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Structural Features and Energetics of Periplasmic Entrance Opening of the Outer Membrane Channel TolC Revealed by Molecular Dynamics Simulation and Markov State Model Analysis Jingwei Weng, and Wenning Wang J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.8b00957 • Publication Date (Web): 15 Feb 2019 Downloaded from http://pubs.acs.org on February 17, 2019

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Journal of Chemical Information and Modeling

Structural Features and Energetics of Periplasmic Entrance Opening of the Outer Membrane Channel TolC Revealed by Molecular Dynamics Simulation and Markov State Model Analysis Jingwei Weng, Wenning Wang* Department of Chemistry, Institute of Biomedical Sciences and Multiscale Research Institute of Complex Systems, Fudan University, Shanghai, China 200433

* To whom correspondence should be addressed: Wenning Wang Tel: +86-21-31243985 Email: [email protected]

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Abstract TolC is a channel protein responsible for substrate translocation across outer membrane, and it is also a part of the tripartite multidrug efflux pumps in Gram-negative bacteria. The crystal structure of TolC shows that the periplasmic entrance is tightly closed in the resting state, while substrate translocation definitely requires the entrance to open. How the occluded periplasmic entrance opens to allow passage of substrates remains elusive. In this work, we constructed a Markov state model from swarms of all-atom molecular dynamics (MD) simulation trajectories, which delineates the energetics of the conformational changes of TolC. Opening of periplasmic entrance results in monotonically increase in free energy, and is accompanied by disruption of inter-protomer interactions, whereas the intra-protomer interactions remain intact. Multi-ion potential of mean force (PMF) profiles for Na+ and Cl− permeation along the channel have been calculated, and the cation/anion permeability

ratio

derived

from

which

are

in

good

agreement

with

electrophysiological experiments. These results not only deepen our understanding on conformational dynamics of isolated TolC, but also provide valuable vision of its functioning state in tripartite efflux pumps.

2


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INTRODUCTION TolC, which was initially named by its mutation that leads to decreased tolerance to bile salts, detergents, and solvents,1, 2 is an outer membrane (OM) channel found in many Gram-negative bacteria. It is important for the cell envelop-spanning efflux of small inhibitory molecules including antibiotics and other antibacterial drugs, so it has been implicated in a global problem of multidrug resistance.3,

4

TolC typically

functions by participating in tripartite efflux pumps, acting as the passive OM channel. The other two components consist of an integral energy-providing inner membrane protein (IMP) and several copies of periplasmic adaptors. TolC could be assembled with different IMPs and adaptors, depending on the type of the substrate transported. Some of the pumps are powered by proton motive force, such as the resistance/nodulation/cell division-type multidrug efflux pump AcrA-AcrB-TolC and the major facilitator-type multidrug efflux pump EmrA-EmrB-TolC. And some others are driven by the energy of ATP hydrolysis, such as the ATP-binding cassette-type macrolide pump MacA-MacB-TolC.5-7 TolC-dependent pumps could also export large proteins such as toxins, proteases and lipases, as exemplified by the Type I secretion tripartite machinery. 8 High-resolution crystal structure has shown that TolC is an elongated homotrimeric hollow cylinder.9 Each protomer contributes four β-strands to constitute a β-barrel domain traversing OM, and donates two long α-helices (H3 and H7) and two pseudo-long helices (consist of short helices H2 plus H4 and H6 plus H8, respectively) to encircle an α-barrel domain protruding far into the periplasm (Fig.

3



1a). The middle part of α-barrel is belted by an α/β mixing equatorial domain, folded by the N- and C-terminal parts of the three protomers. A continuous interior pore is borne inside the α/β-barrels with an average radius of more than 8 Å. The pore is tightened up at the extracellular entrance by extracellular loops, and it is constricted at the periplasmic entrance by the coiled-coils packing of H3/4/7/8 (Fig. 1b). In contrast to the high flexibility of extracellular constriction,10 the periplasmic constriction is only 1.93 Å in radius and stabilized by interlaced hydrogen bonds and salt-bridges across intra-protomer and inter-protomer grooves (Fig. 1c), tightly locking the channel in the closed state.11, 12

Fig. 1. (a) Crystal structure of TolC in the closed state. The channel is rendered by cartoon mode. Helices in one of the three protomers are colored yellow (H2), blue (H3), dark yellow (H4), green (H6), red (H7) and cyan (H8) and the equatorial domain belting the middle part of the α-barrel is colored magenta. (b) Radius profile of the central pore. Residues constricting the extracellular or the periplasmic entrance 4


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are labelled. The zero point of pore axis is placed at the center-of-mass of the channel. (c) The view of the crystal structure of TolC from the periplasmic side. The color scheme used is the same as (a). The inter-protomer and intra-protomer grooves are denoted by purple and yellow areas. (d) PMF of the minimal radius at the periplasmic entrance (rmin). The PMF is calculated based on the MSM-weighted histograms of conformations with a bin width of 0.5 Å. The error bars are estimated by three independent samplings from the Markov states. rmin of the crystal structures of the wild-type TolC (TolCWT), the TolC mutant with Y362F/R367S (TolCYFRS), and the homologous MtrE from N. gonorrhoeae are marked.

The crystal structure of TolC raises a question as to how the α-helices rearrange to open the closed periplasmic entrance to facilitate substrate transport in functioning tripartite pump.9 High-resolution structures of TolC mutants 13, 14 and the homologous MtrE from N. gonorrhoeae 15 provide some snapshots of the opening state. However, the entrance in these structures is only partially opened and still hinders the passage of most substrates. Recent advances in electron microscopy (EM) provided unprecedented structural details on TolC-dependent tripartite pumps, which demonstrate that the periplasmic constriction of TolC is opened in these complexes.7, 16-19

However, EM experiments derived at least two structural models for the

functioning state of TolC, proposed by the same authors,16,

19

and neither of the

models complies with the in vivo cross-linking results20-22 or the experiment of the AcrA-AcrB fusion protein.23 The ambiguity and discrepancy indicate that the question

5



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has yet to be answered. In our previous molecular dynamics (MD) simulation study, we observed constriction opening of TolC mutants, and obtained an assortment of conformations with various degrees of opening of the periplasmic entrance.24 In this work, starting with these conformations, we have performed extensive MD simulations and constructed a Markov state model (MSM) for TolC. MSM analysis has been successfully applied to study conformational changes of proteins,25-30 and has been used here to derive a detailed description for the conformational changes and energetics of TolC. It was found that disruption of the inter-protomer grooves is the main feature of periplasmic entrance opening, providing valuable vision of the functioning state of TolC in tripartite pumps. Moreover, the free energy profiles for the permeation of sodium and chloride ions in free TolC were also obtained.

METHODS System Setup An initial structure picked from protein data bank or from our previous simulations

was

buried

into

a

pre-equilibrated

bilayer

containing

256

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid molecules by the shrinking method,31 and 250 lipid molecules were retained. All residues but His320 were set to their standard protonation states according to the prediction of H++32 at pH 7.5. The predicted pKa for His320 is ~7.8, thereby it was protonated on both Nδ1 and Nε2. The protein-lipid complex was solvated, and Na+ and Cl- ions were added to

6


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maintain physiological salinity (150 mM) and to obtain a neutral total charge for the system. There were totally 173333 atoms in the simulation box with a size of 96×96×183 Å3. The periodic boundary conditions were applied in all three dimensions. The system was then energy-minimized by 100000 steps by using the steepest descent method. A 10 ns equilibration with protein backbone restrained was performed before production run to relax the lipids and the solvent. Simulation Details All MD simulations were performed with Gromacs 4.6.33 Proteins were described by amber99sb force field,34 lipids by Slipid force field,35, 36 and water molecules by TIP3P model.37 Temperature was maintained at 310 K by the Berendsen weak coupling method38 with a time constant of 0.1 ps, and pressure was maintained at 1 bar by the same weak coupling method38 with a time constant of 5 ps accompanied with semi-isotropic pressure coupling. All covalent bond lengths of protein and lipids were constrained by LINCS39 and those of water were constrained by SETTLE algorithm40 so that an integration time step of 2 fs could be used. Electrostatic interactions were calculated using the particle mesh Ewald (PME) method.41 The short-range non-covalent interactions were truncated at 12 Å. The coordinates of proteins and ions were saved every 25 ps. Seeding MD simulations Two rounds of seeding were conducted. The first round includes six 800 ns trajectories initiated from the close state of crystal structure (PDBID: 1EK9), forty-five 150 ns trajectories from five TolC mutant structures with various degrees of

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opening at the periplasmic entrance (after restoring the site-directed mutations), and forty-eight 150 ns trajectories from five snapshots in the targeted MD trajectories. The mutant structures and the targeted MD trajectories were obtained from our previous work.24 Different initial velocities were used for each trajectory. All these simulations were prepared as described in System Setup. The obtained snapshots in the 99 trajectories of the first round were divided into 50 clusters using the K-centers clustering method, and one 150 ns trajectory was initiated from each of the 50 clusters as the second round of seeding. The final data set includes 149 trajectories summing up to 26.25 μs. Among the 124 trajectories initiated from conformations with rmin > 4 Å, 81 of them visited the closed state (defined as rmin < 2 Å) during the simulation (Fig. S1), indicating that the transition between the opening and the closed states is well sampled. Construction and Validation of the Markov State Model Markov state model (MSM) analysis is a powerful tool for turning swarms of short trajectories into a scientifically meaningful model of dynamics.42,

43

MSM

analysis partitions phase space into a number of states, the transitions inside of which are fast but transitions between which are slow. This separation of time scales ensures that the obtained model is Markovian and allows the construction of MSM from many short trajectories. The inter-conversion dynamics between the states can then be propagated to longer time scale:

p(nt )  T  t  p(0) n

(1)

in which T(t) is the transition probability matrix with a time interval t, p(nt) is the

8


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vector of state population at time nt, and p(0) is the vector of initial state population. MSMs in this work were built by the MSMBuilder software 2.644 for partitioning phase space and deriving equilibrium distribution of states.42, 45 Some in-house written python and tcl scripts were also used. Before the construction, two 150 ns trajectories were discarded from the totally 26.25 μs MD trajectories, as each of them holds a particular state which rarely transmitted to other state and hardly appeared in other trajectories. The symmetry of the three protomers of TolC was utilized, which triples our sampling. A subset of atoms including the backbone and Cβ atoms at the periplasmic half of the α–barrel (residue Leu107 to Ile185 and Ser322 to Leu404) was used for the calculation of root-mean-square deviation (RMSD) which measures the geometric distance between a pair of conformations. The hybrid k-centers k-medoids clustering method was used to partition the phase space. The first round of clustering produced 300 clusters and a 298-state model was obtained using the maximum likelihood estimation (two of the clusters were discarded by the BuildMSM command during the construction). The average radius of each cluster is ~ 2 Å. As this model behaves poor in the Chapman-Komolgorov convergence test (also called the residence probability test),46 conformations in 20 clusters featuring closed entrances were re-clustered into 20 clusters, and conformations in 6 of them were further divided into 80 clusters whose average radii are ~ 1.3 Å. Dozens of trial and error tests were made and each adopted different cluster selection and different numbers of clusters for re-clustering. The final model contains 363 states (nine of the clusters were discarded by the BuildMSM command

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in the third round of clustering). It performs much better in terms of implied time scales and the Chapman-Komolgorov convergence test than the 298-state model, though the 363-state and the 298-state models give almost identical free energy profiles (see below). We examined the behavior of the implied time scales to check whether the obtained model is Markovian.47 The implied time scales (τk) are calculated as

k  

t

ln k  t 

(2)

where λk(t) is the kth eigenvalue of the transition matrix T with the lag time t. Each pair of implied time scale and eigenvector describes an aggregate transition between two subsets of states. The eigenvector whose associated eigenvalue is 1 reflects the equilibrium distribution of the states and was ignored in the calculation of implied time scales. If a model is Markovian, the implied time scales should be independent of the lag time. The slowest implied time scale for the 363-state model levels out when the lag time is above 46 ns (Fig. S2a). Therefore we selected the lag time of 46 ns in further calculation and obtained the equilibrium distribution of the 363 states. It is also noticed that the total simulation time is about six times longer than the slowest implied time scale of the system (~4.6 μs, Fig. S2a), indicating our sampling is sufficient. Moreover, the 363-state model is also well validated by the Chapman-Komolgorov convergence test (also called the residence probability test, Fig. S2b).46 The results presented in the main text were derived from the 363-state model, though the 298-state model gives almost identical results (Fig. S3 & S4). Molecular Visualization and Pore Analysis 10


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Molecular Visualization and trajectory analysis were conducted by the molecular visualization program VMD 1.9.2.48 The analysis of pore radius and residue composition at the periplasmic entrance was done by the pore analysis program HOLE.49 Calculation of Potential of Mean Force (PMF) PMFs of the variables concerning the conformational changes in TolC were calculated from the histograms of population-weighted number of conformations. 500 conformations were randomly selected from each Markov state and used for the statistics. The zero point of free energy was always placed at the global minimum. The error bars of PMF were estimated from three independent samplings from the Markov states. To obtain multi-ion PMF associated with ion permeation though the central pore, the number of ions in each 1 Å thick layer parallel to the membrane plane inside the pore was counted along the pore axis, and was then weighted by the equilibrium population of the state to which the conformations belong. The weighted number of each layer was summed up throughout all selected conformations. The multi-ion PMF Wα(z) for ion α is W  z    RT ln

N  z  N ,bulk

(3)

in which R is the gas constant, T is the temperature, Nα(z) is the sum of population-weighted number of ion α in the layer z, and Nα,bulk is the sum of population-weighted number of a selected layer in the bulk solvent distant from the channel. When the layer is located outside of the pore, the number of ions was 11



counted inside a cylinder with a radius of 17 Å and with its axis placed along the pore. The value of radius was selected according the cross-sectional area of TolC, and deviation from the value by several angstroms has a negligible impact (< 0.2 kcal/mol) on the obtained PMFs.

Results and Discussion Energetics of Periplasmic Entrance Opening The MSM containing 363 states was built by using the conformations from totally 26.25 μs MD simulation trajectories (see Methods). The model enables a direct description of the equilibrium conformational ensemble of TolC. To quantitatively describe the energetics of the periplasmic entrance opening, we evaluated the degree of opening by the minimal pore radius at the entrance (rmin) and calculated the potential of mean force (PMF) along rmin using the 363-state model (Fig. 1d). The PMF gives a single energy well between 1 ~ 2 Å of rmin, in good agreement with the closed state captured by the wild-type TolC crystal structure (rmin = 1.94 Å). The N-terminal end of H8 is found to contribute more than 93% of the constriction of the periplasmic entrance, including residues Thr366, Arg367, Thr368, Val370 and Asp371. Conformations with more opened periplasmic entrance are energetically unstable (Fig. 1d). In reference to the minimal radius of the invariant middle part of the central pore (7.9 Å, see Fig. 1b), we take this value as threshold and define the conformations with rmin > 7.9 Å as the fully opening state. In this regard, fully opening of the

12


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periplasmic entrance costs at least 3.6 kcal/mol (Fig. 1d). The relatively low energy cost indicates the presence of transient opening periplasmic entrance in free TolC, though the population of open state is quite low (≈ 0.15%). The most opened crystal structure of a TolC mutant TolCYFRS (Y362F/R367S) with rmin of 3.9 Å14 can be classified to partially opened state (Fig. 1c), and so does the homologous protein MtrE with rmin of 4.2 Å.15 We also used other descriptor to measure the variation of the periplasmic entrance, such as the triangular cross-sectional area spanned by the Cα atoms of Asp374 (TCA374) or that by Gly365 (TCA365), which have been adopted in previous studies.10, 14, 24 The obtained energy profiles have very similar features and the closed state is always stable (Fig. S4).

Gate opening through disruption of the inter-protomer interactions Basically, reorganizations of the inter-protomer grooves or the intra-protomer grooves are two possibilities for TolC to open the periplasmic entrance.9 We analyzed the state of the lower half of the inter-protomer groove using the ensemble-averaged number of contacts formed between H3/4 and the neighboring H7/8 of another protomer (abbreviated as inter-contact hereafter). Similarly, the state of the lower half of intra-protomer groove was analyzed by using the ensemble-averaged number of contacts formed between H3/4 and H7/8 in the same protomer (abbreviated as intra-contact hereafter). The total intra-contacts of the three intra-protomer grooves are almost constant, regardless of the opening of the periplasmic entrance (Fig. 2a, grey curve). The occurrences of the intra-protomer hydrogen bonds also remain

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constant, further corroborating the stability of the intra-protomer grooves (Fig. S5). The inter-protomer grooves, however, are evidently disrupted when the periplasmic entrance opens. The total inter-contacts of the three inter-protomer grooves decrease steadily as rmin goes beyond 2.5 Å, and half of the total inter-contacts are lost when the periplasmic entrance is fully opened (Fig. 2a, blue curve). The inter-protomer disruption also involves disruption of hydrogen bonds. For the seven groups of hydrogen bonds formed across the lower half of each inter-protomer groove, three of them (Asp153-Arg367, Asp162-Ser350, and Glu173-Asn333) dissociate in most conformations with rmin > 7.9 Å (Fig. 2b). The dissociation is energy consuming and will obviously contribute to the energy increase as the periplasmic entrance opens (Fig. 1c). In this view, abolishment of the hydrogen bonds by mutating the related residues would reduce the energy cost and increase the population of opening entrance, which has been observed in TolC mutants, such as R367S,

Y362F/R367S,

Y362F/R367E.12-14,

50

T152V/D153A/R367S,

R367H,

R367E,

and

The other four hydrogen bond groups also dissociate as the

entrance opens, albeit more mildly (Fig. 2b).

Fig. 2. (a) Variation of the ensemble-averaged number of contacts formed between

14


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H3/4 and H7/8 at the lower half of intra-protomer or inter-protomer groove as a function of rmin. The number of contact is defined as the number of all side-chain center-of-mass pairs (i, j), such that residue i belongs to H4 or to the lower half of H3 and residue j belongs to H8 or to the lower half of H7, the separation between which is less than a cutoff of 6.5 Å. (b) Ensemble-averaged occurrences of the hydrogen bonds formed across the lower half of inter-protomer groove as a function of rmin. The occurrence is the mean value of three equivalent hydrogen bonds at different inter-protomer grooves.

Asymmetric entrance opening Inevitably, inter-protomer disruption produces gaps at the inter-protomer grooves. The two-dimensional PMF spanned by rmin and the maximal gap at the periplasmic tip region of the three inter-protomer grooves demonstrates an approximately linear relationship between the two variables (Fig. 3a). In the fully opening state, the maximal inter-protomer gap is at least ~11 Å, and it will allow some substrates to escape from the central pore and to leak into the periplasm. Another notable feature during the loss of inter-contacts is that the process occurs in an asynchronous manner. The inter-protomer groove with the least inter-contacts was mostly disrupted as rmin > 6 Å (Fig. 2a, yellow curve), whereas the groove with the most inter-contacts was largely maintained till rmin > 11 Å (Fig. 2a, red curve). The simultaneous disruption led to asymmetric arrangements at the lower half of the α-barrel, reminiscent of the cases observed in TolC mutants.24 Symmetric

15



arrangements, however, are less populated and less stable, relative to the asymmetric arrangements. By quantifying the structural asymmetry using the difference between the largest and smallest numbers of inter-contacts among the three protomers (Δintercontact), the 2-D PMF shows that the asymmetric opening pathway is energetically favorable and the asymmetric open conformations are generally more stable (Fig. 3b). The symmetrically opened state (an example is shown in Fig. 3b, the black cross) is ~6 kcal/mol higher in energy than the closed state. Using another symmetry parameter to span the 2d-PMF gives a similar value (Fig. S6).

Fig. 3. (a) Two-dimensional PMF spanned by rmin and the maximal gap at the periplasmic tip regions of three inter-protomer grooves. The gap is defined by the minimal distance between the heavy atoms on the periplasmic tip regions of H3/4 (residue Leu137 to Arg158) and those on the neighboring H7/8 (residue Ser354 to Ala375) of another protomer. (b) Two-dimensional PMF spanned by rmin and Δintercontact (see the main text for definition). A small Δintercontact indicates more symmetrical arrangement at the lower half of the α–barrel. The projections of the closed state and the candidate structure of the functioning state of TolC are labeled as

16


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a magenta cross and a black cross, respectively.

Speculations on the functioning state of TolC in tripartite pumps The opening conformations of free TolC provide vision of its functioning state when the channel participates in tripartite pumps. The cryo-EM maps16-19 of AcrA-AcrB-TolC complex have revealed three important features for the functioning state of TolC, including an opening periplasmic entrance, a pseudo-symmetric arrangement of the lower half of α–barrel, and an efficiently sealed-off central pore with no cavity at the interface between the periplasmic end of TolC and the α-hairpins of AcrA (AcrA is the adaptor of the pump). According to the above requirements, we selected a candidate structure from the MD conformation ensemble of free TolC, which has a fully opened entrance (rmin = 7.9 Å), pseudo-symmetrically disrupted inter-protomer grooves (Δintercontact = 6), and relatively small gaps (maximal gap = 12.1 Å) (Fig. 4a and 4b). This structure is ~ 6 kcal/mol higher in energy than the closed state of TolC (Fig. 3b). Symmetric conformations with larger rmin are even more unstable (Fig. 1c) and are thus not selected. A comparison between our candidate structure and the cryo-EM structure models of the tripartite pump at a resolution of ~6 Å (Fig. 4c, d) reveals remarkable differences. Our structure opens the periplasmic entrance by disrupting the inter-protomer grooves (Fig. 4a, b), while the cryo-EM models demonstrated an “iris-like” dilation by maintaining most of inter-protomer hydrogen-bonding networks at the cost of evident intra-protomer rearrangement (Fig. 4d). It is also notable that a

17



third pseudo-atomic structure model of TolC proposed in an earlier EM study (Fig. S8b of reference16, the authors did not provide the related coordinates) may be viewed as a compromise between the above two cases, which bears non-trivial changes in both inter-protomer and intra-protomer grooves.

Fig. 4. Front view (a) and bottom view (b) of the periplasmic end of the selected structure as a candidate of the functioning state of TolC in tripartite pump. The helices of the α–barrel domain are colored according to the protomer they belong to. The color of H7/8 is lighter than H3/4. The gap at each inter-protomer groove is labeled. (c) Bottom view of the closed state TolC in the cryo-EM structure of apo AcrAB-TolC tripartite efflux pump (PDB ID: 5V5S). (d) Bottom view of the fully 18


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opening TolC in the cryo-EM structure of AcrAB-TolC/inhibitor (PDB ID: 5NG5).

The transient or dynamic nature of the AcrA-AcrB-TolC tripartite pump51, 52 may be the underlying cause of so many different AcrA-TolC interfaces and different tripartite complex models.6, 7, 19, 53 Due to the low stabilizing energy, our candidate structure will probably appear in vivo and may also play a functional role in the tripartite pump. Although the large inter-protomer gaps in our structure may cause leakage problem, the AcrA monomers may insert their hairpin tips into the inter-protomer gaps, and part of the α-helices of hairpins may even uncoil to enable the tips to stretch further to the equatorial domain of TolC and to completely plug the gaps. This kind of interface is supported by the structure of homologous MtrCDE multidrug pump from N. gonorrhoeae, where the hairpin tip of the adaptor can be accommodated at various positions (including the equatorial region) within the inter-protomer groove of OM channel without inactivating the pump.22 Further cross-linking experiments may be designed to restrain the inter-protomer grooves or to strengthen the intra-protomer grooves to clarify the functioning state of TolC in tripartite pumps.

Ion permeation through the channel The presence of 150 mM NaCl solution for the simulation system enables the derivation of multi-ion PMFs for the permeation of Na+ and Cl– ions through the central pore (see Methods for more details), which provide a sight at the translocation

19



mechanism of free TolC. The pore is generally attractive to Na+ with its PMF revealing several weak binding sites, the maximal binding energy of which is 0.57 kcal/mol (Fig. 5, blue curve). Almost every binding site definitively corresponds to one group of acidic residues lining the central pore, including Asp275, Asp56, Asp47, Asp249, Asp77, Glu33, Asp23, Asp101, Glu16, Glu385, Asp374 and Asp371, arranged from the extracellular side to the periplasm (Fig. 5), indicating that electrostatic attraction plays a major role. There is a major barrier at either end of the channel. The extracellular barrier at about 64 Å (Fig. 5) can be largely attributed to the narrowing formed by the flexible extracellular loops (Fig. 1b), though the barrier is partially offset by the attractive potentials from Asp56 and Asp275 (Fig. 5). The periplasmic barrier is higher than the extracellular barrier (1.4 vs. 0.9 kcal/mol), and acts as the rate-limiting step of ion permeation. Arg367 on the loop between H7 and H8 is mainly responsible for the barrier by constricting the periplasmic entrance and repelling cations, though the repulsive electric field is partially counteracted by the negatively charge groups of the adjacent aspartate residues involving Asp374 and Asp371 (Fig. 5). In contrast to the case of Na+, the PMF of Cl– demonstrates a completely repulsive energy profile, with no binding site inside of the channel (Fig. 5, orange curve). The outnumbering negatively charged residues relative to the positively charged ones inside the pore are largely responsible for the repulsive curve (Fig. 5). The periplasmic entrance still forms the predominant barrier, mainly through the aspartate residues (Asp374 and Asp371) at the region.

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Fig. 5. Multi-ion PMFs of Na+ and Cl– along the central pore. The zero point of energy is placed in the aqueous phase. Populations of the acidic (magenta curves) and basic (yellow curves) residues lining the pore are also plotted. The central atom of charged group (Arg: Cζ, Lys: Nζ, Asp: Cγ, Glu: Cδ) is used to represent the entire residue.

The rate-limiting role of the periplasmic entrance for both ions is in excellent agreement with electrophysiological experiments. When the sequentially conserved aspartate residues at the region (Asp374 and Asp371) are mutated to alanine residues, the selectivity of TolC reverses from cations to anions.11 The reversed selectivity is probably due to the loss of the electrostatic repulsion between Asp374/371 and Cl-, and the loss of the attraction between Asp374/371 and Na+ at the same time. The multi-ion PMFs also enable an estimation of the relative permeation rate of the ions. 21



Considering that the heights of the predominant barriers for Na+ and Cl– are 1.4 and 3.4 kcal/mol (Fig. 5), respectively, and supposing that the pre-exponential factors are the same for both ions, we obtain a relative cation/anion permeation rate of 26 using the Arrhenius equation. The relative cation/anion permeation rate has been obtained experimentally for a 300:30 mmol/L KCl solution54 and a 1000:100 mmol/L KCl solution,55 which is 16.5 and 30 in the respective experiments. It should be noted that our calculation was performed in the absence of membrane potential. Theoretically, membrane potential perturbs the free energy profile or PMF for ion permeation, thereby changes the rate constants in both directions (and all individual rate constants if the PMF dictates a multi-step permeation model). For example, previous experimental and computational studies have shown that the applied membrane potential could alter the dipole orientation of channel water56 and may change the behavior of ions,57 leading to deviation between calculated and experimental results. In this study, the general agreement between the computational and experimental derived relative permeation rates could be a result of error cancelation, and more detailed simulation with applied membrane potential is needed for a quantitative comparison with experiment and the revelation of the effects of membrane potential.

Conclusions We combined MD simulation and Markov state model analysis to delineate the conformational changes of the OM channel TolC. The closed conformation is the only

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stable state on the free energy landscape and the opening of the periplasmic entrance leads to disruption of the inter-protomer interactions while the intra-protomer interactions are maintained. An unexpected feature is that entrance opening leads to evident gaps between the protomers, which will make the channel leaky to substrates. A new AcrA-TolC interface in the functioning AcrA-AcrB-TolC tripartite pump is proposed, in which these gaps are plugged by the α-hairpin tips from AcrA. The multi-ion PMFs of Na+ and Cl– along the channel have been calculated, revealing that the periplasmic entrance constitutes the main energy barrier for permeation of both ions and determines the ratio of cation/anion permeation rate.

Associated Content The Supporting Information is available free of charge on the ACS Publications website. A figure of the range of variation of rmin in the 149 short trajectories used for MSM construction; a figure of the implied time scales and the Chapman-Komolgorov convergence test of the MSM; a figure of data derived from a 298-state model; a figure of PMFs of the triangular cross-sectional area spanned by the Cα atoms of three Asp374 and that by the Cα atoms of three Gly365; a figure of variation of the occurrences of intra-groove hydrogen bonds as a function of rmin; a figure of two-dimensional PMF spanned by rmin and a variable quantifying the symmetry of the lower half of α–barrel.

Acknowledgements

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This work was supported by the National Key Research and Development Program of China (No. 2016YFA0501702), National Natural Science Foundation of China (21773038, 21473034, 21877017). This research made use of the resources of the computer clusters at the computer centre at Fudan University.

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