Probing Structural Determinants of ATP-Binding Cassette Exporter

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Probing Structural Determinants of ABC Exporter Conformational Transition Using Coarse-grained Molecular Dynamics Zi Wang, and Jie-Lou Liao J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp509178k • Publication Date (Web): 30 Dec 2014 Downloaded from http://pubs.acs.org on January 1, 2015

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The Journal of Physical Chemistry

Probing Structural Determinants of ABC Exporter Conformational Transition Using Coarse-Grained Molecular Dynamics Zi Wang and Jie-Lou Liao* Department of Chemical Physics, University of Science and Technology of China,96 Jinzhai Rd, Hefei, Anhui Province, P. R. China, 230026

ATP-binding cassette (ABC) exporters pump various substrates across the cell membrane by alternating between inward-facing (IF) and outward-facing (OF) conformations of the transmembrane domains (TMDs). However, the structural determinants of the conformational transition and their functional roles are not fully understood. In this study, we carried out coarse-grained molecular dynamics (CG-MD) simulations with umbrella sampling for the multidrug transporter P-glycoprotein from Caenorhabditis elegans in the presence of the membrane and explicit water molecules. The potential of mean force (PMF) is obtained to identify a reliable pathway where the predicted OF and IF structures in good agreement with available experiments. The CG-MD simulations reveal that the different transmembrane (TM) helices play distinct but highly cooperative roles in the large-scale conformational changes. Most notably, the CG-MD trajectories show that the periplasmic gate is closed before the cytoplasmic gate is opened during the OF to IF conformational transition in response to the dissociation of the nucleotide-binding domains (NBDs), capturing the unidirectional feature of substrate translocation through the exporter. The structural and dynamical analyses identify the structural determinants and their functional roles in the structural transition. The present work sheds light on how the mechanical force generated upon the NBD dissociation is transferred to the periplasmic end at a distance over 70Å to close the gate, and subsequently to open the cytoplasmic gate. These results extend our understanding of the ABC transport mechanism.

*Corresponding author. E-mail: [email protected]

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Introduction The ATP-binding cassette (ABC) transporter family comprises the members of importers and exporters that use the energy from ATP hydrolysis to carry a wide variety of substrates across cellular membranes1. In contrast to ABC importers found only in prokaryotes, ABC exporters present ubiquitously in both eukaryotes and prokaryotes are able to recognize and expel many structurally diverse compounds out of cells. Some ABC exporters are implicated in multidrug resistance and represent key targets for drug resistance2-4. The ABC transporters share a common basic architecture, consisting of two nucleotide binding domains (NBDs) and two transmembrane domains (TMDs). The NBDs containing several highly conserved motifs form a tight dimer when bound to ATP and dissociate upon ATP hydrolysis and product release. The TMDs form a substrate translocation pathway. In response to the NBD dimerization/dissociation, the TMDs alternate between inward-facing (IF) and outward-facing (OF) states, allowing the translocation pathway open toward cytoplasm and toward periplasm, respectively5. The ATP-driven conformational changes in the NBDs have been studied extensively over the past decade6. The piece de resistance for understanding ABC transport has been the TMDs5. However, the structural determinants and their functional roles in the TMD conformational transition are not fully understood7. A number of high-resolution crystal structures of full ABC exporters in different conformations have been obtained in recent years7-14, offering snapshots of the ABC exporters during the transport cycle. The ABC exporters have a conserved core of 12 2


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transmembrane (TM) helices, 6 (TM1-TM6) from each TMD subunit (Figure 1). The TMD is connected to the NBD through the NBD-TMD interface, typically the TMD preceding the NBD. Each TMD contributes to this interface mainly through two intracellular coupling helices (ICHs), ICH1 and ICH27-15. These coupling helices are non-covalently interacted with the NBD and oriented roughly parallel to the membrane plane. In particular, ICH2, the linker between TM4 and TM5, as a ball docks into a groove (socket) formed by several α-helices and β-sheets from the NBD, forming a 〝 ball-and-socket 〞 joint11. This structural feature of the NBD-TMD interface is essential for transmitting signal of conformational changes from the NBD to the TMD. Another striking structural feature of the ABC exporters is that ICH2 along with TM4 and TM5 in one subunit reach across and contact the NBD in the other subunit8 (Figure 1). This structural arrangement, which does not exist in an ABC importer, allows TM4 and TM5 in one subunit to tightly interact with TM2 in the other subunit, approximately in a parallel orientation, forming a three helix bundle7-15. An internal (cytoplasmic) gate is thus constructed with two 〝 wings 〞 8, each consisting of TM1-TM3 and TM6 from one subunit and TM4-TM5 from the other subunit

(Fig.1(a)), whereas an external (periplasmic) gate is formed but with two

different wings, each comprising TM1 and TM2 from one subunit and TM3-TM6 from the other (Fig.1(b)). While the internal gate is hinged at the loops between TM3 and TM4 (denoted as L3-4) and between TM5 and TM6 (L5-6) (Figure 1(a)) at the periplasmic side13, the external gate is hinged at ICH1 located on the TMD-NBD interface. The internal and external gates are arranged to face two opposite directions, 3



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i.e., IF and OF, respectively, and are oriented roughly perpendicular to each other5. An ABC exporter can adopt an IF (Figure 1(a)) or an OF conformation (Figure 1(b)). In an IF state, the internal gate is open whereas the external gate is closed, allowing substrates to access from the cell interior, and vice versa in an OF conformation with the extrusion pocket exposed to the periplasm. Structural studies have offered important insight into the mechanism of the ABC export, but detailed molecular understanding of the structural transitions requires a dynamical description of the process. Molecular dynamics (MD) simulations have been employed to study the dynamics of several ABC exporters16-22. A full description of conformational transitions underlying the transport cycle is still beyond the reach of all-atom MD simulations due to the limited time scale of MD23. One way to address this issue is to use a coarse-grained (CG) modeling approach to reduce the system size and remove fastest degrees of freedom24,25. This strategy can be particularly useful for description of large-scale conformational changes in proteins including ABC transporters26,27. However, the state transition of an ABC exporter involves crossing a free-energy barrier. When the barrier is high enough, the conformations located in the barrier region can still be inaccessible on the time scale of CG simulations. Another way is to apply biasing potentials to enhance sampling of conformations with little possibility of access to MD simulations. Recently, a nonequilibrium-driven all-atom MD28 has been used to study the structural transition of a bacterial exporter, MsbA, of which one OF and two IF crystal structures are available9 (termed IF-c and IF-o, 4


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respectively). This work provides an instructive mechanistic picture for the highly cooperative motions of the transporter as a rigid body. However, the free energy profile along the OF→IF transition pathway is incomplete and specific functional roles of different TM helices critical to the conformational transition still remain to be elucidated. Here, we attempt to study the large-scale conformational changes in the TMDs in response to the NBD dissociation and to probe the structural determinants and their functional roles in the conformational transition. In this report, coarse-grained molecular dynamics (CG-MD) simulations combined with the umbrella sampling method are applied to the multidrug transporter P-gp from Caenorhabditis elegans11 as a prototype in the presence of the membrane and explicit water molecules. Methods The crystal structure of the C. elegans P-gp9 (PDB code: 4F4C, termed 4F4C structure), which represents an IF conformation (see Figure 1(a)), was employed for the coarse-grained modeling and simulation using the MARTINI method developed for proteins and lipids24,25. Here, for convenience of discussion, TM7-TM12, ICH3 and ICH4, which correspond to TM1-TM6, ICH1 and ICH2 in the other subunit, are denoted as TM1′-TM6′, ICH1′ and ICH2′, respectively (Figure 1). We notice that TM4′ is discontinuous in the X-ray structure possibly caused by the drug interactions11. Without loss of generality, the discontinuity of this helix is fixed with an elastic network model (ENM)29. The ENM was also used to stabilize the backbone conformations of the protein in the following CG modeling. In addition, the residues 5



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preceding TM1 in the crystal structure are removed for simplicity of discussion. The MARTINI method has been widely and successfully applied for simulations of lipid membranes and membrane proteins24,25,29,30. It maps four heavy atoms on average to a single CG particle with an exception for ring-like molecules whose ring-like fragments are mapped based on the two-to-one approach. In the MARTINI model, four water molecules are also mapped to a CG water particle and one ion is represented by a single CG ion particle. The van der Waals interactions are described using a Lennard-Jones potential function, whereas charged CG particles interact via a Coulomb energy function with a relative dielectric constant of 15 for explicit screening. The nonbonded interactions are cut off at a distance of 1.2nm with a shifted function from 0.9 to 1.2nm for the Lennard-Jones potential and from 0.0 to 1.2nm for the Coulomb potential24,25,30. The long-range electrostatic interactions were treated using the particle mesh Ewald (PME) metthod32. A real space cut-off of 1.2nm and a 0.12nm Fourier grid spacing were used for PME. The CG model of the C. elegans P-gp transporter was inserted into a pre-equilibrated DPPC lipid bilayer modeled with the MARTINI CG force field24. The protein-lipid system was solvated using the standard MARTTINI water model24. The final simulation system contains one C. elegans protein (2352 particles), 7716 DPPC and 38144 water CG particles, resulting in a total of 48212 MARTINI particles. The simulation system was embedded in a rectangular box of dimension 14 nm×19nm×22nm and periodic boundary conditions were applied in all directions (SI, Figure S1). CG-MD simulations with a 20-fs time step were performed using the GROMACS 6


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package, version 4.5.431. The system was weakly coupled to a Berendsen thermostat and barostat maintaining the temperature at 310K and the pressure at 1 atm in all simulations unless otherwise indicated. Following a 10000-step energy minimization, the above 4F4C structure in membrane and aqueous environment was equilibrated. To probe dynamical correlations between two gates in the conformational transition, the center-of-mass (COM) distance, d1, between the two opposing wings, each containing the associated NBD, of the cytoplasmic gate was gradually lowered by successive pushing and equilibration steps. Weak pushing forces and their rates were carefully tested and applied to the NBDs and several residues of TM3-TM3ˊ and TM4-TM4ˊ at the cytoplasmic side, respectively (SI, Figure S2). Here, it should be noted that all external forces were applied only to the NBDs and a small part of the internal gate in this work. The value of d1 is eventually achieved to be 2.5 nm at the end of the pushing simulation. Application of the external forces perturbs the equilibrium of the system, thereby precluding the calculation of a reliable PMF directly from the simulation trajectories. A weighted histogram analysis method (WHAM) proposed by Kumar et al.35 have widely been used to extract equilibrium PMF data from the nonequilibrium simulations. This method computes PMF from a number of simulations conducted on configurations generated from a single MD trajectory. Here, we used the WHAM procedure for the computation of PMF along the COM distance, d1, as a system-specific reaction coordinate. Snapshots of a pushing simulation trajectory were taken to generate the 201 configurations for the umbrella sampling simulations36. In each window, 50-ns CG-MD umbrella sampling simulations were 7



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performed, resulting in a total simulation time of 10.0 µs (effective simulation time24 is ~40.0 µs ). The potential of mean force (PMF) along the defined reaction coordinate from a series of the umbrella sampling simulations was then evaluated using the WHAM analysis to unbias the effect of the biasing umbrella potential. In addition, external forces were also carefully applied only to the NBDs in this research. A number of structures similar to those mentioned above were obtained, and thus a similar PMF (data not shown). The gating dynamical behaviors are also similar to those discussed below. However, the simulation time was much longer as expected. Results and Discussion PMF along the transition pathway. The PMF calculation of the ABC exporter requires the definition of a reaction coordinate designed to monitor the conformational changes in the protein. A widely used reaction coordinate is RMSD of an evolving conformation relative to a target. However, the structural quality of the target is a determining factor for the reliability of the results. In a recent study mentioned above28, the angles between the roll axes of two wings of the internal gate, α, of the external gate, β, and of the two NBDs, γ, were used as the reaction coordinates. These variables are useful for monitoring the collective motion of a rigid body, for example, α and β are used to measure the extent of the opening of the internal and the external gate, respectively. But these angle coordinates seem unable to accurately determine whether a gate is completely closed because it is a couple of gatekeeper residues that usually lock the gates as discussed below. Here, the COM distance, d1, which is an intuitive choice for describing the opening/closing motion of 8


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the cytoplasmic gate, was used as a reaction coordinate to probe the free energy landscape. The COM distances, dNBD, between the two NBDs, d2, between the two wings of the periplasmic gate, and those between TMi and TMi′ (i=1-6) important to monitor the gate motion in response to the NBD movement are also measured over the course of the CG-MD simulations in this study. The PMF along the reaction coordinate, d1, was calculated using the aforementioned CG-MD simulations and the umbrella sampling method. The results are shown in Figure 2. The calculated PMF covers the 2.5-4.4 nm distance range. There exists a small basin around d1=4.3nm, which is associated with the 4F4C X-ray structure (see Figure 2(a)). The relatively high free energy of the 4F4C conformation could reflect the differences between the X-ray and the CG modeled structure where, for example, several substrates tightly bound in the X-ray structure were taken out. The deepest minimum positioned in a basin around 3.6-3.8nm corresponds to an IF conformation (see Figure 2 (b)) resembling the crystal structures of Pg-p13. The PMF results discussed thus far are in qualitative agreement with the previous study28. There exists a minimum located around d1=3.0nm corresponding to a closed IF conformation (termed IF-c, see Figure 2(c)) whose NBDs are loosely contacted, and the cytoplasmic gate is open while the periplasmic gate is closed. Interestingly, this IF-c backbone conformation resembles that of the heterodimeric ABC transporter TM287/TM288 (PDB: 3QF4, termed 3QF4 structure hereafter)12 rather than the above MsAb IF-c structure9. Superposition of the Cα atoms of the computed C. elegans IF-c with those of the 3QF4 X-ray structure gives a root-mean-squared 9



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deviation (RMSD) of 2.8Å, whereas the Cα RMSD with the MsAb IF-c is 10.8 Å. A minimum around d1=2.8nm corresponds to an OF state, in which the NBDs are in close contact with each other. In this conformation, the cytoplasmic gate is closed whereas the periplasmic gate is open. The corresponding backbone conformation (Figure 2 (d)) resembles that of the SAV1866 crystal structure (PDB code: 2HYD)8 with a Cα RMSD of 3.2 Å. In Figure 2, the free energy of the OF state in the absence of nucleotide and substrate is relatively high compared to that of the IF state at the global minimum. It can be anticipated that ATP binding would lower the OF free energy basin and make the OF state more stable. The NBD dimerization could generate some tension in the TMDs as will be discussed below. To elucidate the conformational changes in the TMDs in response to the NBD dissociation, the CG-MD simulations were performed by pulling the calculated C. elegans OF structure back to the IF conformation. Weak pulling forces were applied to the same parts of the protein as in the above pushing simulations (SI, Figure S2). The PMF extracted from the CG-MD trajectory using the umbrella sampling and WHAM methods is in good agreement with that shown in Figure 2. In the remainder of this section, we focus on analyzing gating movements and dynamical roles of the TMD helices from the pulling CG-MD trajectory. Conformational gating and functional roles of the TM helices revealed by the CG-MD simulations. The NBD dissociation initiates a cascade of conformational changes in the protein. Here, the weak pulling forces were employed only to the two NBDs and several residues of TM3 and TM4′ on one side of the internal gate and 10


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TM4 and TM3′ on the other at the cytoplasmic end (SI, Figure S2(c)). Whereas the COM distances, dNBD and dICH2, are used to monitor the relative motions of the two NBDs and ICH2-ICH2′, respectively, the distances, d1 and d2, are computed to measure the movements of the cytoplasmic and periplasmic gates severally. Seen in Figure 3, d2 decreases as d1 increases, i.e., while the cytoplasmic gate is driven to open by the NBD dissociation, the two sides of the periplasmic gate approach each other to close the gate where no external force is applied. The result highlights the cooperative nature of the two gates in the conformational transition. Figure 3 also demonstrates that the relative motion of ICH2 and ICH2′ is positively correlated with that of two NBDs, showing that the NBDs together with the ICHs move as a semi-rigid body during the conformational transition. This observation is consistent with the available X-ray structures7-15 that ICH2/ICH2′ interacts with its associated NBD, forming a〝ball-and-socket〞joint as previously mentioned.

.

To probe the dynamical roles of the TM helices in the conformational gating in response to the NBD dissociation, the COM distances, dic between TMi and TMi′ (i=2-6) at the cytoplasmic end (five residues), and d i p (i=1-6) at the periplasmic end were measured along the CG-MD trajectory (Figure 4(a)-(b))). Overall, d i p is decreased while dic increases during the course of the simulation (see Figure 4(a)-(b)), i.e., when TMi and TMi′ departs from each other at the cytoplasmic side, they approach each other at the periplasmic end. Specifically, TM3-TM3′ and TM4-TM4′ are two innermost helix pairs that manipulate the cytoplasmic gating movements (see Figure 4(a)). While d 4c starts to increase after t=0.6ns, d 3c remains almost constant 11



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before t=1.8ns. The results demonstrate that both TM3-TM3′and TM4-TM4′ pairs close the internal gate during an initial period of the OF to IF transition, but the TM4-TM4′ pair dissociates before the TM3-TM3′ pair does at the cytoplasmic end. As will be discussed later, the earlier TM4-TM4′ dissociation is crucial for the external gate to be closed before the internal gate is opened. It is important to note that although the external forces were applied to pull TM3 and TM3′ as well as TM4 and TM4′ at the cytoplasmic side to depart from each other (SI, Figure S2(c)), the COM distances, d 3c and d 4c remain unchanged for the initial period of time. This finding demonstrates that the pulling force is a weak perturbation that is not able to change the functional role of the helices in the exporter gating. These weak perturbations can thus be used to probe the structural determinants and their functional roles in the conformational transition. At the periplasmic side, whereas TM4 and TM4′ as well as TM3 and TM3′ are the outermost helices, TM1-TM1′ and TM6-TM6′ are two innermost helix pairs of the external gate. In the case of the C. elegans P-gp studied here, the TM1-TM1′ distance,

d1p , remains shorter than the TM6-TM6′ distance, d 6p , during the simulation course, indicating that TM1 and TM1′ approach each other closer, serving as the gatekeeper helices to close the periplasmic gate and eventually lock the gate by their two gatekeeper residues, I106 and A783 (see Figure 5). While TM6 and TM6′ at the periplasmic side take some role in the manipulation of the external gating, these two helices function as a flexible linker between the TMDs and the NBDs, respectively. Seen in Figure 4(a)-(b), the TM helices at the periplasmic side are relatively flexible 12


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compared to at the cytoplasmic side. Interestingly, it is the TM6-TM6′ pair that locks the external gate in some ABC exporters shown in X-ray structures7,12,15. Most likely, the TM1-TM1′ and TM6-TM6′ pairs at the periplasmic side, which are both highly flexible, compete with each other for closing the external gate during the structural transition. Two residues, T197 on TM3 and S856 on TM3′, whose distance variation is shown in Figure 5, are found to be the gatekeepers that lock the cytoplasmic gate before t=1.8ns and open the gate thereafter. I106 on TM1 and A783 on TM1′ mentioned above are the gatekeeper residues that lock the periplasmic gate after t=1.2ns (see Figure 5). Here, a key feature of the exporter gating is identified, that is, the periplasmic gate is closed (t=1.2ns) before the cytoplasmic gate is opened (t=1.8ns, Figure 5) in response to the NBD dissociation. This dynamic feature is crucial for the unidirectionality of the ABC export. If the opening of the internal gate happened prior to the closure of the external gate during the OF to IF conformational transition, the two gates would be both open. This might induce reverse transport of substrates through the cell membrane. At first sight, however, this finding raises a question of how this can happen, as the signal of the conformational changes triggered by the NBD dissociation is first transferred to the cytoplasmic gate region. To address this question, further dynamical and structural analyses are performed. Structural and dynamical analyses demonstrate that the TM4-TM5-TM2′ bundle as well as its counterpart TM4′-TM5′-TM2, the core component of both the internal and the external gates, plays a pivotal role in the exporter gating in response to the 13



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NBD dissociation. The tight interactions between TM5 and TM2′ remain throughout the simulation course. The TM2-TM2′ and TM5-TM5′ pairs behavior therefore in the similar fashion (see d 2c and d 5c in Figure 4(a), and d 2p and d 5p in Figure 4(b)). TM2′ (with TM5-TM4) located at one side of the external gate can rotate around ICH1′ toward TM3′ at the opposite side of the gate. This mode of rotation is functionally important to close the external gate. Examination of the structures from the simulations shows that the OF state (see Figure 2(d)) adopts a large torsional angle for TM2′-ICH1′-TM3′ as well as TM2-ICH1-TM3 and largely bent conformations for TM4/TM4′ and TM5/ TM5′, respectively, compared to the IF states (Figure 2(b)-(c)), generating torsional strain and bending stress. Indeed, the torsional angle of TM2-ICH1-TM3 is reduced by ~90 during the OF to IF transition. This OF conformation is maintained by a network of interactions including those of TM4 with TM3 and TM4′ on the opposite side of the internal gate (vice versa for TM4′), and those of TM3 with TM3′, TM4, and the X-loop of the NBD on the other subunit. Here, the X-loop (TLVGDRG) is a short conserved sequence preceding the ABC signature motif. The NBD dissociation disrupts the above interactions with TM4 and TM4′, respectively, while the TM3-TM3′ contacts remain unbroken until the cytoplasmic gate is open. The disruption of the interactions with TM4/TM4′ leads to the release of the torsional strain, rendering the TM4-TM5-TM2′/TM4′-TM5′-TM2 bundle to rotate around ICH1′/ICH1 toward the opposite side of the external gate as discussed above. Small variations of TM2-TM2′ and TM5-TM5′ at the cytoplasmic end can result in relatively large displacements at the periplasmic end since these helices exceed 70Å 14


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in length. For example, the value of d 2c is increased by ~2.0Å, while d 2p is reduced by ~5.8Å during the closing process of the external gate (Figure 4(a)-4(b)). As TM1 and TM1′ are linked with TM2 and TM2′, respectively, the relative motion of the TM2-TM2′ pair drives TM1 and TM1′ at the periplasmic side to approach each other, allowing the gatekeepers, I106 on TM1 and A783 on TM1′, to close and lock the external gate after t=1.2ns (Figure 5). In addition, the movements of TM5 and TM5′ also drive the neighboring TM6 and TM6′, respectively, to approach each other (Figure 4(b)). As the NBD dissociation continues, the TM4-TM5-TM2′ bundle moves along with its associated NBD against its counterpart on the opposite side of the internal gate, driving TM3′, which is linked to TM2′ via ICH1′, to depart from TM3 at the cytoplasmic side. Eventually, the TM3-TM3′ contacts are broken and the cytoplasmic gate is open. The conformational transition is thus accomplished from the OF to the IF-c state (see Figure 2(c)) where the C-termini of the NBDs are still contacted. As the conformational rearrangements continue, the IF-o state (see Figure 2(b)) is eventually reached. Conclusion ABC exporters extrude various substrates out of the cell membrane through dynamic switching between the OF and IF conformations of the TMDs. However, detailed understanding of dynamical behaviors of the TM helices and their functional roles in the conformational transition is currently lacking. In the present study, the combined CG-MD and umbrella sampling method was used to study the C. elegans 15



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multidrug ABC transporter. The PMF was computed to identify a reliable pathway where the calculated OF and IF structures are in good agreement with the X-ray experimental results. The optimized external forces were applied only to the NBDs and a small part of the internal gate to expedite the NBD dimerization/dissociation process, serving as a weak perturbation to probe the structural determinants of the TMD gating and their functional roles. The pulling CG-MD simulations show that in response to the NBD dissociation, the opening of the internal gate and the closing of the external gate occur in a highly collaborative manner. Interestingly, the trajectories show that the closure of the perplasmic gate happens before the cytoplasmic gate is opened, highlighting the functional importance of the timing of the gating motions for the unidirectional transport of substrates. The structural and dynamical analyses demonstrate that ICH2, ICH2′ and the TMi-TMi′ (i=1-6) pairs play different but highly cooperative roles in the TMD gating movements in response to the NBD dissociation. TM3-TM3′ and TM4-TM4′ are the innermost helix pairs of the cytoplasmic gate, whereas TM1-TM1′ and TM6-TM6′ are the innermost helix pairs of the periplasmic gate, responsible for the closing and opening of the two gates, respectively. TM4 and TM4′ interact with their counterparts on the opposite side of the internal gate, maintaining the periplasmic gate in a tensed conformation in the OF structure discussed above. These interactions are disrupted in response to the NBD dissociation where ICH2 and ICH2′ play a crucial role in the NBD-TMD communication. ICH2 and ICH2′ interact with their associated NBDs in the fashion of the 〝 ball-and-socket 〞 joint, respectively. The mechanical force 16


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generated from the NBD dissociation is transferred through these intracellular coupling helices to drive TM4-TM5 and TM4′-TM5′ to depart from each other at the cytoplasmic end. Subsequently, the aforementioned interactions with TM4 and TM4′ are disrupted, leading to the TM2′-TM4-TM5 bundle along with TM1′ and TM6 and TM2-TM4′-TM5′ with TM1 and TM6′ rotate to each other to close and lock the external gate. While TM4 and TM4′ as two core components of the cytoplasmic gate manipulate the periplasmic gating movements, the TM3-TM3′ pair controls the closing and opening of the internal gate. After the external gate is closed, the tight TM3-TM3′ contacts still remain unchanged until the NBD dissociation further drives the contacts to break and the internal gate to open. The present work demonstrates that the ABC exporter is able to function as a delicate molecular machine. Given the overall similarity in the arrangement of the TM helices, these results suggest a common mechanism of the TMD conformational transition in the ABC exporter family members. In addition, the method applied in this study can be useful to study large-scale conformational changes in other membrane transporters. Acknowledgment. This work was funded by the National Science Foundation of China under Grant Nos. 21073170 and 21273209. We gratefully acknowledge the Supercomputing Center at University of Science and Technology of China (USTC) for computational resources. Supporting Information Available: Two additional figures are provided, the first presenting the coarse-grained model of the system studied in the paper and the second 17



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showing the external forces used in the study. The material is available free of charge through the Internet at http://pubs.acs.org.

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(19) Ferreira, R. J.; Ferreira, M.-J. U.; dos Santos, D. J. V. A. Insights on P-glycoprotein’s Efflux Mechanism Obtained by Molecular Dynamics Simulations. J. Chem. Theory Comput. 2012, 8, 1853-1864. (20) St-Pierre, J. F.; Bunker, A.; Rog, T.; Karttunen, M.; Mousseau, N. Molecular Dynamics Simulations of the Bacterial ABC Transporter SAV1866 in the Closed Form. J. Phys. Chem. B 2012, 116, 2934-2942. (21) Mehmood, S.; Domene, C.; Forest, E.; Jault, J. M. Dynamics of a Bacterial Multidrug ABC Transporter in the Inward- and Outward-Facing Conformations. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 10832-10836. (22) Wen, P. -C.; Verhalen, B.; Wilkens, S.; Mchaourab, H. S.; Tajkhorshid, E. On the Origin of Large Flexibility of P-glycoprotein in the Inward-Facing State. J. Biol. Chem. 2013, 288, 19211-19220. (23) Shaikh, S. A.; Li, J.; Enkavi, G.; Wen, P. -C.; Huang, Z.; Tajkhorshid, E. Visualizing Functional Motions of Membrane Transporters with Molecular Dynamics Simulations. Biochemistry 2013, 52, 569-587. (24) Marrink, S. J.; Risselada, H. J.; Yefimov, S.; Tieleman, D. P.; de Vries, A. H. The MARTINI Force Field: Coarse Grained Model for Biomolecular Simulations. J. Phys. Chem. B 2007, 111, 7812-7824. (25) Monticelli, L.; Kandasamy, S. K.; Periole, X.; Larson, R. G.; Tieleman, D. P.; Marrink, S. J. The MARTINI Coarse-Grained Force Field: Extension to Protein. J. Chem. Theory Comput. 2008, 4, 819-834. (26) Liao, J. -L.; Beratan, D. N. How Does Protein Architecture Facilitate the Transduction of ATP Chemical-Bond Energy into Mechanical Work? The Cases of Nitrogenase and ATP Binding-Cassette Proteins. 2004, 87, 1369-1377. (27)Ward, A. B.; Guvench, O.; Hills Jr., R.D. Coarse Grain Lipid–Protein Molecular Interactions and Diffusion with MsbA Flippase. Proteins, 2012, 80, 2178-2190. (28) Moradi, M.; Tajkhorshid, E. Mechanistic Picture for Conformational Transition of a Membrane Transporter at Atomic Resolution. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 18916-18921. (29) Periole, X.; Cavalli, M.; Marrink, S. J.; Ceruso, M. A. Combining an Elastic 20


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Table of Contents Graphic (TOC GRAPHIC)

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FIGURE

CAPTIONS

Figure 1. Stereoviews of two conformations of ABC exporters. (a) Inward-facing (IF) conformation from the C.elegans multidrug transporter P-glycoprotein (PDB code: 4F4C). The TM1-TM6 helices in the TMD are colored orange. The TM7-TM12 helices are denoted by TM1ˊ-TM6ˊ (blue), and the intracellular coupling helices ICH3 and ICH4 are denoted by ICH1ˊ (green) and ICH2ˊ (red), respectively. See detailed discussion in the main text. (b) Outward-facing (OF) conformation from SAV1866 (PDB code: 2HYD). Figure 2. PMF is expressed as a function of the COM distance, d1, between the two wings of the cytoplasmic gate, each containing the associated NBD. Figure 3. Time evolution of the distances, d1, d2, dNBD and dICH2. Here, d2 is the COM distance between the two wings of the periplasmic gate. dNBD and dICH2 are the COM distances between the two NBDs and between ICH2 and ICH2ˊ, respectively. Figure 4(a) Time evolution of dICH2 and the COM distances, d ic (i=2-6) between TMi and TMiˊ at the cytoplasmic end. Figure 4(b) Time evolution of the COM distances, d ip (i=1-6) between TMi and TMiˊ at the periplasmic end. Figure 5. Time evolution of the Cα-Cα distances, dT197-S856, between the gatekeeper residues, T197 on TM3 and S856 on TM3ˊ, and dI106-A783 between the gatekeeper residues, I106 on TM1 and A783 on TM1ˊ.

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Figure 1.

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Figure 2

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Figure 3.

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Figure 4(a)

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Figure 4(b)

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Figure 5.

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TM-hinge region (L3-4, L5-6) L5-6 L3-4

TM1′

900

T TM2 M

TM6′

TM3 TM1

L5-6

TM3

L3-4 TM6 TM4 TM1

TMD TM2′ TM5

NBD

TTM6 T TM3′ M TM4 2 ICH1′ M TM4′ ICH2 2

TM5′ ICH1 ICH2′

TM2

TM5 IC ICH2 H2

ICH1

(a) (b) (a)

(a) ACS Paragon Plus Environment


16

(a)

(d)

12

PMF(kcal/mol)

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(c) (b)

8

4

0 2.4

2.6

2.8

3.0

3.2

3.4

d1(nm)

3.6


3.8

4.0

4.2

4.4

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dc6

d

c 5

dICH2

dc4 dc3 ACS Paragon Plus Environment

d

c 2

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dp4

dp3 dp5 dp2

dp6 dp1



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Probing Structural Determinants of ATP-Binding Cassette Exporter

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