of SPC/E water36 and 10 sodium ions were added as counter ions to make the system neutral. The whole structure contains 11530 solute atoms and 29105 water molecules. In the active site, E159 is negatively charged. As H192 is adjacent to the negatively charged E159 side chain and γ-phosphate group, the H192 side chain is protonated as shown in Fig. 3 in reference 20. The following calculations were carried out using an NWChem QM/MM module.37 The energy minimization convergence criteria in this work were set to 3.0×10-4 hartree bohr-1 for root-mean-square (RMS) gradient and 1×10-6 hartree for energy (see Table S1-S2 in SI for the RS and PS optimization, respectively). The entire system was partitioned into a QM region treated quantum mechanically and a MM region for the rest of the system using a molecular mechanics method. The QM subsystem (a total of 82 atoms) includes the methyl-triphosphate fragment of ATP,30 a lytic water molecule, Mg2+, two Mg2+-coordinated water molecules, and fragments of the residues including G39, K42, and S43 in the Walker A, Q82 in the Q-loop, E159 in the Walker B, the switch H192 residue in one NBD, and N163 in the D-loop in the other NBD (SI, Figure S2). Whereas the MM region was treated using the AMBER99 force field,38 the QM calculations were performed at the B3LYP39-40 level with the Ahlrich-pVDZ basis set.41 The bonds connecting the QM and MM regions were capped with hydrogen atoms.42 Prior to the QM/MM calculations, a series of 200ps molecular dynamics (MD) annealing simulations were undertaken for the equilibration of the reactant system from 50K to room


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temperature. After the equilibrium stage, the structure was then optimized using a multi-region optimization algorithm.32,42 During the MM optimization, the electrostatic interaction between the QM and MM region were approximated by a set of effective electrostatic potential charges (ESP), which were chosen so that the electrostatic potential outside the QM region was accurately represented.42 The ESP charges were recalculated for each cycle of the optimization. These alternating optimization cycles of the QM and MM regions were carried out until convergence was achieved (SI, Table S1 for the RS optimization). The resulting RS structure and the parameters are shown in Figure 1. Alignment of the RS complex to the 3PUY X-ray structure leads to good agreement with a root mean squared deviation (RMSD) of 0.15Å for the Cα atoms. In the calculated RS structure, the distance, R1, between the terminal phosphorus atom, Pγ, and the bridging oxygen atom, Os , is 1.74Å, and the distance, R2, between Pγ and the oxygen atom, Ow, of the lytic water molecule is 2.74Å. H192 is H-bonded with a nonbridging oxygen atom of the γ-phosphate (the H-bond length is 1.74Å). The sidechain oxygen atom, OE, of E159 and the backbone oxygen of N163 form two H-bonds with the lytic water molecule (the bond lengths are 1.77Å and1.73Å), respectively, orienting the water molecule in the near-attack conformation. The optimized RS structure implies that this glutamate residue could abstract a proton from the lytic water as discussed in reference 20. Following the similar procedure discussed above, the structure for the PS was obtained after an extensive optimization (see Table S2 in SI for the PS optimization) on the basis of the 3PUW structure where the AlF4- group was replaced by one hydrolysis product, H2PO4-. The resulting PS structure (Figure 1) shows excellent agreement with the 3PUW X-ray structure (RMSD 0.11 Å for the Cα atoms). In the computed PS, E159 accepts the proton from the lytic water molecule and H159 is deprotonated and neutralized. Structural analysis demonstrates that the active site of


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Biochemistry

the PS structure is stabilized by tight binding of the products, MgADP-and H2PO4- to residues highly conserved among the ABC transporters. Whereas MgADP- interacts with the protein in the similar fashion to the RS, H2PO4- is H-bonded with the residues including K42, E159 and H192 in one NBD, and N163 in the other. These interactions in the PS, which are also determined in the high-resolution crystallographic study,20 inherit from those in the RS. QM/MM optimization of the NEB path and free energy calculations. The potential of mean force (PMF) along the reaction pathway for ATP hydrolysis in the maltose transporter was calculated using the QM/MM NEB and free energy integration methods.30,32 In the NEB method, a path is constructed with a number of intermediates (images) formed with discrete structures of the system to connect the RS and the PS. Adjacent images are constraint together by harmonic spring forces mimicking an elastic band.43-45 The optimization of the NEB path, mainly the minimization of the force acting on the images, brings the path to the minimum energy pathway. In the present work, a total of 9 images were used for the NEB path and the iterative multiregional QM/MM method discussed above was used for the path optimization. The QM/MM calculations were performed at the B3LYP/6-31+G* level whereas the AMBER99 force field was applied for the MM optimization. The first NEB optimization pass was followed by 150 ps of MD equilibration of the MM region. The final NEB path was obtained by repeating this procedure until it was converged. Single point calculations were then carried out using a

 larger 6-311++G** basis set and the PMF along this NEB optimized path, r min , was obtained by  calculating the free energy difference, F (i j ) , between two consecutive configurations, ri min

 and r jmin , on the NEB path 30

  i j F (i j )  Eqm (r jmin )  Eqm (ri min )  Fqm / mm ,


(1)

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  where E qm (rjmin ) and E qm (ri min ) are the energy of the QM subsystem at ri min and r jmin , respectively. i j Fqm / mm in Eq.(1) is given by

i j Fqm / mm  

Here, ...

qm / mm ,i

1



ln



 ~  min  ~  , R j )  E (r min , Ri )]} i

 d Re xp{ [ E (r j

.

(2)

qm / mm ,i

is an ensemble average over the MM subsystem,  

1 , k B is the Boltzmann k BT

      ~  constant, E (ri min , Ri )  Eqm / mm (ri min , Ri )  Emm ( Ri ) ,30 Ri and R j are the MM coordinates.

To compute eq. (1), a thermodynamic integration method is used by30, 32

i j Fqm / mm 

1



0

d

Fqm / mm ( ) 

.

(3)

   Here, Fqm / mm ( )  Fqm / mm r min ( ), Q( )   1 ln  d Re xp[E~(r min , R)] where r min ( ) and the 

effective charge, Q( ) , are given by

   r min ( )  (1   )ri min  rjmin Q( )  (1   )Qi  Q j ,

(4)

where the  values are taken from 0 to 1 with an interval of 0.1. Using the double-wide sampling method,32 the free energy differences for i -1  i and i  i 1 were obtained simultaneously by sampling at i over 15ps of MD simulation at constant temperature (T=298.15K). Several structural snapshots along the reaction pathway and the PMF are represented in Figures 1 and 2, respectively.


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RESULTS The resulting PMF shown in Figure 2 has a single TS with the activation free energy of 19.2kcal/mol in good agreement with experiments in which the rate constants range from 0.2 to 10.0s-1 (the activation free energy from 16.3 to 18.6kcal/mol).46-49 Compared to the experimental activation free energy of 28.2kcal/mol for ATP hydrolysis in solution (rate constant is 2.2×10-7s-1 at 60℃),50 the calculated barrier height is reduced by 9.0kcal/mol for the reaction in the maltose transporter.


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Figure 1. Structural snapshots of (a) the reactant state, (b) the transition state, (c) the sixth point on the reaction pathway in Figure 2, and (d) the product state from the QM/MM NEB calculations with the important H-bonding interactions illustrated. The atoms, O, C, H, P and N are colored red, green, white, orange, and blue, respectively. Key distances are labeled in Å. A dashed line represents a hydrogen bond or a coordination bond with Mg2+ (green sphere). The atoms, Pγ , Pβ , Ow , Hw, OE and the distances, R1 and R2 are labeled (see details in the main text).


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Figure 2. Potential of mean force (PMF) for ATP hydrolysis in the maltose transporter along the reaction pathway. The transition state is denoted by TS. The variations of R1 and R2 along the reaction pathway are presented in Figure 3. To describe the proton transfer from the lytic water, the distances from the lytic water hydrogen atom, Hw, to the side-chain oxygen atom, OE, of E159, ROE—Hw, and to Ow of the lytic water, ROw—Hw, along the reaction pathway are also plotted in Figure 3, respectively. Starting from the RS with R1=1.74Å and R2=2.74Å, the reaction proceeds uphill to the TS where R1=2.20 Å and R2=2.19 Å. The cleavage of the Pγ–Os bond occurs at the TS where ROE—Hw=1.60 Å and ROw—Hw=1.01Å (Figures 1b and 3), showing that the Ow–Hw bond of the lytic water is not broken yet at the TS. Interestingly, ROE—Hw=1.02Å, ROw—Hw=1.65Å and R1=2.49Å, R2=1.84Å (Figure 1c) at the sixth point right after the TS on the PMF, demonstrating that the proton, H w , and OH- from the lytic water are transferred simultaneously to E159 and the γ-phosphorus atom, respectively. The system then moves downhill to the PS in which R1=2.73 Å and R2=1.67 Å (Figure 1d). 14 Environment ACS Paragon Plus

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Figure 3. Variations of R1 (black line with solid squares), R2 (red line with solid circles), ROE-Ow (green line with solid triangles), and ROw-Hw (blue line with solid triangles) along the reaction pathway. TS denotes the transition state. Structural examination shows that the protein structural changes are rather small during the process from the RS to the TS. The major configurational rearrangements in the complex involve that the γ-phosphorus atom moves by 0.42Å towards the lytic water from its position in the RS, while this water molecule moves to the γ-phosphorus atom only by 0.23 Å (Figure 4).The γphosphate group changes from tetrahedral to planar, together with Os and Ow forming a trigonal


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Figure 4. Overlay of the calculated TS structure (red, green and orange) with the reactant state (cyan). Key distances are labeled in Å. bipyramidal structure. The lytic water molecule in the TS is held by E159 and N163 via two Hbonds, respectively, in a fashion similar to that in the RS. While the length of the H-bond between the lytic water hydrogen and the N163 backbone oxygen is changed from 1.73 Å to 1.67 Å during the passage from the RS to the TS, the H-bond length between the lytic water hydrogen, Hw, and the E159 side-chain oxygen, OE, is decreased from 1.77 Å to 1.60 Å, as shown in Figure 4. The H-bond of H192 with the nonbridging oxygen of the γ-phosphate in the TS is also reduced by 0.15Å compared to that in the RS. Both E159 and H192 thus contribute to the stabilization of the TS. However, these two residues play different roles in the catalysis. While H192 stabilizes the TS via H-bonding with the nonbridging oxygen of the γ-phosphate (rather than interacting with E15917 or with the lytic water molecule21), E159 helps to position the lytic water molecule via the H-bonding for the attack of the γ-phosphorus atom. The


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positioning of the lytic water molecule can provide substantial catalysis in the reaction. 51 Our results are consistent with the biochemical experiments showing that mutation of either E159 or H192 can lead to a reduction in the ATPase activity of the ABC transporters.14-19,21 DISCUSSION It is important to point out that our results are essentially different from the GBC20 (Figure S3) or SAC17 discussed above. Our calculations demonstrate that at the TS the Pγ-Os bond cleavage occurs while the O-H bond of the lytic water molecule is not broken (Figure 1b).This indicates that the Pγ-Os bond breaking rather than the abstraction of the proton from the lytic water by E159 is rate-limiting in ATP hydrolysis. As the hydrogen (or deuterium) atom of the lytic water molecule does not involve directly in the rate-determining step of the reaction, the deuterium KIE for the reaction is secondary. The magnitude of the secondary deuterium KIE is generally smaller than the primary deuterium KIE,29 particularly the isotopic substitution atom (D/H) is far away from the Pγ-Os bond in the case studied here. As discussed above, the kinetic experiments show that the rate constants for ATP hydrolysis in ABC proteins are essentially unchanged in D2O and H2O.21 Our results reported here are consistent with the deuterium KIE experiments. Although E159 abstracts the proton from the water molecule after the TS as the reaction proceeds downhill toward the PS (Figure 1d), this residue contributes to the catalysis by stabilizing the TS via the tight hydrogen bonding with the lytic water molecule rather than by acting as a catalytic base. The calculated TS structure is in good agreement with the X-ray TS analog of the ADP-VO4 complex (PDB: 3PUV)20 as shown in Figure 5. Alignment of both the structures results in a RMSD of 0.11Å for the Cα atoms. An important structural difference is that in the TS analog the V-Os bond length is 1.98 Å while R1 is 2.20 Å in our calculated TS structure (Figure 1b). In the


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VO43- group of the TS analog, VO3- is planar and one oxygen atom occupies the position of the lytic water molecule.20 The TS analog forms an associative pentacovalent structure where the VOs bond is not broken, suggesting that E159 acts as a catalytic base to abstract the proton from the attacking water at the TS.20 However, the GBC inferred from the structural study contradicts the deuterium KIE experiments as discussed above. This might raise the concern of whether vanadate is an ideal TS analog as also pointed out in reference 52. It is of interest to compare our present results for ATP hydrolysis in the ABC protein with those in aqueous solution.30 Herschlag and coworkers ever speculated whether enzymes stabilize transition states that are closely related to the transition states of the corresponding uncatalyzed reactions.50,51 As shown in our previous calculations,30 the PMF for ATP hydrolysis in the aqueous solution has a single transition state with the barrier height of 32.5kcal/mol. Our previous study demonstrates that relatively large configurational rearrangements occur at the active site in the TS for the reaction in the solution.30 In particular, Mg2+ together with its coordinators in the TS is reorganized via moving toward the α-phosphorate by 0.8Å, and Pβ and Os move backward by 0.5Å and 0.8Å, respectively, relative to their positions in the RS. By contrast, the TS rearrangements are rather small in the protein environment. As discussed above, while the major TS rearrangements occur on the P γ atom, the positions of Mg2+, Pβ and Os remain almost unchanged in the protein. This is not unreasonable since the protein provides a relatively rigid environment for ATP hydrolysis compared to in the solution. Interestingly, if the position of the Os atom at the TS in the solution had remained unchanged as in the ABC protein, R1 would be ~2.2Å in the solution almost the same as in the protein. Indeed, the protein pocket where the reaction takes place is rather tight for binding and hydrolyzing the phosphate. For


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Figure 5. Overlay of the calculated TS structure (yellow) with the ADP-VO4 TS analog (PDB code: 3PUV) where O, C, P, N and V are colored red, green, orange, blue, and grey, respectively. The hydrogen atoms are omitted, and only the substrates and several important residues are represented for clarity. Three water molecules, which are labeled by wat1, wat2, and wat3, are represented by the yellow and red balls (Mg2+ is represented by yellow and cyan balls) for the calculated TS and the X-ray TS analog, respectively. The distance between the bridging atom, Os, and V is 1.98 Å and the distance, R1, between Pγ and Os is 2.20 Å.

example, at the RS in the protein Pγ is only 4.6 Å away from the backbone oxygen atom of N163 in the opposite side of the pocket, in which the lytic water molecule is located on the sideway between these two atoms (Figure S2), thereby limiting the motion of the substrate during the course of the reaction. Taken a broader view, ATP/MgATP as well as ADP/MgADP is rather flexible and can adopt a wide variety of conformations in different ATP-dependent proteins


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observed from X-ray structures.53-54 These conformations, of course, are also different from their counterparts in water.55-56Based on these findings, it would not be surprising that enzymes stabilize the transition states that have quantitatively different conformations from that in the solution. Interestingly, however, our present calculations show that the reaction proceeds to the TS from the RS via a concerted (dissociative-like) pathway in the ABC protein, similar to that in the solution,30 i.e., the enzyme does not alter the concerted nature of the TS in the solution similar to the cases for the hydrolysis of phosphate monoester or diester.52,57-58It will be interesting to investigate whether it is common that enzymes do not change the nature of ATP hydrolysis in other enzymes. CONCLUSIONS In this work the QM/MM NEB method is used to evaluate the reaction pathway for ATP hydrolysis in the maltose transporter. The calculated PMF (Figure 2) has a single TS with a 19.2 kcal/mol free energy barrier relative to the RS, in good agreement with the available experimental data for ABC transporters.17,21,46-49The results show that the TS adopts a trigonal bipyramidal geometry and ATP hydrolysis in the ABC protein is concerted (dissociative-like ) in nature similar to that in aqueous solution.30 Our calculations demonstrate that in the TS the PγOs bond cleavage occurs while the bond of the lytic water molecule is not broken, indicating that the cleavage of the Pγ-Os bond rather than the lytic water is rate limiting in the reaction. This finding is consistent with the deuterium KIE experiments for the ABC proteins discussed above. The TS is stabilized by a network of the hydrogen bonds including those between E159 and the lytic water, and H192 and the planar γ-phosphate. Our results clearly show that E159 as well as H192 is a TS stabilizer rather than a catalytic base in the GBC.12-20,25


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The high-resolution crystallographic studies of the maltose transporter provide a structural basis to elucidate the catalysis mechanism.1,12, 20,25-26 However, the GBC inferred from the X-ray studies cannot explain the deuterium KIE experimental data as discussed above. Here, the computer simulations using a high-level QM/MM method clearly show that there is no catalytic base for ATP hydrolysis in the ABC protein, consistent with recent kinetic experiments.21 As the ABC ATPases are highly conserved in sequence and structure, our results for the maltose transporter will be applicable to other members of the ABC protein family.

ASSOCIATED CONTENT Supporting Information. The X-ray structure for the maltose transporter and the schematic illustrations of the calculated structures for the reactant, transition and product states are included. AUTHOR INFORMATION Corresponding Author *Email: [email protected] Notes The authors declare no competing financial interest. Funding This work was funded by the Natural Science Foundation of China under Grant Nos. 21073170 and 21273209. ACKNOWLEDGEMENTS We gratefully acknowledge the Supercomputing Center at University of Science and Technology of China (USTC) for computational resources.


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Catalytic Mechanism of the Maltose Transporter Hydrolyzing ATP

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