- Email: [email protected]
Computer simulations suggest direct and stable tip to tip interaction between the outer membrane channel TolC and the isolated docking domain of the multidrug RND efflux transporter AcrB
Computer Simulations Suggest Direct and Stable Tip to Tip Interaction Between the Outer Membrane Channel TolC and the Isolated Docking Domain of the Multidrug RND Efflux Transporter AcrB Thomas H. Schmidt, Martin Raunest, Nadine Fischer, Dirk Reith, Christian Kandt PII: DOI: Reference:
S0005-2736(16)30115-8 doi: 10.1016/j.bbamem.2016.03.029 BBAMEM 82193
To appear in:
BBA - Biomembranes
Received date: Revised date: Accepted date:
14 December 2015 28 March 2016 31 March 2016
Please cite this article as: Thomas H. Schmidt, Martin Raunest, Nadine Fischer, Dirk Reith, Christian Kandt, Computer Simulations Suggest Direct and Stable Tip to Tip Interaction Between the Outer Membrane Channel TolC and the Isolated Docking Domain of the Multidrug RND Efflux Transporter AcrB, BBA - Biomembranes (2016), doi: 10.1016/j.bbamem.2016.03.029
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ACCEPTED MANUSCRIPT TolC – AcrBDD Interactions
Computer Simulations Suggest Direct and Stable Tip to Tip Interaction Between
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the Outer Membrane Channel TolC and the Isolated Docking Domain of the
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Multidrug RND Efflux Transporter AcrB
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Thomas H. Schmidt1, Martin Raunest2, Nadine Fischer3, Dirk Reith4 & Christian Kandt4*
1: Department of Membrane Biochemistry, Life and Medical Sciences (LIMES) Institute, University
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of Bonn, Carl-Troll-Straße 31, 53115 Bonn, Germany
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2: MLL Münchner Leukämielabor GmbH, Max-Lebsche-Platz 31, 81377 München, Germany
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3: Berlin-Chemie AG ,Glienicker Weg 125, 12489 Berlin, Germany
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4: Bonn-Rhein-Sieg University of Applied Sciences, Department of Electrical/Mechanical Engineering and Tech.Journalism, Grantham-Allee 20, 53757 Sankt Augustin, Germany
*: corresponding author
Email: [email protected] 1
ACCEPTED MANUSCRIPT TolC – AcrBDD Interactions
ABSTRACT
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One way by which bacteria achieve antibiotics resistance is preventing drug access to its target molecule for example through an overproduction of multi-drug efflux pumps of the resistance
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nodulation division (RND) protein super family of which AcrAB-TolC in Escherichia coli is a
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prominent example. Although representing one of the best studied efflux systems, the question of how AcrB – TolC interact is still unclear as the available experimental data suggest that either both
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proteins interact in a tip to tip manner or do not interact at all but are instead connected by a hexamer of AcrA molecules. Addressing the question of TolC – AcrB interaction, we performed a
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series of 100 ns – 1 µs molecular dynamics simulations of membrane-embedded TolC in presence of the isolated AcrB docking domain (AcrBDD). In 5 / 6 simulations we observe direct TolC – AcrBDD
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interaction that is only stable on the simulated time scale when both proteins engage in a tip to tip
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manner. At the same time we find TolC opening and closing freely on extracellular side while remaining closed at the inner periplasmic bottleneck region, suggesting that either the simulated
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time is too short or additional components are required to unlock TolC.
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ACCEPTED MANUSCRIPT TolC – AcrBDD Interactions
INTRODUCTION Reducing the mortality rate from bacterial infections and diseases, antibiotics have become
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cornerstones of modern medicines [1]. Although regarded as highly effective since their first applications in the 1940s, it has become clear that the application of antibiotics also constitutes a
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highly effective means of exerting selection pressure, leading to the emergence of bacterial strains
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now displaying resistance against practically all of the commonly available agents [2–5]. Indeed there is currently no antibiotic in clinical use today to which resistance has not been reported yet
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[5,6]. With bacterial infections again counting among the top five causes of death even in developed countries [7], old antibiotics losing the efficiency faster than new ones can be
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developed [8], and approval rates of new antibiotics continuously declining since the 1980 [9,10], the need to discover and develop new agents is paramount. To reach this goal a detailed
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understanding of the molecular basis underlying antibiotics resistance is crucial.
Possible strategies to discover new antibiotics include the investigation of naturally occurring
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antibacterial attack mechanisms like antimicrobial compounds to exploit their efficiency and bandwidth [11]. Another approach is the study of antibacterial defense mechanisms to identify new drug targets by which bacterial defenses against antibiotics could be disabled. A prominent example of the latter category are multidrug efflux transporter of the resistance nodulation division (RND) protein super family [12,13]. One of the best studied RND transport systems is AcrAB-TolC in Escherichia coli [14–16].
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TolC – AcrBDD Interactions
Figure 1: Spanning the entire periplasmic space, the RND efflux transporter AcrAB-TolC uses the outer membrane factor (OMF) TolC as efflux duct to expel toxic molecules out of the cell (a). Addressing the questions of TolC - AcrB
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interaction and TolC gating, we performed molecular dynamics simulations of the isolated AcrB docking domain alone
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(b) and in presence of membrane-embedded TolC (c).
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Like all RND transporters, AcrAB-TolC is a modular-built membrane protein complex transiently assembled from an inner membrane proton / drug antiporter (AcrB) acting as the pump's engine,
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an outer membrane channel (TolC) acting as efflux duct through which substrate is expelled from the cell, and an adapter protein (AcrA) anchored to the inner membrane stabilizing antiporter and efflux duct interaction and enhancing pump activity [16] (figure 1a). While AcrAB-TolC has been in the intense focus of experimental [13,17–19] and computational research [20,21], the question of how antiporter and efflux duct interact is still unclear as so far the available data suggest two contradicting models of TolC – AcrB interaction: Whereas based on the crystal structures of both proteins [22,23], biochemical cross-linking [24,25] and surface plasmon resonance experiments [26] a direct tip to tip interaction was suggested, electron microscopy studies support the hypothesis that AcrB and TolC do not interact directly but are connected by a hose-like structure 4
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formed by six AcrA molecules [27,28].
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To test if TolC and AcrB can interact directly and if a potential binding affects efflux duct access regulation [29,30], we carried out a series of six independent, unbiased 100ns – 1.05 µs molecular
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dynamics (MD) simulations of phospholipid membrane-embedded TolC in presence of the isolated
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AcrB docking domain (AcrBDD) which we used as a simplified representation of full-length AcrB. After confirming AcrBDD stability in an additional 100 ns MD run of the docking domain on its own,
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we find that in 5 / 6 simulations TolC and AcrBDD engage in direct interaction that is unstable when the binding occurs in a tip to side manner but stable for the simulated time when TolC and AcrBDD
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bind in a tip to tip manner. At the same time we observe no effect on TolC accessibility as the protein opens and closes freely on extracellular side while opening up in the outer periplasmic
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bottleneck region BN-II [30–32] but remaining closed at the inner periplasmic bottleneck BN-I,
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TolC.
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suggesting that either the simulated time is too short or AcrBDD alone is not sufficient to unlock
MATERIALS & METHODS Molecular Dynamics Simulations MD simulations were performed using GROMACS version 4.0.3 [33,34] and the GROMOS96 53A6 force field [35,36]. Standard protonation states were assumed for titratable residues. The LINCS algorithm was applied to constrain all bond lengths permitting an integration time step of 2 fs [37,38]. A temperature of 310 K was maintained separately for protein, lipids (if present), and the water/ion mixture using the Berendsen thermostat [39] with a time constant of 0.1 ps. For
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pressure coupling the Berendsen barostat [39] was employed using a 1 bar reference pressure and a time constant of 4 ps. Whereas the AcrBDD simulation was performed using an isotropic pressure
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coupling scheme, semiisotropic pressure coupling was employed for the TolC + AcrBDD runs to permit bilayer fluctuations in the membrane plane. Electrostatic interactions were calculated
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applied for computing the van der Waals interactions.
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using Particle Mesh Ewald (PME) summation,[40,41] and twin range cutoffs of 1.0 and 1.4 nm were
Simulation Set Up AcrBDD
The model of the isolated AcrBDD was generated by truncating the asymmetric wild type AcrB
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crystal structure 2GIF [42] to residues 182 to 272 and 724 to 812. AcrBDD was then placed at the
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center of a cubic simulation box with an edge length of 10.6 nm, solvated with 36,473 water molecules and 108 sodium and 111 chloride ions yielding a 150 mM NaCl solution and a total
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system charge of zero (figure 1b). After a standard steepest descent energy minimization 100 ns of
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unbiased and unrestrained MD simulation were performed to test the stability of the isolated docking domain.
Simulation Set Up TolC + AcrBDD Using the 1EK9 [22] -based starting structure of our previous study of TolC embedded in a palmitoyl-oleoyl-phosphatidyl-ethanolamine (POPE) membrane [30] as a starting point, we deleted all water and ions and extended the original simulation box to 20.61 nm in Z direction (membrane normal), keeping the original X and Y dimensions of 10.06 nm and 9.97 nm. In the next step, we added AcrBDD in an orientation matching the one in the AcrAB-TolC docking model by Symmons 6
ACCEPTED MANUSCRIPT TolC – AcrBDD Interactions
and co-workers [24]. Subsequently AcrBDD was translocated 1 nm away from TolC parallel to the vector of the membrane normal. As before, the simulation system was solvated with a 150 mM
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NaCl/water solution, comprising 47,423 water molecules as well as 205 sodium and 190 chloride ions yielding a total system charge of zero and an overall system size of 178,028 atoms (figure 1c).
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Following steepest descent energy minimization, four independent, unrestrained and unbiased 150
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ns MD simulations were initiated (run A to D) using different random seed numbers in generating
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the starting velocities. Of these run A was later extended to 1.05 µs.
To test whether the asymmetric monomer conformations in the 2GIF AcrB crystal structure [42]
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influences TolC - AcrBDD interaction, two additional simulations were performed where AcrBDD was initially rotated by +120° and -120° around the membrane normal axis. Following the same
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solvation and energy minimization protocol outlined above, unbiased and unrestrained dynamics
Analysis
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samples were computed for 100 ns each.
AcrBDD Stability To assess AcrBDD stability we calculated Croot mean square deviations (RMSD) after least square fitting to the starting structure and computed Ramachandran distributions over the entire 100 ns simulation times (figure 2, supplemental figure S1). All analyses were compared to previous 100 ns simulations of full length AcrB [43]. In the Ramachandran plots AcrB residues located within an “upper” 1 nm thick slab of the outer membrane-facing side of the protein (figure 2b) are highlighted red. CRMSDs were calculated on protein (figure 2a) and individual atom level in 7
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form of Cdisplacement plots (supplemental figure S1). In case of the latter we also computed the difference in Cdisplacement between AcrBDD in isolation and the full length protein showing
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the maximum AcrBDD displacements. The differences were then mapped onto the simulation
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starting structure.
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TolC – AcrBDD Interaction
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To monitor TolC - AcrBDD interaction we computed for all simulations the buried surface area (BSA) as a function of time (figure 3, 5). Following the same protocol as detailed in [44], the BSA was computed using NACCESS [45]. Horizontal lines in figure 3b and c indicate the TolC – AcrB BSA as
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seen in the Symmons model of the AcrAB-TolC complex [24].
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To identify hotspots of interaction in the TolC-AcrBDD interface we computed for each simulation time-averaged contact frequencies per residue over the entire trajectory, using a respective
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distance cutoff of 0.2 nm (figure 4, 5). Results were mapped onto the simulation structures as formal B-factors and visualized using VMD [46].
TolC Accessibility Tolc accessibility was monitored using the same approach as in [29,30] (figure 6). On extracellular side we calculated for each simulation the distribution of the dihedral angle formed by the carbons of Asp56 in the ß-barrel and Ala270 at the tip of each extracellular loop. On periplasmic side we quantified the opening state of the inner (BN-I) and outer (BN-II) bottleneck computing the
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triangular cross-sectional area (TCA) spanned by α-carbons of Asp374 (BN-I) and Gly365 (BN-II). Vertical and horizontal dashed lines in figure 6 reference TolC and TCA values as observed by X-
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ray crystallography [22,31,32,47–49]. Similar to our previous work on OprM and TolC [29,30], we
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computed 1D and 3D sodium densities to test for preferred sodium binding sites as previous studies revealed an ion dependency of TolC's periplasmic opening state [50]. Calculated over the
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entire simulation time, 1D densities were computed for all simulations, whereas spatial densities were analyzed for the µs run. Using the VolMap plugin in VMD 1.9 [46] at a resolution of 1 ų,
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sodium densities in the TolC periplasmic bottleneck region were calculated at a density level of
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0.01 and 0.1 Na/ų.
Molecular visualizations were generated using VMD 1.9.1 [46], all other illustrations were created
RESULTS
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using Inkscape (figure 1a), Xmgrace & Xara Designer Pro.
AcrBDD Stability To assess the stability of AcrBDD alone and in context of the full-length protein we computed CRMSDs, Cdisplacements and Ramachandran distributions (figure 2, supplemental figure S1). Whereas in full-length AcrB RMSDs of the docking domain range from 0.3 to 0.38 nm throughout 100 ns simulation time (figure 2a, blue), AcrBDD on its own displays an RMSD of 0.41 to 0.46 nm in its 100 ns dynamics sample (figure 2a, red). To detect where conformational changes occur we computed C displacements between the 2GIF X-ray and the simulation end structures of AcrBDD 9
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in isolation (blue graphs) and of previous simulations where AcrBDD was in context of the full length protein (supplemental figure S1 a). Computing the difference between the maximum AcrBDD
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changes seen in the full length protein and the isolated docking domain, we find that on TolCfacing side conformational changes in the isolated AcrBDD are smaller than or equal to the
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maximum changes observed for AcrBDD in the full length protein (supplemental figure S1 b, c).
Comparing the Ramachandran distributions over the 100 ns simulation time we find that AcrBDD
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alone (figure 2c) and AcrBDD as part of full-length AcrB (figure 2d) display similar patterns,
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particularly in the upper 1 nm thick section facing towards the outer membrane (figure 2b-d, red).
Figure 2: To assess its stability we compared dynamics samples of the isolated AcrBDD (red curve in a, panels b & c) with previous full length AcrB simulations [43] (blue curves in a, panel d), computing C root mean square displacements (a) and Ramachandran distributions (b-d), where AcrB residues located within a 1 nm thick slab of the outer membrane-facing side of the protein are highlighted red (b).
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TolC - AcrBDD Interaction
Figure 3: TolC - AcrBDD buried surface area (BSA) computed for each of the four production runs (runA-D) and the two
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control simulations where AcrBDD was rotated by ± 120° around the membrane normal. For each simulation we show the configuration displaying the maximum BSA (a), the total amount of BSA (b) as well as the individual contribution of
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each monomer (c). In total we observed three types of TolC - AcrBDD binding events: tip to tip binding (yellow), tip to
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side binding (cyan) and no binding (grey). In (c) T-A, T-B, T-C and A-A, A-B, A-C indicate monomers A, B, C in TolC and
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AcrBDD respectively. Horizontal lines in (b) and (c) indicate the interface area as seen in the Symmons model [24].
To quantify TolC-AcrBDD interaction and monitor potential binding events we computed for each simulation the TolC-AcrBDD buried surface area (BSA) as a function of time (figure 3). Illustrated by simulation snapshots of maximum interface area (figure 3a), the BSA is shown in total between the TolC and AcrBDD trimers (figure 3b) as well as broken down to the individual contributions of each monomer (figure 3c). Horizontal lines in figure 3b and c indicate the BSA seen in the AcrAB-TolC Symmons model [24]. In total we observe three types of TolC-AcrBDD interactions: (A) a stable tip to tip binding in 4 / 6 simulations (figure 3, yellow panels) with runA exhibiting the highest and most homogeneous BSA values; (B) a transient tip to side binding in 1 / 6 simulations where TolC's
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periplasmic mouth engages the monomer C and B flank of AcrBDD for about 40 ns (figure 3, cyan); and (C) no binding at all in 1 / 6 simulations (figure 3, grey). Further inspection shows that in cases
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(B) and (C), due to the periodic boundary conditions of the simulations, AcrBDD drifted to the other side of the membrane, leading to physiologically irrelevant interactions with TolC's extracellular
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loops or the POPE phospholipids of the model membrane (supplemental figure S2).
Figure 4: TolC - AcrBDD interface residues as determined by close contact frequencies computed over each simulation
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and mapped onto the AcrBDD and TolC starting structures. Results are shown for the production runs (left) and the control simulations (right) where AcrBDD was rotated by ± 120° around the membrane normal. Panel colours indicate tip to tip (yellow) and tip to side (cyan) interaction. Run B was excluded from the analysis as no physiologically relevant TolC - AcrBDD interaction occurred in that simulation.
To identify residues involved in the spontaneously formed TolC – AcrBDD interfaces we computed for each simulation with a binding event (figure 3, yellow & cyan panels) time-averaged, perresidue contact frequencies between both proteins. Calculated over the entire length of each simulation, the results were then mapped onto the simulation starting structures (figure 4). 12
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Whereas the tip to side binding in run C clearly sticks out (figure 4, cyan panel), the tip to tip bindings share a common pattern of contact that, although pronounced to varying degrees in the
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four simulations (maximum in run A), outlines the same set of interface residues (figure 4, yellow
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panels).
Figure 5: TolC
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AcrBDD interactio n in run A extended to 1.1 µs
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as illustrated
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by
total
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(a) and per monomer buried surface area analysis (b), interface snapshots of the simulation end conformation (c) and TolC - AcrBDD close contact frequencies computed over the entire simulation and mapped onto the starting structures
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(d). In (b), (c) and (d) T/A, T/B, T/C and A/A, A/B, A/C indicate monomers A, B, C in TolC and AcrBDD respectively. Horizontal lines in (a) and (b) indicate the interface area as seen in the Symmons model [24].
Displaying the highest and most homogeneous BSA levels as well as the most pronounced patterns of close contact interface residues, the initially 150 ns long run A was extended to a total length of 1.05 µs (figure 5). Throughout that time TolC-AcrBDD binding became continuously tighter with the BSA surpassing the interface area of 43.03 nm² of the Symmons model after 380 ns (figure 5a). At the same time the initially homogenous interaction pattern between the two proteins (monomer A with A, B with B and C with C) becomes asymmetric with for example TolC monomer A additionally
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interacting with AcrBDD monomer B or TolC-B with AcrBDD-C (figure 5b). Inspection of the simulation end conformation shows that whereas AcrBDD-A and C exhibit a similar TolC binding
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characteristic as in the Symmons model, AcrBDD-B adopts a shifted configuration where its DC hairpin is located in between the helix 7/8 loop of TolC-B and the helix 3/4 loop of TolC-A instead of
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the gap between both helix-loops of monomer B (figure 5c). In terms of interface residues the
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results of the contact frequency analysis (figure 5d) are in line with our findings for the initial set of
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simulations (figure 4).
TolC Accessibility
To monitor and quantify TolC accessibility throughout the simulations, we monitored the channel's
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opening state computing (a) the distributions of the dihedral angle formed between the ß-barrel
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and the tip of each extracellular loop; and (b) the triangular cross-sectional area (TCA) spanned by
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the inner and outer periplasmic bottlenecks BN-I and BN-II (figure 6).
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In the runs with tip to tip interaction the distributions range from -40° to 170° (figure 6a, yellow panels) and from -90° to 160° in the simulation where tip to side interaction occurred (figure 6a, cyan panel). Clearly exceeding the range of ~ 90°-110° observed in TolC wild type and mutant crystal structures [22,31,32,47–49], the distributions from our simulations predominantly exhibit maxima located at values either overlapping with or higher than the experimentally observed range. In this context the tip to side simulation is an exception as here the monomer B distribution peaks at -60° due to a physiologically irrelevant interaction of AcrBDD with the periodic image of TolC's extracellular loop in monomer B (supplemental figure S2).
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ACCEPTED MANUSCRIPT TolC – AcrBDD Interactions Figure 6: TolC
ty
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accessibili as
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monitored through
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the dihedral
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angle formed between
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each extracellular loop and the ß-barrel (a) and the triangular cross sectional area (TCA) spanned by the -carbons of Gly365 and Asp374 forming the outer (BNI) and inner (BNII) periplasmatic bottlenecks (b). Panels (c) and (d) show the
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outcome of the same analyses for the 1.05 µs simulation. Green TCA regions indicate opening states as observed in [30] whereas yellow and cyan background colors indicate tip to tip and tip to side interactions of TolC and AcrB DD.
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Dashed vertical lines in (a) and (c) mark dihedrals observed experimentally [22,31,32,47–49], whereas horizontal
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dashed lines in (b) and (d) represent the BNI and BNII TCAs of the 1EK9 X-ray structure [22].
On periplasmic side the TCA in the outer bottleneck (BN-II) increases rapidly at the beginning of each simulation covering a TCA range from 1.9 nm² to 2.9 nm² by the end of the simulations (figure 6b, green graphs). In the inner bottleneck BN-I the TCA changes little staying close to its initial 0.6 nm² as seen in the 2EK9 starting structure [22] (figre 6b, red graphs).
In the run A simulation extended to 1.05 µs the distributions range from 60° to 180° with maxima located at 150° and 165° (figure 6c). On periplasmic side the BN-I TCA fluctuates around the experimentally observed value, whereas the BN-II TCA explores a range from 1.4 to 3.2 nm² 15
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throughout the additional simulation time (figure 6d).
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Analogue to [29,30] we computed 1D and 3D sodium densities (supplemental figure 3) to test for preferred Na+ binding sites. As in our previous work on TolC [30] we find in all simulations
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heightened sodium densities at the lipid head groups an in the periplasmic bottleneck region
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(supplemental figure S3a). Inspecting this region further in the 1 µs simulation, we find the resulting spatial sodium density resembling the ones reported in previous TolC simulations [30]
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when BNII is open (supplemental figure S3b).
DISCUSSION
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Addressing the questions of direct TolC – AcrB interaction and TolC access regulation, we
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conducted a series of six unbiased and independent 100 ns – 1 µs molecular dynamics simulations of membrane-embedded TolC in presence of the isolated AcrB docking domain whose stability was
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assessed in an additional 100 ns run. With AcrBDD alone displaying similar conformational dynamics as in context of full-length AcrB, we observe that TolC and AcrBDD either engage in a transient tip to side or a tip to tip interaction that is stable on the simulated time scale. At the same time we find that in all simulations TolC opens and closes freely on extracellular side while opening up in the outer periplasmic bottleneck region BN-II at Gly-365 but remaining closed in the inner bottleneck BN-I at Asp-374 in all simulations. We begin this section discussing the limitations of our approach before proceeding to our findings and their biological implications.
Limitations of our Approach
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The computer simulations reported here are mainly limited by three key factors: (1) the AcrB model used; (2) the representation of the proteins' micro-environment and (3) the amount of
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conformational sampling achieved.
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To test for possible interactions between two proteins, including both molecules in their entirety
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would be the best approach. However, with TolC and AcrB being membrane proteins exhibiting a defined spatial orientation, reducing system complexity and computational cost by truncating e.g.
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one protein to functionally relevant parts is a reasonable strategy as long as the e.g. domain is sufficiently stable to represent the full length protein. With AcrBDD on its own displaying a similar
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Ramachandran pattern and thus a high degree of conformational similarity with AcrBDD connected to the full length protein (figure 2), we consider our reduced AcrB model a reasonable starting
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point to study TolC – AcrB interaction. One should keep in mind however that in this approach the
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potential observation of functionally relevant protein responses to complexation is for the most
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part restricted to TolC.
In vivo AcrAB-TolC's micro-environment is characterized by two phospholipid membranes of heterogeneous and, in case of the outer membrane, highly asymmetric lipid composition (figure 1a). Moreover TolC is not only membrane-embedded but also spans the peptidoglycan layer located in between the outer and inner membrane. In our simulations we omit the peptidoglycan layer and resort to a homogeneous POPE bilayer as model membrane. While clearly a simplification of the in vivo conditions, we nevertheless consider our model a reasonable starting point given that (a) peptidoglycan and lipopolysaccharide models are still in their infancy and were not available for E. coli by the time this study was conducted [51–58] and (b) previous simulation
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studies of outer membrane proteins have successfully made biologically relevant predictions using similar simplifications [29,30,59–63]. These studies include our own previous works on TolC and
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OprM to which the present study is a direct successor using a similar experimental setup for better
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comparability.
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Whether the amount of conformational sampling obtained in an MD simulation study is sufficient depends on the questions the study seeks to answer. Here we address the questions if TolC and
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AcrB can interact directly and if this interaction affects the regulation of TolC accessibility. To this end we performed six independent and unbiased MD runs using three different starting
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orientations with simulation times ranging from 100 ns to 1.05 µs. Given that we observed two types of direct interaction in 5 / 6 simulations (figure 3, 5) and in all simulations a pattern of TolC
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accessibility (figure 6) similar to previous studies of bilayer-embedded outer membrane factors
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to answer.
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[29,30], we consider the amount of conformational sampling adequate for the question we sought
Functional implications TolC – AcrB Interactions
Over the years AcrAB-TolC has been the focus of numerous studies, investigating different aspects of RND efflux pump structure and functional mechanism, including the question of how AcrB, AcrA and TolC assemble into a functional complex. So far the available data suggest two contradicting models of TolC – AcrB interaction: Whereas based on the crystal structures of both proteins [22,23], biochemical cross-linking [24,25,64], minimum inhibitors concentrarion measurements
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[65] and surface plasmon resonance experiments [26] a direct tip to tip interaction was suggested, electron microscopy studies support the hypothesis that AcrB and TolC do not interact directly but
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are connected by a hose-like structure formed by six AcrA molecules [27,28].
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To test if TolC and AcrB can engage in direct interaction we performed MD simulations of TolC and
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AcrBDD which we used as a simplified representation of full length AcrB (figure 1). As indicated by the BSA analyses (figure 3) binding events occur in 5 / 6 simulations, leading to an unstable tip to
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side and stable tip to tip interactions in four simulations. Interestingly we observe this stability regardless of how far the binding process has progressed: once an initial contact has been
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established TolC and AcrBDD do remain together whereas the tip to side complex falls apart after 40 ns. If our simulations are correct this finding suggests that under the simulated conditions TolC and
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AcrBDD can engage in direct interaction that is only stable on the observed 100 ns – 1.05 µs time
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scale (figure 3, 5) when AcrBDD binds to TolC in a tip to tip manner - which is compatible with the type of TolC – AcrB interaction proposed in [22–26]. While one could argue that the observed
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binding behaviour is merely random, the fact that (a) tip to tip interaction occurs four times whereas tip to side complexation is seen only once and (b) only in tip to tip complexations TolC and AcrBDD stay together speaks against this argument. As our simulations further indicate the formation of a common pattern of TolC - AcrBDD interface residues in the runs with tip to tip interaction (figure 4, 5), a potential way to experimentally test our computer prediction could be to for example introduce cross-linkers in the observed TolC - AcrBDD interface and check whether the two proteins come off separately or conjoined in a subsequent preparation step involving gel electrophoresis or mass spectrometry.
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TolC Access Regulation With distributions (figure 6a, c) clearly exceeding the experimentally observed range in TolC
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crystal structures [22,31,32,47–49] (figure 6) our findings indicate that TolC is opening and closing
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freely on extracellular side suggesting the absence of an access regulation mechanism on this side of the protein. These results are in line with our previous work of outer membrane factors
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reporting the same behavior [29,30]. Whether the shift of maxima towards larger dihedrals and thus more open loop conformations in the 1.05 µs simulation (figure 6c) could be interpreted as a
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possible response of TolC to the increasingly tighter binding of AcrBDD (figure 5a) signifying a potential preparation for transport cannot be decided based on the current data, as only a single
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microsecond trajectory was generated.
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On periplasmic side the TCA analysis delivers a consistent picture in all simulations (figure 6 b, d):
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whereas the TCA at Gly-365 increases rapidly by a factor higher than 3.5 in all simulations, the TCA at Asp-374 fluctuates around the value seen in the wild type crystal structure from which the
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simulations were initiated [22]. If our simulations are correct this finding suggests that TolC opens up at the outer bottleneck BN-II but remains closed at the inner bottleneck BN-II which is in agreement with our previous studies [29,30]. Finding the same TCA profiles regardless whether AcrBDD has bound or not, this behavior suggests that either the simulated time was too short or AcrBDD alone is not sufficient to unlock TolC, requiring instead the presence of additional components absent in our simulations. In that light one could argue that our in silico findings are also compatible with the hypothesis of no direct interaction between TolC and AcrB [27,28] in so far that while direct interaction is in principle possible but does not lead to a functional efflux pump. Experimental ways to test our predictions could include double spin lable EPR experiments
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to monitor TolC accessibility on the extracellular side. Another option could be TolC activity tests where the efflux duct is reconstituted in a membrane separating a two component system with
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one of them containing AcrBDD plus transport substrate detectable by e.g. fluorescence
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spectroscopy or pH measurements.
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CONCLUSIONS
Addressing the questions if TolC and AcrB can interact directly and if such an interaction affects
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TolC access regulation, we carried out a series of 100 ns – 1.05 µs MD simulations of membraneembedded TolC in presence of the isolated AcrB docking domain. In 5 / 6 runs we find that TolC
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and AcrBDD engage in direct interaction that is stable throughout the simulations when the binding
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occurs in a tip to tip manner. Opening and closing freely on extracellular side, TolC opens in the outer periplasmic bottleneck region but remains closed at the inner periplasmic bottleneck,
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suggesting that either simulated time is too short or AcrBDD alone is not sufficient to unlock TolC.
Acknowledgements This work was financially supported by the Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen within the NRW Rückkehrer-Programm; the Deutsche Forschungsgemeinschaft and the Bundesministerium für Bildung und Forschung.
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TolC – AcrBDD Interactions
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Graphical Abstract
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The authors declare no conflicts of interest.
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Highlights AcrAB-TolC is a prominent multi-drug efflux transporter in Escherichia coli representing one of the major causes of antibiotics resistance. Experimental evidence supports two contradicting types of interaction between AcrB and TolC: (a) a direct tip-to-tip interaction and (b) an indirect interaction mediated by AcrA Molecular dynamics simulations of TolC in presence of the isolated AcrB docking domain suggest a direct and stable tip-to-tip interaction between both proteins. At the same time TolC opens and closes freely on extracellular side while remaining closed at the outer perisplasmic bottleneck BN II
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S0005-2736(16)30115-8 doi: 10.1016/j.bbamem.2016.03.029 BBAMEM 82193
To appear in:
BBA - Biomembranes
Received date: Revised date: Accepted date:
14 December 2015 28 March 2016 31 March 2016
Please cite this article as: Thomas H. Schmidt, Martin Raunest, Nadine Fischer, Dirk Reith, Christian Kandt, Computer Simulations Suggest Direct and Stable Tip to Tip Interaction Between the Outer Membrane Channel TolC and the Isolated Docking Domain of the Multidrug RND Efflux Transporter AcrB, BBA - Biomembranes (2016), doi: 10.1016/j.bbamem.2016.03.029
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ACCEPTED MANUSCRIPT TolC – AcrBDD Interactions
Computer Simulations Suggest Direct and Stable Tip to Tip Interaction Between
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the Outer Membrane Channel TolC and the Isolated Docking Domain of the
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Multidrug RND Efflux Transporter AcrB
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Thomas H. Schmidt1, Martin Raunest2, Nadine Fischer3, Dirk Reith4 & Christian Kandt4*
1: Department of Membrane Biochemistry, Life and Medical Sciences (LIMES) Institute, University
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of Bonn, Carl-Troll-Straße 31, 53115 Bonn, Germany
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2: MLL Münchner Leukämielabor GmbH, Max-Lebsche-Platz 31, 81377 München, Germany
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3: Berlin-Chemie AG ,Glienicker Weg 125, 12489 Berlin, Germany
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4: Bonn-Rhein-Sieg University of Applied Sciences, Department of Electrical/Mechanical Engineering and Tech.Journalism, Grantham-Allee 20, 53757 Sankt Augustin, Germany
*: corresponding author
Email: [email protected] 1
ACCEPTED MANUSCRIPT TolC – AcrBDD Interactions
ABSTRACT
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One way by which bacteria achieve antibiotics resistance is preventing drug access to its target molecule for example through an overproduction of multi-drug efflux pumps of the resistance
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nodulation division (RND) protein super family of which AcrAB-TolC in Escherichia coli is a
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prominent example. Although representing one of the best studied efflux systems, the question of how AcrB – TolC interact is still unclear as the available experimental data suggest that either both
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proteins interact in a tip to tip manner or do not interact at all but are instead connected by a hexamer of AcrA molecules. Addressing the question of TolC – AcrB interaction, we performed a
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series of 100 ns – 1 µs molecular dynamics simulations of membrane-embedded TolC in presence of the isolated AcrB docking domain (AcrBDD). In 5 / 6 simulations we observe direct TolC – AcrBDD
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interaction that is only stable on the simulated time scale when both proteins engage in a tip to tip
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manner. At the same time we find TolC opening and closing freely on extracellular side while remaining closed at the inner periplasmic bottleneck region, suggesting that either the simulated
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time is too short or additional components are required to unlock TolC.
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INTRODUCTION Reducing the mortality rate from bacterial infections and diseases, antibiotics have become
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cornerstones of modern medicines [1]. Although regarded as highly effective since their first applications in the 1940s, it has become clear that the application of antibiotics also constitutes a
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highly effective means of exerting selection pressure, leading to the emergence of bacterial strains
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now displaying resistance against practically all of the commonly available agents [2–5]. Indeed there is currently no antibiotic in clinical use today to which resistance has not been reported yet
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[5,6]. With bacterial infections again counting among the top five causes of death even in developed countries [7], old antibiotics losing the efficiency faster than new ones can be
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developed [8], and approval rates of new antibiotics continuously declining since the 1980 [9,10], the need to discover and develop new agents is paramount. To reach this goal a detailed
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understanding of the molecular basis underlying antibiotics resistance is crucial.
Possible strategies to discover new antibiotics include the investigation of naturally occurring
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antibacterial attack mechanisms like antimicrobial compounds to exploit their efficiency and bandwidth [11]. Another approach is the study of antibacterial defense mechanisms to identify new drug targets by which bacterial defenses against antibiotics could be disabled. A prominent example of the latter category are multidrug efflux transporter of the resistance nodulation division (RND) protein super family [12,13]. One of the best studied RND transport systems is AcrAB-TolC in Escherichia coli [14–16].
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TolC – AcrBDD Interactions
Figure 1: Spanning the entire periplasmic space, the RND efflux transporter AcrAB-TolC uses the outer membrane factor (OMF) TolC as efflux duct to expel toxic molecules out of the cell (a). Addressing the questions of TolC - AcrB
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interaction and TolC gating, we performed molecular dynamics simulations of the isolated AcrB docking domain alone
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(b) and in presence of membrane-embedded TolC (c).
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Like all RND transporters, AcrAB-TolC is a modular-built membrane protein complex transiently assembled from an inner membrane proton / drug antiporter (AcrB) acting as the pump's engine,
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an outer membrane channel (TolC) acting as efflux duct through which substrate is expelled from the cell, and an adapter protein (AcrA) anchored to the inner membrane stabilizing antiporter and efflux duct interaction and enhancing pump activity [16] (figure 1a). While AcrAB-TolC has been in the intense focus of experimental [13,17–19] and computational research [20,21], the question of how antiporter and efflux duct interact is still unclear as so far the available data suggest two contradicting models of TolC – AcrB interaction: Whereas based on the crystal structures of both proteins [22,23], biochemical cross-linking [24,25] and surface plasmon resonance experiments [26] a direct tip to tip interaction was suggested, electron microscopy studies support the hypothesis that AcrB and TolC do not interact directly but are connected by a hose-like structure 4
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formed by six AcrA molecules [27,28].
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To test if TolC and AcrB can interact directly and if a potential binding affects efflux duct access regulation [29,30], we carried out a series of six independent, unbiased 100ns – 1.05 µs molecular
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dynamics (MD) simulations of phospholipid membrane-embedded TolC in presence of the isolated
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AcrB docking domain (AcrBDD) which we used as a simplified representation of full-length AcrB. After confirming AcrBDD stability in an additional 100 ns MD run of the docking domain on its own,
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we find that in 5 / 6 simulations TolC and AcrBDD engage in direct interaction that is unstable when the binding occurs in a tip to side manner but stable for the simulated time when TolC and AcrBDD
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bind in a tip to tip manner. At the same time we observe no effect on TolC accessibility as the protein opens and closes freely on extracellular side while opening up in the outer periplasmic
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bottleneck region BN-II [30–32] but remaining closed at the inner periplasmic bottleneck BN-I,
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TolC.
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suggesting that either the simulated time is too short or AcrBDD alone is not sufficient to unlock
MATERIALS & METHODS Molecular Dynamics Simulations MD simulations were performed using GROMACS version 4.0.3 [33,34] and the GROMOS96 53A6 force field [35,36]. Standard protonation states were assumed for titratable residues. The LINCS algorithm was applied to constrain all bond lengths permitting an integration time step of 2 fs [37,38]. A temperature of 310 K was maintained separately for protein, lipids (if present), and the water/ion mixture using the Berendsen thermostat [39] with a time constant of 0.1 ps. For
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pressure coupling the Berendsen barostat [39] was employed using a 1 bar reference pressure and a time constant of 4 ps. Whereas the AcrBDD simulation was performed using an isotropic pressure
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coupling scheme, semiisotropic pressure coupling was employed for the TolC + AcrBDD runs to permit bilayer fluctuations in the membrane plane. Electrostatic interactions were calculated
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applied for computing the van der Waals interactions.
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using Particle Mesh Ewald (PME) summation,[40,41] and twin range cutoffs of 1.0 and 1.4 nm were
Simulation Set Up AcrBDD
The model of the isolated AcrBDD was generated by truncating the asymmetric wild type AcrB
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crystal structure 2GIF [42] to residues 182 to 272 and 724 to 812. AcrBDD was then placed at the
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center of a cubic simulation box with an edge length of 10.6 nm, solvated with 36,473 water molecules and 108 sodium and 111 chloride ions yielding a 150 mM NaCl solution and a total
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system charge of zero (figure 1b). After a standard steepest descent energy minimization 100 ns of
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unbiased and unrestrained MD simulation were performed to test the stability of the isolated docking domain.
Simulation Set Up TolC + AcrBDD Using the 1EK9 [22] -based starting structure of our previous study of TolC embedded in a palmitoyl-oleoyl-phosphatidyl-ethanolamine (POPE) membrane [30] as a starting point, we deleted all water and ions and extended the original simulation box to 20.61 nm in Z direction (membrane normal), keeping the original X and Y dimensions of 10.06 nm and 9.97 nm. In the next step, we added AcrBDD in an orientation matching the one in the AcrAB-TolC docking model by Symmons 6
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and co-workers [24]. Subsequently AcrBDD was translocated 1 nm away from TolC parallel to the vector of the membrane normal. As before, the simulation system was solvated with a 150 mM
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NaCl/water solution, comprising 47,423 water molecules as well as 205 sodium and 190 chloride ions yielding a total system charge of zero and an overall system size of 178,028 atoms (figure 1c).
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Following steepest descent energy minimization, four independent, unrestrained and unbiased 150
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ns MD simulations were initiated (run A to D) using different random seed numbers in generating
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the starting velocities. Of these run A was later extended to 1.05 µs.
To test whether the asymmetric monomer conformations in the 2GIF AcrB crystal structure [42]
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influences TolC - AcrBDD interaction, two additional simulations were performed where AcrBDD was initially rotated by +120° and -120° around the membrane normal axis. Following the same
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solvation and energy minimization protocol outlined above, unbiased and unrestrained dynamics
Analysis
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samples were computed for 100 ns each.
AcrBDD Stability To assess AcrBDD stability we calculated Croot mean square deviations (RMSD) after least square fitting to the starting structure and computed Ramachandran distributions over the entire 100 ns simulation times (figure 2, supplemental figure S1). All analyses were compared to previous 100 ns simulations of full length AcrB [43]. In the Ramachandran plots AcrB residues located within an “upper” 1 nm thick slab of the outer membrane-facing side of the protein (figure 2b) are highlighted red. CRMSDs were calculated on protein (figure 2a) and individual atom level in 7
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form of Cdisplacement plots (supplemental figure S1). In case of the latter we also computed the difference in Cdisplacement between AcrBDD in isolation and the full length protein showing
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the maximum AcrBDD displacements. The differences were then mapped onto the simulation
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starting structure.
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TolC – AcrBDD Interaction
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To monitor TolC - AcrBDD interaction we computed for all simulations the buried surface area (BSA) as a function of time (figure 3, 5). Following the same protocol as detailed in [44], the BSA was computed using NACCESS [45]. Horizontal lines in figure 3b and c indicate the TolC – AcrB BSA as
PT
ED
seen in the Symmons model of the AcrAB-TolC complex [24].
CE
To identify hotspots of interaction in the TolC-AcrBDD interface we computed for each simulation time-averaged contact frequencies per residue over the entire trajectory, using a respective
AC
distance cutoff of 0.2 nm (figure 4, 5). Results were mapped onto the simulation structures as formal B-factors and visualized using VMD [46].
TolC Accessibility Tolc accessibility was monitored using the same approach as in [29,30] (figure 6). On extracellular side we calculated for each simulation the distribution of the dihedral angle formed by the carbons of Asp56 in the ß-barrel and Ala270 at the tip of each extracellular loop. On periplasmic side we quantified the opening state of the inner (BN-I) and outer (BN-II) bottleneck computing the
8
ACCEPTED MANUSCRIPT TolC – AcrBDD Interactions
triangular cross-sectional area (TCA) spanned by α-carbons of Asp374 (BN-I) and Gly365 (BN-II). Vertical and horizontal dashed lines in figure 6 reference TolC and TCA values as observed by X-
IP T
ray crystallography [22,31,32,47–49]. Similar to our previous work on OprM and TolC [29,30], we
CR
computed 1D and 3D sodium densities to test for preferred sodium binding sites as previous studies revealed an ion dependency of TolC's periplasmic opening state [50]. Calculated over the
US
entire simulation time, 1D densities were computed for all simulations, whereas spatial densities were analyzed for the µs run. Using the VolMap plugin in VMD 1.9 [46] at a resolution of 1 ų,
MA N
sodium densities in the TolC periplasmic bottleneck region were calculated at a density level of
ED
0.01 and 0.1 Na/ų.
Molecular visualizations were generated using VMD 1.9.1 [46], all other illustrations were created
RESULTS
AC
CE
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using Inkscape (figure 1a), Xmgrace & Xara Designer Pro.
AcrBDD Stability To assess the stability of AcrBDD alone and in context of the full-length protein we computed CRMSDs, Cdisplacements and Ramachandran distributions (figure 2, supplemental figure S1). Whereas in full-length AcrB RMSDs of the docking domain range from 0.3 to 0.38 nm throughout 100 ns simulation time (figure 2a, blue), AcrBDD on its own displays an RMSD of 0.41 to 0.46 nm in its 100 ns dynamics sample (figure 2a, red). To detect where conformational changes occur we computed C displacements between the 2GIF X-ray and the simulation end structures of AcrBDD 9
ACCEPTED MANUSCRIPT TolC – AcrBDD Interactions
in isolation (blue graphs) and of previous simulations where AcrBDD was in context of the full length protein (supplemental figure S1 a). Computing the difference between the maximum AcrBDD
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changes seen in the full length protein and the isolated docking domain, we find that on TolCfacing side conformational changes in the isolated AcrBDD are smaller than or equal to the
US
CR
maximum changes observed for AcrBDD in the full length protein (supplemental figure S1 b, c).
Comparing the Ramachandran distributions over the 100 ns simulation time we find that AcrBDD
MA N
alone (figure 2c) and AcrBDD as part of full-length AcrB (figure 2d) display similar patterns,
AC
CE
PT
ED
particularly in the upper 1 nm thick section facing towards the outer membrane (figure 2b-d, red).
Figure 2: To assess its stability we compared dynamics samples of the isolated AcrBDD (red curve in a, panels b & c) with previous full length AcrB simulations [43] (blue curves in a, panel d), computing C root mean square displacements (a) and Ramachandran distributions (b-d), where AcrB residues located within a 1 nm thick slab of the outer membrane-facing side of the protein are highlighted red (b).
10
ACCEPTED MANUSCRIPT TolC – AcrBDD Interactions
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TolC - AcrBDD Interaction
Figure 3: TolC - AcrBDD buried surface area (BSA) computed for each of the four production runs (runA-D) and the two
ED
control simulations where AcrBDD was rotated by ± 120° around the membrane normal. For each simulation we show the configuration displaying the maximum BSA (a), the total amount of BSA (b) as well as the individual contribution of
PT
each monomer (c). In total we observed three types of TolC - AcrBDD binding events: tip to tip binding (yellow), tip to
CE
side binding (cyan) and no binding (grey). In (c) T-A, T-B, T-C and A-A, A-B, A-C indicate monomers A, B, C in TolC and
AC
AcrBDD respectively. Horizontal lines in (b) and (c) indicate the interface area as seen in the Symmons model [24].
To quantify TolC-AcrBDD interaction and monitor potential binding events we computed for each simulation the TolC-AcrBDD buried surface area (BSA) as a function of time (figure 3). Illustrated by simulation snapshots of maximum interface area (figure 3a), the BSA is shown in total between the TolC and AcrBDD trimers (figure 3b) as well as broken down to the individual contributions of each monomer (figure 3c). Horizontal lines in figure 3b and c indicate the BSA seen in the AcrAB-TolC Symmons model [24]. In total we observe three types of TolC-AcrBDD interactions: (A) a stable tip to tip binding in 4 / 6 simulations (figure 3, yellow panels) with runA exhibiting the highest and most homogeneous BSA values; (B) a transient tip to side binding in 1 / 6 simulations where TolC's
11
ACCEPTED MANUSCRIPT TolC – AcrBDD Interactions
periplasmic mouth engages the monomer C and B flank of AcrBDD for about 40 ns (figure 3, cyan); and (C) no binding at all in 1 / 6 simulations (figure 3, grey). Further inspection shows that in cases
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(B) and (C), due to the periodic boundary conditions of the simulations, AcrBDD drifted to the other side of the membrane, leading to physiologically irrelevant interactions with TolC's extracellular
CE
PT
ED
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loops or the POPE phospholipids of the model membrane (supplemental figure S2).
Figure 4: TolC - AcrBDD interface residues as determined by close contact frequencies computed over each simulation
AC
and mapped onto the AcrBDD and TolC starting structures. Results are shown for the production runs (left) and the control simulations (right) where AcrBDD was rotated by ± 120° around the membrane normal. Panel colours indicate tip to tip (yellow) and tip to side (cyan) interaction. Run B was excluded from the analysis as no physiologically relevant TolC - AcrBDD interaction occurred in that simulation.
To identify residues involved in the spontaneously formed TolC – AcrBDD interfaces we computed for each simulation with a binding event (figure 3, yellow & cyan panels) time-averaged, perresidue contact frequencies between both proteins. Calculated over the entire length of each simulation, the results were then mapped onto the simulation starting structures (figure 4). 12
ACCEPTED MANUSCRIPT TolC – AcrBDD Interactions
Whereas the tip to side binding in run C clearly sticks out (figure 4, cyan panel), the tip to tip bindings share a common pattern of contact that, although pronounced to varying degrees in the
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four simulations (maximum in run A), outlines the same set of interface residues (figure 4, yellow
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US
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panels).
Figure 5: TolC
-
AcrBDD interactio n in run A extended to 1.1 µs
ED
as illustrated
PT
by
total
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(a) and per monomer buried surface area analysis (b), interface snapshots of the simulation end conformation (c) and TolC - AcrBDD close contact frequencies computed over the entire simulation and mapped onto the starting structures
AC
(d). In (b), (c) and (d) T/A, T/B, T/C and A/A, A/B, A/C indicate monomers A, B, C in TolC and AcrBDD respectively. Horizontal lines in (a) and (b) indicate the interface area as seen in the Symmons model [24].
Displaying the highest and most homogeneous BSA levels as well as the most pronounced patterns of close contact interface residues, the initially 150 ns long run A was extended to a total length of 1.05 µs (figure 5). Throughout that time TolC-AcrBDD binding became continuously tighter with the BSA surpassing the interface area of 43.03 nm² of the Symmons model after 380 ns (figure 5a). At the same time the initially homogenous interaction pattern between the two proteins (monomer A with A, B with B and C with C) becomes asymmetric with for example TolC monomer A additionally
13
ACCEPTED MANUSCRIPT TolC – AcrBDD Interactions
interacting with AcrBDD monomer B or TolC-B with AcrBDD-C (figure 5b). Inspection of the simulation end conformation shows that whereas AcrBDD-A and C exhibit a similar TolC binding
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characteristic as in the Symmons model, AcrBDD-B adopts a shifted configuration where its DC hairpin is located in between the helix 7/8 loop of TolC-B and the helix 3/4 loop of TolC-A instead of
CR
the gap between both helix-loops of monomer B (figure 5c). In terms of interface residues the
US
results of the contact frequency analysis (figure 5d) are in line with our findings for the initial set of
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simulations (figure 4).
TolC Accessibility
To monitor and quantify TolC accessibility throughout the simulations, we monitored the channel's
ED
opening state computing (a) the distributions of the dihedral angle formed between the ß-barrel
PT
and the tip of each extracellular loop; and (b) the triangular cross-sectional area (TCA) spanned by
CE
the inner and outer periplasmic bottlenecks BN-I and BN-II (figure 6).
AC
In the runs with tip to tip interaction the distributions range from -40° to 170° (figure 6a, yellow panels) and from -90° to 160° in the simulation where tip to side interaction occurred (figure 6a, cyan panel). Clearly exceeding the range of ~ 90°-110° observed in TolC wild type and mutant crystal structures [22,31,32,47–49], the distributions from our simulations predominantly exhibit maxima located at values either overlapping with or higher than the experimentally observed range. In this context the tip to side simulation is an exception as here the monomer B distribution peaks at -60° due to a physiologically irrelevant interaction of AcrBDD with the periodic image of TolC's extracellular loop in monomer B (supplemental figure S2).
14
ACCEPTED MANUSCRIPT TolC – AcrBDD Interactions Figure 6: TolC
ty
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accessibili as
CR
monitored through
US
the dihedral
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angle formed between
ED
each extracellular loop and the ß-barrel (a) and the triangular cross sectional area (TCA) spanned by the -carbons of Gly365 and Asp374 forming the outer (BNI) and inner (BNII) periplasmatic bottlenecks (b). Panels (c) and (d) show the
PT
outcome of the same analyses for the 1.05 µs simulation. Green TCA regions indicate opening states as observed in [30] whereas yellow and cyan background colors indicate tip to tip and tip to side interactions of TolC and AcrB DD.
CE
Dashed vertical lines in (a) and (c) mark dihedrals observed experimentally [22,31,32,47–49], whereas horizontal
AC
dashed lines in (b) and (d) represent the BNI and BNII TCAs of the 1EK9 X-ray structure [22].
On periplasmic side the TCA in the outer bottleneck (BN-II) increases rapidly at the beginning of each simulation covering a TCA range from 1.9 nm² to 2.9 nm² by the end of the simulations (figure 6b, green graphs). In the inner bottleneck BN-I the TCA changes little staying close to its initial 0.6 nm² as seen in the 2EK9 starting structure [22] (figre 6b, red graphs).
In the run A simulation extended to 1.05 µs the distributions range from 60° to 180° with maxima located at 150° and 165° (figure 6c). On periplasmic side the BN-I TCA fluctuates around the experimentally observed value, whereas the BN-II TCA explores a range from 1.4 to 3.2 nm² 15
ACCEPTED MANUSCRIPT TolC – AcrBDD Interactions
throughout the additional simulation time (figure 6d).
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Analogue to [29,30] we computed 1D and 3D sodium densities (supplemental figure 3) to test for preferred Na+ binding sites. As in our previous work on TolC [30] we find in all simulations
CR
heightened sodium densities at the lipid head groups an in the periplasmic bottleneck region
US
(supplemental figure S3a). Inspecting this region further in the 1 µs simulation, we find the resulting spatial sodium density resembling the ones reported in previous TolC simulations [30]
ED
MA N
when BNII is open (supplemental figure S3b).
DISCUSSION
PT
Addressing the questions of direct TolC – AcrB interaction and TolC access regulation, we
CE
conducted a series of six unbiased and independent 100 ns – 1 µs molecular dynamics simulations of membrane-embedded TolC in presence of the isolated AcrB docking domain whose stability was
AC
assessed in an additional 100 ns run. With AcrBDD alone displaying similar conformational dynamics as in context of full-length AcrB, we observe that TolC and AcrBDD either engage in a transient tip to side or a tip to tip interaction that is stable on the simulated time scale. At the same time we find that in all simulations TolC opens and closes freely on extracellular side while opening up in the outer periplasmic bottleneck region BN-II at Gly-365 but remaining closed in the inner bottleneck BN-I at Asp-374 in all simulations. We begin this section discussing the limitations of our approach before proceeding to our findings and their biological implications.
Limitations of our Approach
16
ACCEPTED MANUSCRIPT TolC – AcrBDD Interactions
The computer simulations reported here are mainly limited by three key factors: (1) the AcrB model used; (2) the representation of the proteins' micro-environment and (3) the amount of
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conformational sampling achieved.
CR
To test for possible interactions between two proteins, including both molecules in their entirety
US
would be the best approach. However, with TolC and AcrB being membrane proteins exhibiting a defined spatial orientation, reducing system complexity and computational cost by truncating e.g.
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one protein to functionally relevant parts is a reasonable strategy as long as the e.g. domain is sufficiently stable to represent the full length protein. With AcrBDD on its own displaying a similar
ED
Ramachandran pattern and thus a high degree of conformational similarity with AcrBDD connected to the full length protein (figure 2), we consider our reduced AcrB model a reasonable starting
PT
point to study TolC – AcrB interaction. One should keep in mind however that in this approach the
CE
potential observation of functionally relevant protein responses to complexation is for the most
AC
part restricted to TolC.
In vivo AcrAB-TolC's micro-environment is characterized by two phospholipid membranes of heterogeneous and, in case of the outer membrane, highly asymmetric lipid composition (figure 1a). Moreover TolC is not only membrane-embedded but also spans the peptidoglycan layer located in between the outer and inner membrane. In our simulations we omit the peptidoglycan layer and resort to a homogeneous POPE bilayer as model membrane. While clearly a simplification of the in vivo conditions, we nevertheless consider our model a reasonable starting point given that (a) peptidoglycan and lipopolysaccharide models are still in their infancy and were not available for E. coli by the time this study was conducted [51–58] and (b) previous simulation
17
ACCEPTED MANUSCRIPT TolC – AcrBDD Interactions
studies of outer membrane proteins have successfully made biologically relevant predictions using similar simplifications [29,30,59–63]. These studies include our own previous works on TolC and
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OprM to which the present study is a direct successor using a similar experimental setup for better
CR
comparability.
US
Whether the amount of conformational sampling obtained in an MD simulation study is sufficient depends on the questions the study seeks to answer. Here we address the questions if TolC and
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AcrB can interact directly and if this interaction affects the regulation of TolC accessibility. To this end we performed six independent and unbiased MD runs using three different starting
ED
orientations with simulation times ranging from 100 ns to 1.05 µs. Given that we observed two types of direct interaction in 5 / 6 simulations (figure 3, 5) and in all simulations a pattern of TolC
PT
accessibility (figure 6) similar to previous studies of bilayer-embedded outer membrane factors
AC
to answer.
CE
[29,30], we consider the amount of conformational sampling adequate for the question we sought
Functional implications TolC – AcrB Interactions
Over the years AcrAB-TolC has been the focus of numerous studies, investigating different aspects of RND efflux pump structure and functional mechanism, including the question of how AcrB, AcrA and TolC assemble into a functional complex. So far the available data suggest two contradicting models of TolC – AcrB interaction: Whereas based on the crystal structures of both proteins [22,23], biochemical cross-linking [24,25,64], minimum inhibitors concentrarion measurements
18
ACCEPTED MANUSCRIPT TolC – AcrBDD Interactions
[65] and surface plasmon resonance experiments [26] a direct tip to tip interaction was suggested, electron microscopy studies support the hypothesis that AcrB and TolC do not interact directly but
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are connected by a hose-like structure formed by six AcrA molecules [27,28].
CR
To test if TolC and AcrB can engage in direct interaction we performed MD simulations of TolC and
US
AcrBDD which we used as a simplified representation of full length AcrB (figure 1). As indicated by the BSA analyses (figure 3) binding events occur in 5 / 6 simulations, leading to an unstable tip to
MA N
side and stable tip to tip interactions in four simulations. Interestingly we observe this stability regardless of how far the binding process has progressed: once an initial contact has been
ED
established TolC and AcrBDD do remain together whereas the tip to side complex falls apart after 40 ns. If our simulations are correct this finding suggests that under the simulated conditions TolC and
PT
AcrBDD can engage in direct interaction that is only stable on the observed 100 ns – 1.05 µs time
CE
scale (figure 3, 5) when AcrBDD binds to TolC in a tip to tip manner - which is compatible with the type of TolC – AcrB interaction proposed in [22–26]. While one could argue that the observed
AC
binding behaviour is merely random, the fact that (a) tip to tip interaction occurs four times whereas tip to side complexation is seen only once and (b) only in tip to tip complexations TolC and AcrBDD stay together speaks against this argument. As our simulations further indicate the formation of a common pattern of TolC - AcrBDD interface residues in the runs with tip to tip interaction (figure 4, 5), a potential way to experimentally test our computer prediction could be to for example introduce cross-linkers in the observed TolC - AcrBDD interface and check whether the two proteins come off separately or conjoined in a subsequent preparation step involving gel electrophoresis or mass spectrometry.
19
ACCEPTED MANUSCRIPT TolC – AcrBDD Interactions
TolC Access Regulation With distributions (figure 6a, c) clearly exceeding the experimentally observed range in TolC
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crystal structures [22,31,32,47–49] (figure 6) our findings indicate that TolC is opening and closing
CR
freely on extracellular side suggesting the absence of an access regulation mechanism on this side of the protein. These results are in line with our previous work of outer membrane factors
US
reporting the same behavior [29,30]. Whether the shift of maxima towards larger dihedrals and thus more open loop conformations in the 1.05 µs simulation (figure 6c) could be interpreted as a
MA N
possible response of TolC to the increasingly tighter binding of AcrBDD (figure 5a) signifying a potential preparation for transport cannot be decided based on the current data, as only a single
ED
microsecond trajectory was generated.
PT
On periplasmic side the TCA analysis delivers a consistent picture in all simulations (figure 6 b, d):
CE
whereas the TCA at Gly-365 increases rapidly by a factor higher than 3.5 in all simulations, the TCA at Asp-374 fluctuates around the value seen in the wild type crystal structure from which the
AC
simulations were initiated [22]. If our simulations are correct this finding suggests that TolC opens up at the outer bottleneck BN-II but remains closed at the inner bottleneck BN-II which is in agreement with our previous studies [29,30]. Finding the same TCA profiles regardless whether AcrBDD has bound or not, this behavior suggests that either the simulated time was too short or AcrBDD alone is not sufficient to unlock TolC, requiring instead the presence of additional components absent in our simulations. In that light one could argue that our in silico findings are also compatible with the hypothesis of no direct interaction between TolC and AcrB [27,28] in so far that while direct interaction is in principle possible but does not lead to a functional efflux pump. Experimental ways to test our predictions could include double spin lable EPR experiments
20
ACCEPTED MANUSCRIPT TolC – AcrBDD Interactions
to monitor TolC accessibility on the extracellular side. Another option could be TolC activity tests where the efflux duct is reconstituted in a membrane separating a two component system with
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one of them containing AcrBDD plus transport substrate detectable by e.g. fluorescence
US
CR
spectroscopy or pH measurements.
MA N
CONCLUSIONS
Addressing the questions if TolC and AcrB can interact directly and if such an interaction affects
ED
TolC access regulation, we carried out a series of 100 ns – 1.05 µs MD simulations of membraneembedded TolC in presence of the isolated AcrB docking domain. In 5 / 6 runs we find that TolC
PT
and AcrBDD engage in direct interaction that is stable throughout the simulations when the binding
CE
occurs in a tip to tip manner. Opening and closing freely on extracellular side, TolC opens in the outer periplasmic bottleneck region but remains closed at the inner periplasmic bottleneck,
AC
suggesting that either simulated time is too short or AcrBDD alone is not sufficient to unlock TolC.
Acknowledgements This work was financially supported by the Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen within the NRW Rückkehrer-Programm; the Deutsche Forschungsgemeinschaft and the Bundesministerium für Bildung und Forschung.
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TolC – AcrBDD Interactions
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Graphical Abstract
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The authors declare no conflicts of interest.
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ACCEPTED MANUSCRIPT TolC – AcrBDD Interactions
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Highlights AcrAB-TolC is a prominent multi-drug efflux transporter in Escherichia coli representing one of the major causes of antibiotics resistance. Experimental evidence supports two contradicting types of interaction between AcrB and TolC: (a) a direct tip-to-tip interaction and (b) an indirect interaction mediated by AcrA Molecular dynamics simulations of TolC in presence of the isolated AcrB docking domain suggest a direct and stable tip-to-tip interaction between both proteins. At the same time TolC opens and closes freely on extracellular side while remaining closed at the outer perisplasmic bottleneck BN II
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