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Yun Huang, Hao-chen Xu, Jie-lou Liao. Coarse-Grained Free-Energy Simulations of Conformational State Transitions in an Adenosine 5′-Triphosphate-binding Cassette Exporter[J]. Chinese Journal of Chemical Physics , 2020, 33(6): 712-716. doi: 10.1063/1674-0068/cjcp1908149
Citation: Yun Huang, Hao-chen Xu, Jie-lou Liao. Coarse-Grained Free-Energy Simulations of Conformational State Transitions in an Adenosine 5′-Triphosphate-binding Cassette Exporter[J]. Chinese Journal of Chemical Physics , 2020, 33(6): 712-716. doi: 10.1063/1674-0068/cjcp1908149

Coarse-Grained Free-Energy Simulations of Conformational State Transitions in an Adenosine 5′-Triphosphate-binding Cassette Exporter

doi: 10.1063/1674-0068/cjcp1908149
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  • Corresponding author: Jie-lou Liao, E-mail: liaojl@ustc.edu.cn
  • Received Date: 2019-08-06
  • Accepted Date: 2019-08-30
  • Publish Date: 2020-12-27
  • ATP-binding cassette exporters transport many substrates out of cellular membranes via alternating between inward-facing and outward-facing conformations. Despite extensive research efforts over the past decades, understanding of the molecular mechanism remains elusive. As these large-scale conformational movements are global and collective, we have previously performed extensive coarse-grained molecular dynamics simulations of the potential of mean force along the conformational transition pathway [J. Phys. Chem. B 119 , 1295 (2015)]. However, the occluded conformational state, in which both the internal and external gate are closed, was not determined in the calculated free energy profile. In this work, we extend the above methods to the calculation of the free energy profile along the reaction coordinate, $d_1$$-$$d_2$, which are the COM distances between the two sides of the internal ($d_1$) and the external gate ($d_2$). The potential of mean force is thus obtained to identify the transition pathway, along which several outward-facing, inward-facing, and occluded state structures are predicted in good agreement with structural experiments. Our coarse-grained molecular dynamics free-energy simulations demonstrate that the internal gate is closed before the external gate is open during the inward-facing to outward-facing transition and vice versa during the inward-facing to outward-facing transition. Our results capture the unidirectional feature of substrate translocation via the exporter, which is functionally important in biology. This finding is different from the previous result, in which both the internal and external gates are open reported in an X-ray experiment [Proc. Natl. Acad. Sci. USA 104 , 19005 (2007)]. Our study sheds light on the molecular mechanism of the state transitions in an ATP-binding cassette exporter.
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Coarse-Grained Free-Energy Simulations of Conformational State Transitions in an Adenosine 5′-Triphosphate-binding Cassette Exporter

doi: 10.1063/1674-0068/cjcp1908149

Abstract: ATP-binding cassette exporters transport many substrates out of cellular membranes via alternating between inward-facing and outward-facing conformations. Despite extensive research efforts over the past decades, understanding of the molecular mechanism remains elusive. As these large-scale conformational movements are global and collective, we have previously performed extensive coarse-grained molecular dynamics simulations of the potential of mean force along the conformational transition pathway [J. Phys. Chem. B 119 , 1295 (2015)]. However, the occluded conformational state, in which both the internal and external gate are closed, was not determined in the calculated free energy profile. In this work, we extend the above methods to the calculation of the free energy profile along the reaction coordinate, $d_1$$-$$d_2$, which are the COM distances between the two sides of the internal ($d_1$) and the external gate ($d_2$). The potential of mean force is thus obtained to identify the transition pathway, along which several outward-facing, inward-facing, and occluded state structures are predicted in good agreement with structural experiments. Our coarse-grained molecular dynamics free-energy simulations demonstrate that the internal gate is closed before the external gate is open during the inward-facing to outward-facing transition and vice versa during the inward-facing to outward-facing transition. Our results capture the unidirectional feature of substrate translocation via the exporter, which is functionally important in biology. This finding is different from the previous result, in which both the internal and external gates are open reported in an X-ray experiment [Proc. Natl. Acad. Sci. USA 104 , 19005 (2007)]. Our study sheds light on the molecular mechanism of the state transitions in an ATP-binding cassette exporter.

Yun Huang, Hao-chen Xu, Jie-lou Liao. Coarse-Grained Free-Energy Simulations of Conformational State Transitions in an Adenosine 5′-Triphosphate-binding Cassette Exporter[J]. Chinese Journal of Chemical Physics , 2020, 33(6): 712-716. doi: 10.1063/1674-0068/cjcp1908149
Citation: Yun Huang, Hao-chen Xu, Jie-lou Liao. Coarse-Grained Free-Energy Simulations of Conformational State Transitions in an Adenosine 5′-Triphosphate-binding Cassette Exporter[J]. Chinese Journal of Chemical Physics , 2020, 33(6): 712-716. doi: 10.1063/1674-0068/cjcp1908149
  • Adenosine 5$ ' $-triphosphate (ATP)-binding cassette (ABC) exporters are molecular machines that translocate a wide variety of substrates out of biological membranes [1-3]. ABC exporters share a common architecture with two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs) that form a pathway for substrate translocation (see FIG. 1). As the NBDs dimerize upon ATP binding, they dissociate after ATP hydrolysis and the release of the hydrolytic products. The NBD dimerization/dissociation drives large-scale conformational changes for unidirectional transport of substrates out of the membrane by alternating between inward-facing (IF) and outward-facing (OF) conformations in the TMDs.

    Figure 1.  Stereoviews of two conformations of an ABC exporter (a) inward-facing conformation, in which the internal gate is open whereas the external gate is closed, (b) outward-facing conformation, in which the internal gate is closed whereas the external gate is open.

    The TMDs comprise twelve transmembrane helices (TMs), each has six TMs (TM$ i $ and TM$ i' $, $ i $ = 1$ - $6, respectively in FIG. 1). The TMD connects its associated NBD with two intracellular coupling helices, ICH1 and ICH2, which are oriented roughly parallel to the membrane plane. An exporter is characterized by a domain-swapped arrangement, i.e., TM4 and TM5 in one subunit reach across and contact the NBD in the other subunit [4, 5]. This structural arrangement allows TM4 and TM5 in one subunit to interact tightly with TM2$ ' $ in the other subunit (approximately in a parallel orientation), facilitating the formation of a TM4-TM5-TM2$ ' $ (TM4$ ' $-TM5$ ' $-TM2) bundle. The TMDs comprise two gates, an internal gate facing the cytoplasmic side inward facing and an external gate facing the periplasmic side (outward facing), which are arranged roughly perpendicular to each other. 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 the OF conformation with the extrusion pocket exposed to the periplasm [6]. While both gates are closed, the protein is located at the occluded (OC) state. Despite substantial efforts over the past decades, mechanistic understanding of ABC exporter gating movements at the molecular level is not fully understood [7, 8].

    A current understanding of NBD-coupled conformational transitions in the TMDs was based on the full-length crystal structures of the bacterial ABC exporter, MsbA, including one OF conformation (PDB code: 3B60; termed 3B60 structure hereafter) and two IF conformations (PDB codes: 3B5X and 3B5W, termed IF-closed (IF-c) and IF-open (IF-o), whose NBDs were found significantly twisted [9]. Thereby, it was proposed that the NBD twisting motion drives the IF$ \leftrightarrow $OF transition of MbA. However, recent structural studies of MsbA [10, 11] have challenged this mechanistic view. Although remarkable progress in the structural biology for ABC exporters has been made in the past decade, the nature of the transition pathway in these proteins remains largely unknown [8].

    In principle, detailed elucidation of the molecular mechanism of ABC exporter action requires the evaluation of free energy profile along a certain "reaction coordinate" (i.e., potential mean force, PMF) [6, 8, 12]. These free energy landscapes typically contain distinct conformational states separated by high barriers that prevent an efficient sampling of configuration space by standard all-atom MD simulations. To address this issue, one way is to apply a biased or nonequilibrium method to enhance sampling of conformations with little possibility of access to standard all-atom MD simulations. Moradi and Tajkhorshid used the nonequilibrium-driven all-atom MD to study the conformational transition of MsbA with the aforementioned crystal structures, particularly the IF-c one as the references [12]. Their work offers an interesting mechanistic picture for the highly cooperative movements of the transporter as a rigid body. However, the obtained free energy profile cannot identify the OF and OC states, demonstrating that their calculations were unable to capture the OF$ \leftrightarrow $IF transition. Most likely, the reaction coordinate, $ \alpha $, which was used in their work [12], is defined as the angle between two sides of the internal gate, but it is too coarse-grained for describing the gating movements of the protein, as the complete closure of the (internal) gate is often controlled by a few gatekeeper residues [6]. Furthermore, careful examination shows that the IFc structure (PDB code: 3B5X) [9] used in their calculation [12] represents a state, in which the internal and external gate are both open (see FIG. 2(a)). This is contradictory against the unidirectionality (i.e., from intracellular to extracellular) of transport substrates by an exporter.

    Figure 2.  Views of the external gate from the periplasmic entrance. (a) The X-ray IF-c structure of MsbA (PDB code: 3B5X) [9], the C$ _\alpha $$ - $C$ _\alpha $ distance between the two gatekeeper residues at the external gate is 18.6 Å, showing that the external gate is open (see discussion in the main text). (b) A typical IF structure, for example, the C. elegan IF structure (PDB code: 4F4C) [13], the C$ _\alpha $$ - $C$ _\alpha $ distance between the gatekeeper residues at the external gate is 5.5 Å. (c) The supposition of 3B5X (red) and 4F4C (green). (d) The supposition of the above 3B5X (red) and the IF-o structure of MsAb (blue) (PDB code: 3B5W) [9], where the C$ _\alpha $$ - $C$ _\alpha $ distance between the gatekeeper residues is $ \sim $6.5 Å.

    Alternatively, as interdomain conformational changes in ABC exporters are global and collective movements [6, 14], a coarse-grained (CG) approach is especially useful to reduce the system size and to remove fastest degrees of freedom [15, 16]. In our previous study, we carried out CG-MD free-energy simulations of the OF$ \leftrightarrow $IF transition in response to the NBD dissociation in a multidrug transporter P-glycoprotein for probing the structural determinants [6]. Although the OF$ \leftrightarrow $IF transition can be captured in our calculations, the OC state was not yet identified in the free energy profile (see FIG. 2 in Ref.[6]) and detailed understanding of the large-scale conformational changes remains to be completed. In this work, we aim to address these issues using the bacterial ABC exporter, MsbA, as a prototype in the presence of the lipid bilayer and explicit water molecules. The CG-MD umbrella sampling method is employed for the PMF calculations. Our PMF calculations have identified the pathway for the state transitions between OF, OC, and IF, and the predicted structures are consistent with structural experiments.

  • All simulations in this work were performed with the GROMACS 5.0.5 package [17]. The full-length cryo-EM structure of MsbA, which adopts an IF conformation, was taken from the Protein Data Bank (PDB code: 5TV4) [10] serving as the starting structure for our simulations. MsbA acts as a homodimeric exporter to transport lipopolysaccharide (LPS) responsible for inducing a potent inflammatory response during bacterial infection to the outer membrane of gram-negative bacteria. We used the Martini method [15, 16, 18], which has been applied successfully to a large variety of proteins, lipid membranes, DNAs and RNAs [19-26] to model MsbA together with an elastic network model for the stabilization of the backbone conformation of the CG protein [27]. The resulting MsbA CG model was inserted into a pre-equilibrated POPC lipid bilayer modeled with the Martini force field [15, 18]. The MsbA-lipid system was solvated in polarizable Martini water [28, 29] in a rectangular box of 18 nm$ \times $14 nm$ \times $14 nm (FIG. 3). The final system comprises totally 130898 MARTINI particles, including one MsbA protein (2546 particles), 990 POPCs, and 116472 polarizable water CG particles (FIG. 3). In the Martini model, the van der Waals interactions are described using a Lennard-Jones potential function, whereas charged CG particles interact via a Coulomb energy function with the dielectric constant, $ \varepsilon_{{\rm r}} $ = 15. The cutoff of 1.2 nm was used for the Lennard-Jones potential, which was smoothly shifted to zero between 0.9 and 1.2 nm. The long-range electrostatic interactions were treated using the particle mesh Ewald (PME) method with a real space cutoff of 1.2 nm and a 0.12 nm Fourier grid spacing [30]. The simulations performed with the PW model have a van der Waal cut-off of 1.2 nm and a dielectric constant, $ \varepsilon_{{\rm r}} $ = 2.5. In all cases, the Verlet scheme for the neighbor list update and the PME method for the electrostatic interactions, with a grid spacing of 0.12 nm, are used. The CG-MD simulations were executed with a time step of 20 fs under a periodic boundary condition. The above MsbA-lipid system was weakly coupled to the Berendsen thermostat ($ \tau_{{\rm T}} $ = 2 ps) and the Parrinello-Rahman barostat ($ \tau_{{\rm p}} $ = 12 ps) fixed at 310 K and 1 bar in all simulations unless otherwise indicated. Following a 20000-step energy minimization, the above coarse-grained protein in the membrane and aqueous environment was equilibrated.

    Figure 3.  The CG system containing the MsbA exporter (spheres colored red), DOPC lipid bilayer (spheres colored cyan, blue and orange), and water molecules (spheres colored dark blue), a total of 130898 MARTINI particles are used to compute the PMF described in the main text.

    The CG-MD simulation and umbrella sampling (US) with the weighted histogram analysis method (WHAM) [31] were then employed to calculate the PMF along the reaction coordinate.

  • The calculations of the free energy profile often require the definition of a "reaction coordinate" designed to monitor the conformational changes in the protein. A frequently used reaction coordinate is the root-mean-squared deviation (RMSD) of an evolving structure relative to its target. However, the quality of the target structure is a determinant factor for the reliability of the results. As mentioned above, $ \alpha $, defined as the angle between the roll axes of two sides of the internal gate, was used as the reaction coordinate to investigate the conformational changes in the MsbA protein [12]. This variable is useful to measure the extent of the opening/closure of the internal gate as a rigid-body. However, this angle coordinate seems unable to determine whether the gate is closed completely because it usually is a couple of gatekeeper residues that lock the gate [6] as mentioned above. The OF state was thus not determined in the resulting PMF and the OF$ \leftrightarrow $IF transition was unable to be described in their study (see FIG. 4 in Ref.[12]). Intuitively, the COM distance, $ d_1 $, was employed as a reaction coordinate to probe the free energy landscape in our previous study [6]. The thus-obtained PMF captures the OF$ \leftrightarrow $IF transition and the predicted OF and IF structures are in good agreement with structural experiments [6]. Unfortunately, the OC state was not well identified and the OF$ \leftrightarrow $OC transition cannot be described in the calculations (see FIG. 2 in Ref.[6]). Careful examination shows that during the OF$ \leftrightarrow $OC transition, the COM distance, $ d_2 $, which describes the opening/closure extent of the external gate (FIG. 1), is decreased by $ \sim $5.0 Å while $ d_1 $ is increased only by $ \sim $2.0 Å (2.8$ - $3.0 nm). Therefore, to address the above issue, we use $ d_1 $$ - $$ d_2 $ as the reaction coordinate to evaluate the free energy profile.

    Figure 4.  PMF presented as a function of $ d_1 $$ - $$ d_2 $. Here, $ d_1 $ and $ d_2 $ are defined as the COM distances between the two sides of the internal gate (each containing the associated NBD) and the external gate, respectively.

    In the following discussion, the CG-MD simulation and umbrella sampling with the weighted histogram analysis method (WHAM) were then employed to calculate the potential of mean force (PMF) along the reaction coordinate [6]. To this end, we applied weak pulling forces only to the NBDs and a small portion of the TMDs at the cytoplasmic end of the internal gate. Consequently, the COM distances, $ d_1 $ and $ d_2 $, were gradually changed via the successive pulling and equilibration steps. Structural snapshots were then taken out from the CG-MD trajectory to generate the configurations for the umbrella sampling simulations. This COM pulling method has been widely applied in various complex biological systems [6, 31, 32, 33]. In this study, we computed PMF along the reaction coordinate, $ d_1 $$ - $$ d_2 $, generating 141 windows. For each window, 60 ns CG-MD umbrella sampling simulations were executed. A total of effective simulation time [32] was $ \sim $33.8 $ {{\rm \mu }} $s in our calculations.

    The resulting PMF along the reaction coordinate, $ d_1 $$ - $$ d_2 $, is presented in FIG. 4. The PMF has several minima, whose atomistic structures can be obtained using the back mapping method [34]. These minimum points a$ - $d represent the OF, OC, IFc and IF-o state, respectively. Except for the OC state, all other structures predicted from our calculations are similar to those from our previous study [6] and are in good agreement with structural experiments. Alignment of the calculated OC structure (i.e., FIG. 4(b)) to the 5TTP structure [10], which adopts an OC conformation, leads to a root-mean-squared deviation (RMSD) of 2.8 Å (3.0 Å RMSD with the 4S0F structure [35]), in good agreement with experiments. In addition, the free energies of the b, c, and d states relative to that of a are 2.3 kcal/mol, $ - $2.4 kcal/mol, and $ - $5.0 kcal/mol, respectively.

    Our PMF simulations demonstrate that the OF and IF state transition occurs via an OC state. This is crucial for the unidirectionality of the transport function of an ABC exporter. If the internal and external gate both open, it might cause reverse transport of substrates through the membrane. Intriguingly, the IFc backbone conformation (i.e., in FIG. 4(c)) is similar to that of the heterodimeric ABC exporter TM287/TM288 (PDB code: 3QF4) (RMSD of 3.3 Å for Cα atoms) [36] rather than that of the MsbA IFc (PDB code: 3B5X) (C$ _\alpha $ RMSD of 10.5 Å) [9]. Careful examination of the 3B5X structure shows that although it was acclaimed to be at an IF state, its external gate is actually open (see FIG. 2), demonstrating that this structure corresponds to a state in which the internal and external gate are both open.

  • Quantification of the free energy landscape is of importance to elucidate the molecular mechanism of the state transitions in an ABC exporter [8]. In a previous study as mentioned above, the nonequilibrium-driven MD method was applied to calculate the PMF for the MsbA exporter, but no OF and OC states were determined in their calculations [12]. Our previous study evaluated the PMF for a C. elegans exporter, however, we were unable to identify the OC state [6]. In this present work, we used the CG-MD and umbrella sampling approaches with the weighted histogram method to evaluate the free energy profile treating the difference of the COM distances, $ d_1 $$ - $$ d_2 $ as the reaction coordinate. We thus obtained the PMF that identified a reliable pathway, along which the transitions from the OF to the IF state via the OC conformation were captured. The calculated OF, OC, and IF structures are in good agreement with X-ray experiments. To the best of our knowledge, this is the first time that the PMF is calculated to capture all these conformational transitions for an ABC exporter.

    Detailed analysis of the structural changes along the transition pathway (FIG. 4) demonstrates that during the transition from the OF to IF state driven by the NBD dissociation, the external gate is closed before the internal gate is opened, and vice verse in the transition from the IF to OF state. This feature is essential for the unidirectionality of the substrate transport via an ABC exporter. The determination of the OC state in the PMF is important for understanding the mechanism of the OF$ \leftrightarrow $IF transition. It should be pointed out that no state with both gates open was found in our PMF calculations, indicating that such conformations would be located at high free-energy levels.

  • This work was supported by the National Natural Science Foundation of China (No.21073170 and No.21273209). We gratefully acknowledge the Supercomputing Center at University of Science and Technology of China for computational resources.

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