Insights into the Inhibitory Mechanism of D13-9001 ... - ACS Publications

Article pubs.acs.org/JPCB

Insights into the Inhibitory Mechanism of D13-9001 to the Multidrug Transporter AcrB through Molecular Dynamics Simulations Zhicheng Zuo, Jingwei Weng,* and Wenning Wang* Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials, Department of Chemistry, and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, People’s Republic of China S Supporting Information *

ABSTRACT: The resistance-nodulation-cell division transporter AcrB is responsible for energy transduction and substrate recognition in the tripartite AcrAB-TolC efflux system in Escherichia coli. Despite a broad substrate specificity, only a few compounds have been cocrystallized with AcrB inside the distal binding pocket (DBP), including doxorubicin (DOX) and D13-9001. D13-9001 is a promising efflux pump inhibitor that potentiates the efficacy of a wide variety of antibiotics. To understand its inhibition effect under the framework of functional rotating mechanism, we performed targeted and steered molecular dynamics simulations to compare the binding and extrusion processes of this inhibitor and the substrate DOX in AcrB. The results demonstrate that, with respect to DOX, the interaction of D13-9001 with the hydrophobic trap results in delayed disassociation from the DBP. Notably, the detachment of D13-9001 is tightly correlated with the side-chain reorientation of Phe628 and large-scale displacement of Tyr327. Furthermore, the inhibitor induces much more significant conformational changes at the exit gate than DOX does, thereby causing higher energy cost for extrusion and contributing to the inhibitory effect in addition to the tight binding at DBP.

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structures and biochemical studies,10−18 a functional rotating mechanism has been proposed that each protomer cycles consecutively through the three conformational states during substrate efflux. The interstate transition leads to evident relative movements among the subdomains of the porter domain of AcrB.5,19 Accordingly, the porter domain of each protomer accommodates a differently shaped substrate translocation tunnel in it, while the three TolC-docking domains enclose a central funnel from which the substrate moves up to the TolC lumen (Figure 1b). From the available AcrB/ substrate complex structures, two binding pockets have been identified inside the porter domain: the deep/distal binding pocket (DBP) located between the PN2 and PC1 subdomains and the proximal binding pocket (PBP) lying between PC1/ PC2 cleft (Figure 1b), which are segregated by a functionally relevant switch-loop.14,20−22 Structural and biochemical researches suggest that substrates bind to the PBP of the access protomer at the first stage of translocation, and subsequently move to the DBP in the A → B transition.13,14 During the B → E conversion, the shrinking of DBP expels the substrates toward the central funnel bypassing the putative exit gate (Figure 1, parts b and c).

INTRODUCTION Multidrug resistance (MDR) in bacteria has posed a growing threat to treatment of infectious diseases.1 A major contributor to bacterial MDR is the overexpression of multidrug efflux pumps that export a broad variety of structurally and/or chemically unrelated antimicrobials out of cells.1 One of the most-studied efflux systems in Gram-negative bacteria is the tripartite AcrAB-TolC pump from Escherichia coli, which comprises an internal membrane transporter AcrB of resistance-nodulation-cell division (RND) family, an outer membrane exit duct TolC, and a periplasmic adaptor AcrA bridging AcrB and TolC.2−4 The assembled transport machine, spanning the entire bacterial cell envelope, is powered by the electrochemical proton gradient across the internal membrane. AcrB is responsible for both energy transduction and substrate recognition, thus playing a central role in the functioning of the tripartite complex.5,6 The high-resolution structure of AcrB was first solved as a homotrimer in a threefold symmetric form,7−9 with each protomer composed of a transmembrane (TM) domain, a porter domain, and a TolC-docking domain (Figure 1a), whereas subsequent crystallographic studies10−15 demonstrated that the three protomers assume distinct conformational states, dubbed, respectively, as access (A), binding (B), and extrusion (E), or alternatively, as loose (L), tight (T), and opening (O) (Figure 1, parts a and b). On the basis of the asymmetric © 2016 American Chemical Society

Received: December 7, 2015 Revised: February 20, 2016 Published: February 22, 2016 2145

DOI: 10.1021/acs.jpcb.5b11942 J. Phys. Chem. B 2016, 120, 2145−2154

The Journal of Physical Chemistry B

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Figure 1. Simulation systems. (a) AcrB trimer in the phospholipid membrane/solvent environment. The access, binding, and extrusion protomers are colored cyan, pink, and lime, respectively. The inhibitor D13-9001 inside the DBP is depicted in yellow sphere model highlighted by the blue rectangle box, and the phosphorus atoms of lipids are rendered as magenta vdW spheres. (b) Top view of the porter domains from the periplasm. The DBP lining and gating residues are shown as magenta and green stick models, respectively. The substrate translocation tunnels (depicted by yellow tubes) inside AcrB are calculated by CAVER3.0. (c) Superimposed D13-9001 (yellow stick) and doxorubicin (red stick) within the DBP. The hydrophobic trap enclosed by Phe136, Phe178, Phe610, Phe615, and Phe628 is outlined with dashed line, which branches off the substrate translocation tunnel (solid line) toward the exit gate (green surface). (d) Chemical structure of inhibitor D13-9001 (ABI). (e) Chemical structure of doxorubicin (DOX).

Figure 2. Shrinking of the distal binding pocket and the resulting ligand dissociation in the ABI-bound (a and b) and DOX-bound (c and d) systems. (a and c) Temporal evolution of the radius of gyration (Rg) of the DBP. (b and d) Temporal evolution of the ligand displacement from the DBP.

D13-9001, 1 5 , 2 8 and pyranopyridine series, such as MBX2319.29 Unfortunately, very few of them have advanced to preclinical stage due to toxicity issues. Among them, D139001 (Figure 1d) has been tested to be a promising lead compound with potent inhibitory activity, high solubility, and low cytotoxicity.15,28 In contrast with the broad-spectrum inhibitor PAβN, D13-9001 specifically antagonizes AcrAB and MexAB (a homologue in Pseudomonas aeruginosa),1,15 referred to as AcrAB/MexAB-specific inhibitor (ABI). Importantly, ABI is the only efflux pump inhibitor cocrystallized with the native RND transporters AcrB and MexB thus far, and is hardly

Efflux pump inhibitors are of great interest in combination chemotherapy, given that their use as adjuvants is expected to potentiate the activities of many existing antibiotics by raising the level of antibiotic accumulation inside the pathogens.23 Moreover, the inhibitors can be exploited to examine the presence and contribution of efflux determinants in clinical isolates.24 Different classes of inhibitors have been reported and characterized in the last 2 decades, including peptidomimetics, such as phenylalanylarginine-β-naphthylamide (PAβN),25 arylpiperazines, like 1-(1-naphthylmethyl)-piperazine (NMP),26,27 pyridopyrimidine derivatives, represented by 2146



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Figure 3. Disassociation of ABI from the DBP. (a) Close view of ABI bound in the DBP in the initial structure. (b) Snapshot of ABI in the DBP at 3.5 ns in one of the targeted MD trajectories. (c) Close view of ABI and the DBP in the end structure of the targeted MD trajectory. (d) Temporal evolution of Phe628 χ1 angle. (e) Temporal evolution of the distance between the mass center of Tyr327 side chain and the Cα atom of Phe628.

exported by these transporters.15 The crystal structures reveal that the hydrophilic piperidine acetoaminoethylene ammonioacetate (PAEA) moiety as well as the ethyl tetrazole group almost overlaps with DOX and minocycline inside the DBP, while the lipophilic tert-butyl thiazolyl aminocarboxyl pyridopyrimidine (TAP) moiety of ABI is deeply inserted into a narrow phenylalanine-rich cave (called hydrophobic trap) that branches off the substrate translocation tunnel (Figure 1c−e).15 A wealth of structural and biochemical information has made remarkable contributions to our understanding of the working mechanism of AcrB; however, many details of the mechanism, especially the action mechanism of efflux pump inhibitors, remain elusive. Molecular dynamics (MD) simulation has become a powerful tool in exploring protein conformational changes and in rationalizing existing experimental data. To this end, various MD simulations have been performed to address the mechanistic knowledge gaps of AcrB in terms of conformational cycling and substrate extrusion,30−36 porter domain opening and closing motions,37 substrate recognition by the porter domain,38−42 proton transfer pathways in the TM domain,5,36,43,44 and energetics of substrate translocation along the uptake pathways.45,46 In this contribution, we explored the detailed inhibition mechanism of ABI toward AcrB by comparing the binding and extrusion processes of this inhibitor and a typical substrate DOX (Figure 1e). Through free energy estimation, we demonstrate that van der Waals interactions dominate the difference of the binding affinities of ABI and DOX. In the induced ABE → BEA transition simulated with targeted MD method, a longer dwelling period of ABI inside the distal pocket was observed compared to DOX.

Furthermore, the ensuing steered MD simulations show a strong resistance to ABI extrusion at the gate to the central funnel.

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RESULTS It Is More Difficult for ABI To Dissociate from the Binding Site in DBP Than for DOX. We first performed 5 ns targeted MD simulations to induce one-step functional rotation of AcrB trimer (viz. from ABE state to BEA state) in the ABI and DOX bound systems, respectively. In both systems, the ABE → BEA transition leads to progressive closure of the DBP with ABI or DOX bound (Figure 2, parts a and c), thereby propelling ligand disassociation therefrom (Figure 2, parts b and d). The displacement profiles of the ligands show that ABI (Figure 2b) and DOX (Figure 2d) moved similar distances at the end of the simulations. However, the inhibitor left the DBP at ∼3.5 ns, systematically postponed by ∼1 ns (i.e., one-fifth of the simulation time) relative to the substrate. This observation is independent of the selection of simulation length or force constant (Table S1 and Figure S1, Supporting Information), implying that the molecular determinants for ABI and DOX unbinding are per se different. Further inspection of the trajectories demonstrates that the disassociation process of ABI is strongly dependent on the collective movements of Phe628 and Tyr327, which are two residues constituting the lower part of the hydrophobic trap. The side chain of Phe628 kept its original orientation (characterized by the torsional angle χ1 at about −170°) until ∼3.5 ns (Figure 3, parts a and d), before which ABI hardly moved in the DBP (Figure 2b). Afterward, the side chain of 2147



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Table 1. Free Energy Components of ABI and DOX Binding to AcrB in ABE and BEA States (kcal/mol) ABE state

BEA state

AcrB−ABI a

b

ΔE

mean

ΔEvdW ΔEele ΔEMMd ΔGsol,pol ΔGsol,np ΔGsole ΔGele,totf ΔGbindg

−77.89 509.91 432.02 −470.19 −9.03 −479.22 39.72 −47.20

AcrB−DOX c

AcrB−ABI

AcrB−DOX

STD

mean

STD

mean

STD

mean

STD

(3.70) (19.17) (19.06) (17.71) (0.33) (17.70) (4.46) (4.01)

−49.38 −674.75 −724.13 696.69 −6.51 690.18 21.94 −33.95

(3.78) (30.68) (29.41) (25.64) (0.44) (25.42) (6.18) (5.02)

−55.45 521.33 465.87 −486.95 −7.03 −493.98 34.38 −28.10

(4.78) (20.57) (21.10) (21.85) (0.49) (21.63) (5.40) (3.52)

−38.51 −730.10 −768.61 746.36 −6.05 740.31 16.25 −28.30

(4.14) (25.34) (25.74) (22.33) (0.48) (21.98) (7.17) (5.60)

a

ΔE = Ecomplex − Ereceptor − Eligand. The single trajectory protocol (STP) was used, and the entropic contribution was not taken into account. EvdW, van der Waals energy in vacuo; Eele, Coulombic electrostatic energy in vacuo; Gsol,pol, polar solvation energy; Gsol,np, nonpolar solvation energy. b Averaging over a total of 2000 snapshots from the unbiased simulations (taken at an interval of 10 ps). cStandard deviation in parentheses. d Molecular mechanics energy: ΔEvdW + ΔEele (the internal potential terms cancel out in the STP). eSolvation energy (computed via the GB model): ΔGsol, pol + ΔGsol,np. fTotal net electrostatic energy: ΔEele + ΔGsol,pol. gTotal binding free energy: ΔEMM + ΔGsol = ΔEvdW + ΔGsol,np + ΔGele,tot.

(−47.2 ± 4.0 vs −48.8 ± 4.8 kcal/mol).42 This binding affinity is apparently stronger than that of DOX by 13.3 kcal/mol (−47.2 ± 4.0 vs −33.9 ± 5.0 kcal/mol; Table 1). The interactions between the TAP moiety of ABI and the residues lining the hydrophobic trap of AcrB (involving Phe178, Phe628, Phe615, Phe136, and Phe610, etc.) are the major contributions to the affinity difference (Table S2, Supporting Information). After switching to the BEA state, the affinities of both ABI and DOX to AcrB were reduced, facilitating their disassociation from the distal pocket. The binding affinity of ABI is remarkably lowered by 19.1 kcal/mol (from −47.2 ± 4.0 to −28.1 ± 3.5 kcal/mol; Table 1), mainly resulting from the loss of the strong interactions with the hydrophobic trap, especially Phe178 and Phe628 (Table S2, Supporting Information). The binding strength of DOX is decreased moderately by ∼6 kcal/mol (from −33.9 ± 5.0 to −28.3 ± 5.6 kcal/mol; Table 1). This is because the loss of interactions is partly compensated by enhanced interactions of DOX with several polar residues outside of the DBP, such as Arg620 and Asp174 (Table S2, Supporting Information). Among the individual energy terms (Table 1), the van der Waals interactions (ΔEvdW) predominates the binding free energy in ABE state, while the nonpolar solvation term (ΔGsol,np) contributes marginally to ligand binding. The overall electrostatics (ΔGele,tot), however, disfavors complex formation, regardless of the net positive or negative charges carried by the ligands (ABI, −1; DOX, +1; Figure 1, parts d and e). The results confirm the notion that ligand association with the poorly solvated binding pocket is dominantly driven by van der Waals interactions.50 Upon transition to the BEA state, van der Waals interactions are markedly weakened, especially in the ABI-bound system (Table 1). ABI Has To Overcome a Higher Barrier to Cross the Exit Gate Compared to DOX. Although ABI and DOX moved considerably (5−7 Å) toward the exit gate during the targeted MD simulations (Figure 2, parts b and d; Figure S1, Supporting Information), the extrusion process is far from completion since there is still a long distance between the ligand molecules and the central funnel. The possible factors limiting the extrusion have been previously discussed, including the time scale amenable to computer simulations, the cooperativity associated with multisubstrate binding, and the influence of AcrA on the dynamics of AcrB.30,34,35 To investigate the entire extrusion process, we proceeded to

Phe628 flipped upward with χ1 switching to ∼−60°, pushing the TAP moiety of ABI (Figure 3, parts b and d) and resulting in the detachment of ABI from its binding site (Figure 2b). Almost simultaneously, the side chain of Met573 which lies next to Phe628 reoriented and pointed away from the hydrophobic trap (Figure 3, parts b and c), and Tyr327 underwent large-scale displacement with the distance of its indole group to the Cα atom of Phe628 decreasing by 6−7 Å (Figure 3e). In contrast to the ABI-bound system, the initial arrangement of Tyr327 and Phe628 in the DOX-bound system is already similar to the final state observed in the ABI-bound system (Figure S2a−c, Supporting Information). Throughout the simulations, χ1 of Phe628 constantly fluctuated around −60° (Figure S2d, Supporting Information) and the distance between Phe628 and Tyr327 declined by only 2−3 Å (Figure S2e, Supporting Information). It does not, however, mean that Phe628 and Tyr327 are functionally irrelevant to DOX transport in that they would participate in the preparation period or the very initial stage of substrate dissociation.33,41 We noticed that the squeezing motion of Phe136 and Phe615 (Figure S2, parts a−c and f, Supporting Information) is instead more responsible for DOX disassociation, as the motion is very closely correlated with the movement of DOX (Figure S2g, Supporting Information). This observation is generally consistent with previous targeted MD simulations with DOX,30 despite a different initial binding pose here. ABE → BEA Transition Reduces the Binding Affinities for Both ABI and DOX. On the other hand, we estimated the binding free energies of ABI and DOX to AcrB before and after functional rotation using the molecular mechanics−generalized Born surface area (MM−GBSA) method (Table 1).47−49 To obtain more physical insights, we partitioned the total binding free energy (ΔGbind) into three components (marked in bold in Table 1), viz., the van der Waals interaction energy (ΔEvdW), the nonpolar solvation energy (ΔGsol,np), and the overall electrostatic energy (ΔGele,tot), which is the sum of the electrostatic term (ΔEele) in molecular mechanics energy and the polar/electrostatic contribution to solvation energy (ΔGsol,pol). Moreover, we performed per-residue free energy decomposition (Table S2, Supporting Information), allowing for an identification of key residues to binding. The calculated binding free energy of ABI to AcrB in ABE state (Table 1) is comparable to that reported by Vargiu et al. 2148



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Figure 4. Extrusion through the exit gate. (a) Average pulling force profiles for ABI (green line) and DOX (orange line) extrusion. Force peaks associated with unbinding from the DBP and crossing the exit gate are labeled pI and pII, respectively. The displacement is defined as the inverse of the distance between the mass center of ABI or DOX to the centroid of the Cα atoms of Asn747 in chain A (binding protomer) and Asp788s in chain B (extrusion protomer) and C (access protomer). The centroid is located in the central funnel. The inverse is used to direct the eye. (b) Variations of the distance between the Cα atoms of Gln124 and Tyr758 during ABI and DOX extrusion. The horizontal dashed line denotes the interatom distance calculated from the structure of extrusion protomer. (c) Close view of ABI bound inside the extrusion tunnel at the force peaks pI and pII from one trajectory. (d) Close view of DOX bound inside the extrusion tunnel at the force peaks pI and pII from one trajectory. The ligands binding at the edge of DBP are represented by yellow sticks, with adjacent residues and gating residues colored by atom type (C, pink; N, blue; O, red), and are colored green when passing the exit gate (in cyan). The intermolecular hydrogen bonds are denoted by dashed lines.

accordance with the similar binding affinities of ABI and DOX to AcrB after ABE → BEA transition derived from the MM−GBSA calculations (Table 1). Remarkably, the height of pII in the ABI-bound system is much higher than that in the DOX-bound system by ∼150 pN, indicating that ABI has to encounter much stronger resistance than DOX when passing through the gate region. The difference at pII also implies a potential role of exit gate in sorting substrates. For comparison, steered MD simulations using the same steering velocity and spring constant were also conducted for ABI- and DOX-bound systems when AcrB stays in the ABE state. Relative to the BEA state, the maximal pulling forces increase by 300 and 150 pN for ABI- and DOX-bound systems, respectively (Figures S4 and S5, Supporting Information), indicating that the ABE → BEA transition indeed facilitates the dissociation process. Moreover, DBP shows higher affinity for ABI than DOX as larger force is required to pull ABI out of the binding pocket, in good agreement with the targeted MD and MM−GBSA calculations. Further examination of the pulling trajectories shows that the exit gate reacts differently to ABI and DOX extrusion. With the ligands approaching the gate, the inter-Cα distances between Gln124 and Tyr758 in the ABI and DOX systems increased by

conduct steered MD simulations on both ABI- and DOXbound systems, in which the bound ligands were pulled out of the now extrusion protomer toward the central funnel at a constant velocity of 0.005 Å/ps with a spring constant of 10.0 kcal/mol. The results were averaged over 36 trajectories with different initial velocities for each system (see Materials and Methods for details). Four times slower of the steering velocity provides generally similar results (Figure S3, Supporting Information). The average pulling force profiles are plotted for both systems (Figure 4a; Figure S4, Supporting Information). Though the magnitude of pulling force is often overestimated by steered MD simulations, peaks in force profile still provide valuable insights into the energy barriers faced in the process. The figures show that both systems feature two pronounced force peaks, indicating two main barriers against further translocation. The first peak (pI) is associated with the complete ligand detaching from the DBP, disrupting the remnant interactions between ligand and DBP. The second peak (pII) emerges as the ligand is crossing the exit gate confined by the residues Gln124, Gln125, and Tyr758 (Figure 4, parts c and d). The difference between the pI heights of the two systems is relatively small (∼50 pN), which is in 2149



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the DBP and may be an important factor for ABI being a good competitive inhibitor of AcrB. Another important observation in this study is that the extrusion of ABI brings about much more significant conformational changes at the exit gate with respect to DOX (Figure 4). Accordingly, the larger conformational rearrangements of the region result in higher energy cost for ABI extrusion. Therefore, in addition to the competitive binding at the DBP, the resistance to passing through the exit gate could also account for the inhibition activity of ABI. Consequently, ABI dissociating from the DBP would possibly block the exit gate, thus hindering the efflux of other compounds as well as the functional rotation cycle. The perturbation of ABI to the exit gate is not surprising considering its large molecular size. However, some of the AcrB substrates also have large molecular sizes, such as rifampicin and erythromycin.13 It is reasonable to expect that these high-molecular-weight substrates would also induce considerable conformational changes at the exit gate during the extrusion process. However, the genuine energy cost for gate crossing includes contributions from the ligand− protein interactions in addition to the protein conformational changes. Therefore, substrate−protein interactions at the gate region may partly compensate for the energy cost of gate opening. The above inhibition mechanism of ABI to AcrB most likely can be applied to MexB since ABI binds to MexB in a very similar fashion as that to AcrB in the crystal structure.15 The difference lies in the PAEA moiety, which extends toward a different direction in MexB than that in AcrB. However, according to our simulations, the PAEA moiety does not play a key role during the dissociation process. Due to the structural diversity of various efflux pump inhibitors, it is expected that the mechanisms of action could be different. The inhibition mechanisms of a few RND pump inhibitors have also been studied by MD simulations,39,42 although only the crystal complex structure with ABI is available among them.15 For example, simulations predicted that PAβN and NMP bind at the DBP of AcrB with relatively low affinities and may interfere the substrate translocation by straddling the switch-loop between the DBP and the PBP.39 MBX2319 was predicted by MD simulation to bind to the hydrophobic trap in the DBP as ABI does with high affinity,42 and experiments showed that MBX2319 and ABI have approximately the same decreases in minimum inhibitory concentration (MIC),29,52 suggesting that they have the same mechanism of inhibition. However, as shown in this study and other MD simulations, the way the inhibitor inhibits AcrB function may lie in many aspects during binding and extrusion process, and there are probably subtle differences in the inhibition mechanism that are not easily to be revealed. Further detailed experimental and computational studies are required to decipher the working mechanisms of various inhibitors. In conclusion, on the basis of previous experimental studies, this work has provided a deeper understanding at atomic level of the inhibition effect of ABI under the framework of functional rotating mechanism. The simulation results suggest that the mechanism of ABI action originates from the high energy cost of dissociation from the DBP and crossing of exit gate.

3.5 and 1.5 Å (relative to that in the extrusion protomer) on average, respectively (Figure 4b). This indicates that the translocation of ABI into the central funnel entails larger conformational perturbation to the gating region as compared to DOX, thereby explaining the source of a higher rupture force for ABI to cross the exit gate (Figure 4a). Intriguingly, ABI experienced a bending conformational change during gate crossing (Figure 4c) in 29 out of 36 independent trajectories, though the conformational change did not show relevance to the gating motion (Figure S6, Supporting Information).

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DISCUSSION AND CONCLUSIONS The last 2 decades have seen continuous efforts in developing multidrug resistance efflux pump inhibitors. To date, the pyridopyrimidine derivative, D13-9001 (ABI), is the only inhibitor that has been cocrystallized with the RND-type multidrug transporters AcrB and MexB.15 It is surprising that this compound binds to the same pocket as substrates do but is hardly exported by the transporters.15,51 Although its potent efficacy has been generally attributed to the tight binding at the DBP, the detailed mechanism, especially related to the dynamic aspects underlying the inhibition, remains largely unclear. In this work, we compared the extrusion processes of ABI and DOX at an atomic level through various MD simulations. The targeted MD simulations demonstrate that the dissociation of DOX from the DBP is highly correlated with the closure motion of the Phe136-Phe615 pair (Figure S2, Supporting Information), whereas the departure of ABI strongly depends on the side-chain flipping of Phe628 and the large-scale displacement of Tyr327 (Figure 3). It is noteworthy that the conformational changes of the DBP during ABI dissociation do not follow the zipper-like motion observed in the DOX-bound system in the previous study30 and in the current work (Figure S7, Supporting Information). In the ABI-bound system, the Phe136-Phe615 pair kept closing from the beginning of the simulation (Figure S7a, Supporting Information), but ABI hardly left the binding site until the movements of Tyr327 and Phe628 started at a later stage of the simulation (Figure 3). Therefore, the order of conformational changes and the dissociation mechanism are most likely dependent on various binding modes of ligands. We also note that Tyr327 is located at the hinge region between the TM2 helix and the PN2 subdomain, a site most likely of importance for coupling the upward displacement of TM2 and the inward rotation of PN2 during the B → E transition.5 This notion has been supported by a recent study in which both the single mutation Y327A and the double mutation Y327A/ T329A were shown to result in various increased susceptibilities to all the tested drugs.45 The longer dwelling time of ABI inside the DBP is reminiscent of the behavior of DOX in the AcrB F610A mutant.33 In the mutant transporter, DOX was observed to slide down (by ∼5 Å) toward the hydrophobic trap, resulting in stagnated movement during subsequent functional rotation.33 Notably, the contribution from the hydrophobic trap to the binding free energy (ΔGbind) of ABI is 29%, comparable with that of DOX in the F610A mutant of 28%, and higher than that of 21% for DOX in the wild-type AcrB (Table S3, Supporting Information). Moreover, binding of another substrate, the minocycline (MIN), also shows a relatively low contribution of only 12% from the hydrophobic trap.42 Thus, the interaction strength of ligand with the hydrophobic trap may determine whether or not the bound ligand could easily dissociate from

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MATERIALS AND METHODS System Setup. The starting coordinates of the AcrB complex with the inhibitor D13-9001 (ABI) were derived from 2150



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the crystal structure 3W9H,15 and the initial configuration of AcrB in complex with the drug doxorubicin (DOX) was taken from the crystal structure 4DX714 after removing the DOX dimer inside the proximal binding pocket (PBP) of the access (A) protomer. The principal axis of AcrB was prealigned along the z-axis direction (i.e., membrane normal) through the orient plug-in in VMD.53 The construction of the two protein/ membrane systems was facilitated by the CHARMM-GUI Membrane Builder.54 Three titratable residues, Asp407, Asp408, and Lys940, in the transmembrane (TM) domain were protonated according to ref 5, and standard protonation states were adopted for the other ionizable residues. The “replacement” scheme55 was employed to pack the 1-palmitoyl2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer around the TM zone of AcrB. The dimensions of the membrane plane (x−y) were set to be 150 Å × 150 Å, and the thickness of the water layers from either boundaries of AcrB along the z-axis be 20 Å. The K+ and Cl− were chosen to generate a physiological salt solution of 0.1 M. The above settings result in a simulation box of approximately 150 × 150 × 173 Å3 for each system. Finally, the water molecules located in the TM central hole of AcrB were removed and replaced with 20 POPC lipids, leading to a periodic box comprising ∼382 000 atoms. Conventional Molecular Dynamics Simulations. All the simulations were performed with the package NAMD2.956 using the AMBER force fields ff12SB and lipid1457,58 for the protein and lipids, respectively, and the TIP3P model59 for the water. For the ABI and DOX, the atomic restrained electrostatic potential (RESP) charges were derived by the module “antechamber” of AmberTool1457 after a geometric optimization performed with Gaussian09,60 and the other parameters were taken from the force field GAFF.61 The periodic boundary conditions were applied in the simulations. The covalent bonds involving hydrogen atoms were constrained using the SHAKE algorithm.62 The time step of integration was set to 1 fs in the stages of equilibration and in the biased simulations (i.e., targeted and steered MD simulations below), and 2 fs for the unconstrained runs. The long-range electrostatic interactions were treated via the particle-mesh Ewald (PME) method63 with a grid spacing of 1 Å in each dimension. Meanwhile, the short-range nonbonded interactions were truncated at 12 Å, with the smoothing function switched on since 9 Å. Apart from the two types of biased simulations that were run in the NVT ensemble, the remaining ones were conducted in the NPT ensemble. The temperature was kept at 303.15 K through the Langevin thermostat, and the pressure was maintained at 1.013 bar using the Nosé−Hoover Langevin piston control.64 The simulation system underwent a sequence of steps to reach stability following the collective variable-based restraining scheme,54,56 and then the production run without constraints was extended up to 40 ns. Targeted Molecular Dynamics Simulations. The proposed functional rotation for substrate transport by AcrB was enforced by means of targeted MD approach.30,65−67 Targeted MD induces conformational transition between two known states by applying an external force. The force exerted on each atom is given by the gradient of the guiding potential: k [RMSD(t ) − RMSD*(t )]2 UTMD = 2N

where the force constant k is scaled down by the number N of targeted atoms, RMSD(t) is the instantaneous best-fit rootmean-square deviation (RMSD) of the current coordinates from the target coordinates RMSD*(t). RMSD*(t) evolves linearly from the initial RMSD at the first targeted MD step to the final RMSD at the last step. The starting structures of the targeted MD simulations for the two systems were taken from the final snapshots in their respective unbiased MD trajectories, in which the protonation states of Asp407, Asp408, and Lys940 in each protomer were changed to the next states prior to simulations. We performed a series of targeted MD simulations with different force constants and simulation time (Table S1, Supporting Information). The results presented in the main text refer to the simulations of 5 ns with the steering forces imposed on the heavy atoms of AcrB using a spring constant of 0.5 kcal/mol/Å2 per atom. A total of four targeted MD simulations were performed for each system with different initial velocities. After completion of targeted MD simulations, each run was continued by an additional 5 ns simulation with gradually reduced restraints on protein backbone and by another 5 ns unrestrained simulation. Steered Molecular Dynamics Simulations. The above targeted MD simulations lead to limited ligand displacements from the DBP. To investigate the complete extrusion process, we continued to employ the steered MD technique to pull the ligands inside the now extrusion protomer toward the central funnel enclosed by the TolC-docking domains. In each system, the ligand was pulled at a constant velocity along an instantaneously adjusted direction from its center of mass to the centroid of the Cα atoms of the residues Asn747 in chain A, Asp788 in chain B and C. Meanwhile, the Cα atoms of Ser79, Met575, Asn747, and Asp788 of each protomer were restrained with a force constant of 1.0 kcal/mol/Å2 to counterbalance the external force applied to the system. These residues were found to exhibit low fluctuations in the unbiased simulations, with the former two located in the porter domain and the latter two in the TolC-docking domain. Through a series of preliminary tests, a pulling velocity of 0.005 Å/ps along with a spring constant of 10 kcal/mol/Å2 was adopted, given that this combination appears to achieve a fine balance between the accuracy and computational efficiency. A total of 36 independent simulations for each system were performed, in which the initial structures were extracted from the respective unbiased trajectories after the targeted MD simulations. The simulation time is set to 6.5 ns to ensure that the ligands enter into the central funnel. Binding Free Energy Calculations. Computational methods that combine molecular mechanics and continuum solvation models, such as molecular mechanics−Poisson− Boltzmann surface area (MM−PBSA) and MM−GBSA have been widely exploited in free energy calculations of biological macromolecules.47−49 MM−GBSA is computationally more economical than MM−PBSA and has been proved to give a comparable or even better accuracy in ranking ligand affinities, although the latter is theoretically more rigorous.68−70 MM− GBSA was applied to estimate the binding affinities of ABI and DOX to AcrB from the unbiased MD trajectories. In MM− PBSA or MM−GBSA, the binding free energy of a receptor− ligand complex is formulized as ⟨ΔG bind⟩ = ⟨Gcomplex ⟩ − ⟨Greceptor ⟩ − ⟨G ligand⟩

(2)

where ⟨···⟩ denotes an average over a set of snapshots along an equilibrium trajectory, and G is the (absolute) free energy of

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each species composed of the gas-phase MM energy (EMM), the solvation energy (ΔGsol), and the solute conformation entropy (TΔS). Hence the free energy difference can be rewritten as ⟨ΔG bind⟩ = ⟨ΔEMM⟩ + ⟨ΔGsol⟩ − ⟨T ΔS⟩

(3)

The first two terms in eq 3 comprise various energetic contributions from different interaction types, expressed as ⟨ΔEMM⟩ = ⟨ΔE int⟩ + ⟨ΔE vdW ⟩ + ⟨ΔEele⟩

(4)

⟨ΔGsol⟩ = ⟨ΔGsol,pol⟩ + ⟨ΔGsol,nonpol⟩

(5)

where ΔEint, ΔEvdW, and ΔEele are the changes of the internal strain energy, van der Waals energy, and Coulombic interaction energy upon binding, respectively, and ΔGsol,pol and ΔGsol,nonpol represent the polar and nonpolar contributions to solvation energy, respectively. The above energy terms were calculated via the module MMPBSA.py of AmberTools14.71 The entropic contribution was not taken into account due to the difficulty in convergence through the normal-mode analysis.39 However, the omission of this term does not affect the qualitative analysis of the results here, as discussed previously.39,42 More details regarding the MM−GBSA calculations have been described in recent works.39,42,72

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.5b11942. A figure of the time evolutions of the distances of ABI and DOX from the DBP in the targeted MD simulations, a figure depicting the details of DOX dissociation from the DBP, a figure of the conformational changes at the exit gate as DOX and ABI pass through in the BEA state, a figure depicting the detailed conformational changes of the DBP during targeted MD simulations of ABI and DOX bound systems, a table of targeted MD simulation details, a table of per-residue contributions to the binding free energies by the MM−GBSA calculations, and a table of weights of the hydrophobic trap contributing to ΔGbind (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the National Major Basic Research Program of China (2014CB910201, 2011CB808505), National Science Foundation of China (21473034, 21403036), Specialized Research Fund for the Doctoral Program of Higher Education (20130071140004), and Science & Technology Commission of Shanghai Municipality (08DZ2270500). We thank the supercomputer center of Fudan University for their allocation of computer time.

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REFERENCES

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