Enrofloxacin Permeation Pathways across the Porin OmpC - The

Article pubs.acs.org/JPCB

Cite This: J. Phys. Chem. B 2018, 122, 1417−1426

Enrofloxacin Permeation Pathways across the Porin OmpC Jigneshkumar Dahyabhai Prajapati,† Carlos José Fernández Solano,† Mathias Winterhalter,‡ and Ulrich Kleinekathöfer*,† †

Department of Physics and Earth Sciences and ‡Department of Life Sciences and Chemistry, Jacobs University Bremen, 28759 Bremen, Germany S Supporting Information *

ABSTRACT: In Gram-negative bacteria, the lack or quenching of antibiotic translocation across the outer membrane is one of the main factors for acquiring antibiotic resistance. An atomic-level comprehension of the key features governing the transport of drugs by outer-membrane protein channels would be very helpful in developing the next generation of antibiotics. In a previous study [J. D. Prajapati et al. J. Chem. Theory Comput. 2017, 13, 4553], we characterized the diffusion pathway of a ciprofloxacin molecule through the outer membrane porin OmpC of Escherichia coli by combining metadynamics and a zero-temperature string method. Here, we evaluate the diffusion route through the OmpC porin for a similar fluoroquinolone, that is, the enrofloxacin molecule, using the previously developed protocol. As a result, it was found that the lowest-energy pathway was similar to that for ciprofloxacin; namely, a reorientation was required on the extracellular side with the carboxyl group ahead before enrofloxacin reached the constriction region. In turn, the free-energy basins for both antibiotics are located at similar positions in the space defined by selected reaction coordinates, and their affinity sites share a wide number of porin residues. However, there are some important deviations due to the chemical differences of these two drugs. On the one hand, a slower diffusion process is expected for enrofloxacin, as the permeation pathway exhibits higher overall energy barriers, mainly in the constriction region. On the other hand, enrofloxacin needs to replace some polar interactions in its affinity sites with nonpolar ones. This study demonstrates how minor chemical modifications can qualitatively affect the translocation mechanism of an antibiotic molecule.

1. INTRODUCTION As several studies suggest,1−3 multidrug-resistant superbugs might claim millions of lives globally in the near future. For instance, the resistance of Escherichia coli bacteria to fluoroquinolones has increased to a threatening level in recent decades.4−8 Antibiotic influx across the outer membrane (OM) has been recognized as one of the factors playing a critical role in this resistance.9 As has been confirmed by several experiments,10−14 the general-diffusion OM channels of E. coli, for example, the porins OmpF and OmpC, especially facilitate antibiotic diffusion toward the periplasmic space. Consequently, the down-regulation of these major porins is associated with a decrease in the intracellular accumulation of fluoroquinolones.15−19 At the same time, higher osmotic stress due to the presence of antibiotics favors the expression of OmpC over OmpF.16,20 Similarly, milieus containing high levels of nutrients, for example those in mammalian intestines, promote the expression of the OmpC porin.21−23 Therefore, a proper understanding of antibiotic permeation through OmpC is biologically relevant and might be very helpful in guiding the development of next-generation antibiotics.24 The translocation of antibiotics into Gram-negative bacteria is a key problem in discovering new drugs for treating the infections they cause.25,26 Molecular-dynamics (MD) simulations became a valuable tool for drug discovery in general27 and in particular when it comes to ion permeation and substrate translocation through © 2018 American Chemical Society

OM pores. These simulations include, for example, studies of OmpF,28−30 OmpC,24,31 OprP,32−34 OprO,35 and OprD.36,37 Recently, these simulations include an improved description of OMs, that is, lipopolysaccharide (LPS)-phospholipid membranes.38−40 Moreover, substrate transport through OM channels is often studied in combinations of experiments and simulations.41−43 Metadynamics simulations44 have been used to illustrate the permeation of various classes of antibiotics through OmpF43,45−51 and, in fewer investigations, through OmpC.24,45,52 The early studies24,45−50 were conducted on the basis of the escaping-free-energy-minima protocol;53 that is, the simulations were stopped after the first permeation event from the extracellular side to the periplasmic side of the pore (or vice versa). The respective estimated first-transition paths were considered the most favorable ones. However, these studies were only able to provide a qualitative and incomplete picture of antibiotic diffusion pathways as their sampling was very limited by the accessible simulation time a couple of years ago; that is, the simulations were performed for at most 100 ns. In the last few years, more extensive simulations have been performed,43,51,52 in which free-energy surfaces (FESs) were reconstructed as functions of predefined collective variables Received: December 21, 2017 Revised: January 5, 2018 Published: January 6, 2018 1417

DOI: 10.1021/acs.jpcb.7b12568 J. Phys. Chem. B 2018, 122, 1417−1426

Article

The Journal of Physical Chemistry B

Figure 1. (A) OmpC trimer64 (PDB ID: 2J1N) shown in cartoon representation. The loops L2, L3, and L4 are highlighted in yellow, magenta, and cyan, respectively. The important negatively charged (D105, E109, and D113) and positively charged (K16, R124, R74, and R37) residues located in the constriction region are shown in stick representation. (B) 2D structure of the enrofloxacin molecule in its zwitterionic form. (C) System setup for well-tempered metadynamics simulations of enrofloxacin permeation through the OmpC porin. An OmpC trimer (cartoon representation) inserted in a POPE lipid bilayer (surface representation) is shown together with enrofloxacin molecules (van der Waals representation) placed at the EC and PP sides of the porin. Water is shown in surface representation, and ions are shown as balls.

electrophysiology experiments,63 but a similar dissociation rate. As a result, the estimated flux through OmpC at −100 mV is lower for enrofloxacin (0.5 ± 0.07 molecules/s) than for ciprofloxacin (3.0 ± 0.3 molecules/s).63 However, the estimated fluxes through OmpF based on single-channel electrophysiology are very similar for both molecules (i.e., 8.0 ± 1.0 and 7.0 ± 0.8 molecules/s for enrofloxacin and ciprofloxacin, respectively).63 Thus, this subtle difference in the permeation rate of enrofloxacin through OmpC deserves further studies concerning the main differences in the diffusion mechanisms at the molecular level. With the outcome of this study, we provide very detailed insight at the atomistic level into the permeation paths of two antibiotics, which moreover enables us to explain the differences observed in the electrophysiology experiments. The paper is organized as follows: Section 2 is mainly devoted to descriptions of the system setup and simulation protocol for studying enrofloxacin translocation across the OmpC porin of E. coli. In Section 3, we discuss the results and compare them with those obtained for ciprofloxacin permeation. Finally, we conclude with some remarks in Section 4.

(CVs), that is, the distances and orientations of the antibiotics with respect to the channel axes. The main goal of these studies was to establish a theoretical foundation for the different kinetic rates of antibiotics in comparison to electrophysiology experiments. Nevertheless, these studies were mainly focused on antibiotic transport in the vicinity of or inside the constriction region, and little information was provided about the full permeation process. On the other hand, cocrystal structures of three antibiotics complexed with the OmpF porin were recently determined using X-ray crystallography.54 Notably, this study shows three different affinity sites, that is, one for each antibiotic molecule, which are located at the OmpF vestibules. Therefore, there is still a need to characterize the full diffusion routes of available antibiotics in order to establish useful structure−function relationships for various porins. Recently, we have proposed a computational protocol55 to characterize the antibiotic permeation pathways across OM channels that combines metadynamics53,56−58 and a zerotemperature string method.59,60 As a result, we have been able to construct the minimum free-energy permeation path for the ciprofloxacin antibiotic along the OmpC porin. To our knowledge, this is the first study that fully characterizes an antibiotic permeation pathway through an OM channel and identifies the most relevant affinity sites along the pathway. Here, we study the permeation process for enrofloxacin, namely, a fluoroquinolone molecule very similar to ciprofloxacin, in order to establish the influence of chemical modifications to a drug on membrane translocation. It has been shown that enrofloxacin is less efficient against E. coli than ciprofloxacin on the basis of its higher minimum inhibitory concentration.20,61,62 Moreover, enrofloxacin translocation across OmpC has a lower association rate constant than ciprofloxacin, as estimated on the basis of single-channel

2. MATERIAL AND METHODS 2.1. System Setup and Metadynamics Simulations. This work focuses on the porin OmpC from the Gram-negative bacterium E. coli.21,64,65 The porin OmpC is classified as a nonspecific protein channel because it allows the passage of ions and metabolites up to 600 Da without any clear specificity. OmpC has a sequence similarity of around 60% with OmpF, and 74% of the residues along the pore lumen are identical64,66 in both porins. As shown in Figure 1A, the porin OmpC is a trimer in which each monomer forms a hollow 16-stranded βbarrel. All the monomers share exactly the same primary and tertiary structure. The constriction region is formed by an 1418


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were deposited every 4 ps, and the initial height was set to 0.72 kcal/mol, leading to a deposition rate of 0.18 kcal/(mol·ps). The bias factor was set to 30, which was equivalent to a tuning temperature of 8700 K. At the same time, the movement of the antibiotic was restricted inside a monomer by applying halfharmonic walls. Some FES artifacts near the boundaries were relieved with the help of a previously suggested fix.83 The bias was stored on a mesh grid with a grid size of 0.01 Å, which allows for efficient on-the-fly estimation of bias forces by interpolation. A total of 16 walkers were used with nine and seven of them started from the EC and PP mouths, respectively. The total simulation time was set to 4 μs (250 ns for each walker), which corresponds to a deposition of 1 million Gaussian hills. The metadynamics simulations were performed using the PLUMED plugin version 2.2.384 together with the GROMACS package.72 An MD time step of 5 fs was used with the help of virtual hydrogen sites.85−87 2.2. Estimation of the FES and Translocation Pathway. After achieving a reasonable convergence in the multiple-walker WTmetaD simulation (see the SI, section S3), the FES was reconstructed as a function of the CVs z and zij using the Tiwary and Parrinello reweighting technique82 as implemented in an in-house code55 with a grid size of 0.5 and 0.4 Å. A reasonable assumption is that antibiotics roughly follow pathways of minimal free energy during the permeation process. Thus, identifying diffusion routes is very similar to finding minimum free-energy paths (MFEPs) on the FES. Because metadynamics allows for estimating the FES and deriving its gradient, MFEPs can be computed efficiently using the string method in the available CVs,59,60 which was implemented in an in-house code.55 The lowest-energy translocation pathway was estimated as a concatenation of MFEPs that connect the EC and PP sides, winding through the constriction region. 2.3. Unbiased MD Simulations. Several energy basins along the reconstructed FES were identified and unbiased MD simulations starting from configurations located at these energy basins were performed to evaluate their metastability.55 Four independent unbiased MD simulations were run for 25 ns each for every basin, in which different initial velocities were assigned to the enrofloxacin molecule according to the Maxwell− Boltzmann distribution, and an interaction energy was estimated from the nonbonded pairwise interactions between the enrofloxacin and porin atoms within a cutoff distance of 12 Å. We defined this interaction as the average sum of the electrostatic and van der Waals contributions for those configurations along the trajectories in which the antibiotic molecule remained inside the respective basin. Moreover, those residues located at a distance less than 4 Å from any atom belonging to the antibiotic molecule were identified in each basin, and the respective interaction energies per residue were also determined. 2.4. Metadynamics Simulations in a Restricted CV Space. As in the case of ciprofloxacin,55 an enrofloxacin molecule can adopt two main orientations in the mouth of the constriction region at the EC side, with either the amino or carboxyl group in front with respect to the channel axis. In turn, these conformations determine two different pathways in the permeation process. To explore the energy barriers and topologies of these pathways in the eyelet region of the OmpC porin in more detail, we performed two additional multiple-walker WTmetaD simulations by biasing the CV z in a reduced region of the (z, zij) space. To this end, the antibiotic’s

inward-folded L3 loop. In the constriction region, a strong transversal electric field31 is created as a consequence of the negatively charged residues on the L3 loop (D105, E109, and D113) and the positively charged residues on the opposite side of the pore wall (K16, R37, R74, and R124) that are separated only by a short distance. The loops L2 and L4 bend over the barrel wall of an adjacent monomer and are held in place by salt-bridges and hydrogen bonds. An overall hourglass shape describes each monomer, in which the narrow constriction region separates the extracellular (EC) and periplasmic (PP) vestibules. All the amino acids were treated in their standard protonation states except residue D299, which was protonated in order to stabilize the fluctuations of the L3 loop in the constriction region.55 At pH 7, the enrofloxacin molecule has a zwitterionic configuration with a zero net charge and also a permanent dipole moment (see Figure 1B). The reported pKa values for the carboxyl and the amino groups are 5.94 and 8.7, respectively.67 The aromatic quinoline scaffold contains polar fluorine and carbonyl groups as well as a nonpolar cyclopropyl ring. An ethyl substitution on the N atom from the amino group is the only difference compared with the structure of the ciprofloxacin molecule. The initial force-field parameters for enrofloxacin were taken from the CGenFF database68−70 and further optimized using the ffTK toolkit71 (see the SI, Section S1). A similar system setup as in our previous work55 was deployed as shown in Figure 1C. In brief, an OmpC trimer was inserted in a fully hydrated POPE bilayer and neutralized with potassium cations that were placed near the edge of the simulation box and restrained by applying harmonic constraints. Two different systems were created by placing an enrofloxacin molecule in the mouth of a given monomer either at the EC or the PP side. Both systems were composed of 135 247 atoms, which included 295 POPE lipids, 26 618 TIP3P waters, and 42 K+ ions. The systems were equilibrated by performing MD simulations with the GROMACS package version 5.1.272 and the CHARMM36 force field73,74 in several consecutive steps.55 We employed the same force field for the lipid and protein atoms, bond-distance constraint, and treatment for nonbonded interactions as those described in our previous work.55 A large variety of enhanced sampling methods44,75−81 have been proposed for exploring and quantifying free-energy surfaces (FESs) in a limited number of reaction coordinates, often referred to as collective variables (CVs). Here we focus on metadynamics,53,56−58 a popular and insightful sampling method that iteratively builds a bias potential to keep the system away from already visited regions and thereby increases the rate of transitions between metastable FES basins. The present permeation process is described in terms of two linear CVs,55 labeled z and zij, which specify the position and orientation of the enrofloxacin molecule with respect to the channel axis. In Section S2 of the SI, the atoms selected for defining the CVs are shown. The convergence of the FES is a critical criterion that needs to be satisfied, otherwise the results might be meaningless. Thus, we adopt a two-stage strategy in which a well-converged FES was initially obtained by only biasing the CV z in a multiple-walker well-temperedmetadynamics (WTmetaD) simulation (see the SI, Section S3) and then recomputed as a function of the CVs z and zij by the aid of the Tiwary−Parrinello reweighting technique.82 The same WTmetaD parameters as those described in our previous work have been employed.55 Briefly, Gaussian hills 1419


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Figure 2. (A) Reweighted FES as a function of the CVs z and zij. The lowest-energy pathway is depicted by a black line. (B) Free energy along the lowest-energy pathway for enrofloxacin and ciprofloxacin. The variable s ∈ [0, 1] is used to parametrize the pathway. (C) Reweighted free-energy landscapes as a function of the CVs z and zij from two independent multiple-walker WTmetaD simulations performed in restricted CV spaces. The key metastable states are labeled in all the panels.

position was restricted by the application of half-harmonic walls at z = −10 and +10 Å. Additional half-harmonic walls at zij = −4 and +4 Å were applied in each simulation to restrict the antibiotic orientations to those with either the amino or carboxyl group ahead. In total, ten walkers were used, in which the initial positions of enrofloxacin were located at z ≈ −10 Å and at z ≈ +10 Å for five walkers each. The simulation for each walker was carried out for 100 ns, leading to total simulation time of 1 μs. The same above detailed parameters for the metadynamics simulations were used.

3. RESULTS AND DISCUSSION The CV z describes enrofloxacin’s position along the channel axis.55 Between the EC (z < −5 Å) and PP (z > 5 Å) vestibules, the constriction region is approximately located at z ∈ [−5, 5] Å. At the same time, the CV zij provides information about enrofloxacin’s orientation with respect to the channel axis.55 Their limiting values, −8 Å (when the carboxyl group is ahead in the z direction) and 8 Å (when the amino group is ahead in the z direction), correspond to parallel and antiparallel orientations with respect to the channel axis. Note that the CVs z and zij are identical to those of the ciprofloxacin molecule studied earlier because the same atoms are involved in defining them.55 Figure 2A depicts the reweighted FES in the (z, zij) space obtained using the Tiwary−Parrinello algorithm.82 The lowest-energy pathway is depicted as a black line on this FES. The enrofloxacin and ciprofloxacin pathways share remarkable similarities (see the SI, Figure S5A). Namely, the enrofloxacin molecule is captured in the EC mouth (z ≈ −24 Å) with a conformation in which the amino group is ahead, pointing toward the eyelet region. A reorientation is required, however, in the EC vestibule before the molecule reaches the constriction region with its carboxyl group ahead. Significantly, the main FES basins for enrofloxacin and ciprofloxacin are located at similar positions in the CV space, enabling us to

Figure 3. (A) Distribution of enrofloxacin conformations in the CV space (z, zij) from unbiased MD simulations initiated in the different energy basins (see Figure 2A,B). Different colors are used for each basin. (B) Representative conformations of the enrofloxacin molecule in each basin. The arrows indicate the transitions into neighboring basins as enrofloxacin translocates from the EC side to the PP side. Loops L3 and L4 from monomer 1 are highlighted in magenta and gray, respectively, and loop L2 from monomer 2 and loop L4 from monomer 3 are highlighted in yellow and cyan, respectively. The enrofloxacin molecule is shown in stick representation with its carboxyl and amino groups in red and blue, respectively. For simplicity, the hydrogen atoms are omitted. The remainder of the antibiotic is shown in the color corresponding to that of the respective basin. The enrofloxacin molecule has been constrained to stay inside monomer 1.

adopt the same notation as in our previous work.55 As a main difference, the overall enrofloxacin permeation pathway exhibits energy barriers larger than those for the ciprofloxacin molecule (see Figure 2B), especially in the constriction region. This 1420


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clearly indicates a slower diffusion for enrofloxacin permeation, in agreement with experiments.63 Unbiased MD simulations were performed in those energy basins identified in Figure 2. In Figure 3A, the distribution of the enrofloxacin conformations from unbiased MD simulations are mapped onto the (z, zij) space. Some conformations from simulations initiated in basin 1 are able to escape outside the monomer, which supports the idea of an entropic barrier at the mouth of the pore. Only a low-energy barrier separates basin 3b from basin 2, seen in the fact that some conformations from simulations started in energy basin 3b are able to emigrate to basin 2 but not vice versa. A similar behavior was found for the ciprofloxacin molecule55 (see the SI, Figure S5B). In contrast to ciprofloxacin, the enrofloxacin molecule often moves back to basin 3 when it is initially located in basin 4. Moreover, basin 5 has low energy barriers to the other basins, and hence, very fast transitions to either basin 4 or basins located at the PP vestibule

Figure 4. Average interaction energies for the enrofloxacin and ciprofloxacin molecules in each energy basin. The error bars represent the standard deviations.

Figure 5. Interaction energies per residue for the enrofloxacin and ciprofloxacin molecules in each energy basin. The residues are classified into five categories: acidic (red), basic (blue), polar (magenta), aromatic (orange), or nonpolar (black). The residues underlined with solid and dotted lines are from the loops L2 and L4 of monomers 2 and 3, respectively. The most prominent residues are indicated by the symbol † and depicted in the Figure 6. Moreover, other relevant residues are indicated by the symbol *. 1421


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Table 1. Prominent Interactions Found between Enrofloxacin Functional Groups and the OmpC Porin Residues in Each Energy Basina energy basin

interaction types

enrofloxacin interacting groups

porin residues

1

salt bridges hydrogen bonds

−NH(C2H5)+ −COO−

D171 T208

π-stacking hydrophobic contact 2

salt bridges hydrogen bonds

−CO −F quinoline ring quinoline, cyclopropyl, and piperazinyl rings −COO− −NH(C2H5)+

3

4

salt bridges hydrogen bonds hydrophobic contact salt bridges hydrogen bonds

π-stacking salt bridges hydrogen bonds

interaction types hydrophobic contact salt bridges hydrogen bonds

N155 Q175 F161, Y211 P156, A205, A209, A210, A172

5

6

−NH(C2H5)+ −COO− −NH(C2H5)+

R174, R246 S157, G158*, E159*, N167, G169* (loop L4, monomer 3) N70 N67 (loop L2, monomer 2) E109 R174, R246 Y22

hydrophobic contact salt bridges

7

hydrogen bonds hydrophobic contact salt bridges

−NH(C2H5)+

Y22, V29, F110, I337

−COO− −NH(C2H5)+

R124 N70

−COO− −CO −F

Q59 S117 N67 (loop L2, monomer 2) W72 R74, R124 Q123

−NH(C2H5) −F 3b

energy basin

+

quinoline ring −COO− −COO−

hydrogen bonds

8

π-stacking salt bridges hydrogen bonds π-stacking

enrofloxacin interacting groups

porin residues

−NH(C2H5)+ −NH(C2H5)+

Y22 Y22, V29, F110, I337

−COO− −NH(C2H5)+

R37, R74 Y22

−CO −NH(C2H5)+

R37, R74 Y22, V29, F110, I337

−NH(C2H5)+ −COO− −NH(−C2H5)+ cyclopropyl ring

D105 K51 V106*, L107*, P108*, D109*, F110*, G111* Y94, Y98

−NH(C2H5)+ −COO− −NH(C2H5)+

D105 K308 V106*, L107*, P108*

−CO quinoline ring −COO− −COO−

R272 Y305 K308 Q266

−F quinoline ring

Q345 Y305

For −NH(C2H5)+, the polar interactions, i.e., the salt bridges and hydrogen bonds, involve the −NH fragment, and the hydrophobic contacts are formed through the −C2H5 fragment. Asterisks (*) denote residues in which the backbone atoms take part in the interactions. a

major differences in the interaction energies for basins 1, 2, 3, and 8. However, smaller interaction energies are observed in basins 5 and 7 for enrofloxacin compared with those of ciprofloxacin. In particular, the interaction for basin 5 of enrofloxacin is much more unfavorable than that for the analogous basin of ciprofloxacin, which strongly supports the finding that enrofloxacin needs to overcome higher energy barriers inside the constriction region and that this basin exhibits poor metastability (see the previous discussion). In Figure 5, the interaction energies per residue are shown for enrofloxacin and ciprofloxacin in each energy basin. The charged and polar residues are found to be the dominating factors for stabilizing the antibiotics in their basins. Furthermore, aromatic and nonpolar residues make significant contributions, and the interactions involving these residues are very often mediated by backbone atoms instead of by side chains. The relevant porin residues involved in the enrofloxacinaffinity sites are listed in Table 1, and representative enrofloxacin conformations are shown in Figure 6. Enrofloxacin adopts conformations similar to those of ciprofloxacin in the affinity sites as they share a wide number of common residues. For example, π-stacking interactions between the benzene ring of enrofloxacin and some aromatic residues (W72, F161, Y211, and Y305) are observed in the affinity sites corresponding to basins 1, 3, 7, and 8, which are also known to play a critical role in the target mechanism of fluoroquinolone antibiotics.88

are observed. It is worth mentioning that all transitions between minima are in accordance with the predicted translocation pathway. In Figure 3B, the representative enrofloxacin conformations are depicted in Euclidean space for the different energy basins. As in the ciprofloxacin-translocation mechanism,55 two orientations are available when approaching the constriction region from the EC vestibule. In basin 3b, the amino group flips, pointing to the L3 loop, while the carboxyl group remains in a similar position to that in basin 2. In basin 3, the amino group remains in a similar position to that in basin 2, but the carboxyl group flips, pointing toward the L3 loop. Our results strongly suggest that enrofloxacin is crossing the constriction region mainly starting from basin 3. Additional multiple-walker WTmetaD simulations in the restricted regions of the CV space provide evidence that the discrimination between pathways passing through minima 3 and 3b is even more drastic for enrofloxacin than for ciprofloxacin (see Figure 2C). By performing a detailed analysis of the trajectories generated by the unbiased MD simulations, we were able to characterize the affinity sites associated with each energy basin. In order to compare the affinity sites of enrofloxacin and ciprofloxacin, the interaction energies between these molecules and the protein residues were estimated for each energy basin (see Figure 4). The unbiased MD simulations performed for the ciprofloxacin molecule in our previous study55 were used for estimating the respective interaction energies. As can be seen, there are no 1422


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Figure 6. Representative conformations of the enrofloxacin molecule in each energy basin. The contributing porin residues and enrofloxacin molecule are shown in stick representation. For enrofloxacin, the C atoms are depicted in orange, O atoms in red, N atoms in blue, F atoms in cyan, and H atoms in ice blue. For the amino acids, the C atoms are shown in green, O atoms in red, and N atoms in blue.

orientations to favor more suitable interactions. These observations clearly emphasize the key role that nonpolar residues play in the transport of antibiotics through nonspecific pores. Finally, we would like to highlight some prominent correlations between previously reported experimental results and our numerical findings. Baslé et al.64 pointed out that OmpC and OmpF mainly differ by a few amino acids located along the pore lumen. Kojima and Nikaido89 demonstrated how a mutation of any of these residues can modify a substrate’s diffusion through OmpC so that it behaves like it is diffusing through OmpF and vice versa. In the case of OmpC, the list of residues is composed of V29, N67, E68, W72, D171, L173, R246, D18, D135, and K317. Here, we have found that residues V29, N67, E68, W72, D171, L173, and R246 make favorable interactions with both ciprofloxacin and enrofloxacin molecules in different affinity sites. In particular, residue V29, belonging to the hydrophobic pocket, is responsible for varying the enrofloxacin orientation in basins 3b, 4, and 5 with respect to the ciprofloxacin one. Moreover, the mutation of residue R124 in mutant-type OmpC3390 was found to drastically reduce antibiotics flux relative to that of wild-type OmpC.24,52 Again, we have found that this residue makes favorable interactions with both ciprofloxacin and enrofloxacin molecules in basins 4 and 5.

Figure 7. Comparison between the ciprofloxacin and enrofloxacin conformations for the affinity sites associated with the energy basin 4 in the OmpC porin. The colors are the same as in Figure 6 except for the hydrophobic pocket formed by residues Y22, F110, L20, V29, and I337 in which the C atoms are depicted in cyan.

Moreover, there exists a similar cooperative effect among different monomers because the affinity sites for basins 2 and 3 require some residues belonging to loops L2 and L4 of neighboring monomers. The main differences are observed in basins 3b, 4, and 5 for the interactions involving the amino group as the ethyl substitution decreases the ability to form hydrogen bonds or salt bridges. Instead, the enrofloxacin molecule forms nonpolar contacts between the ethyl group and some aliphatic and aromatic residues. In the constriction region, the amino group of enrofloxacin orients itself toward the hydrophobic pocket formed by the residues L20, Y22, V29, F110, and I337. As shown in Figure 7, the enrofloxacin and ciprofloxacin molecules are located at similar positions in the affinity sites associated with basin 4 but have different

4. CONCLUSIONS We have been able to describe the primary features of enrofloxacin translocation across the OmpC porin of E. coli by using a previously defined theoretical approach.55 The lowestenergy pathway for enrofloxacin is similar to that for 1423


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ciprofloxacin, the energy basins for both antibiotics are located at similar positions in the two-dimensional CV space, and their affinity sites share a wide range of common porin residues. These facts are very reasonable in view of the minor difference between the enrofloxacin and ciprofloxacin structures, namely, an ethyl substitution in the amino group. However, this substitution leads to some relevant modifications. In a comparison of its affinity sites with those of ciprofloxacin, enrofloxacin replaces some interactions between its amino group and polar residues by hydrophobic interactions with aliphatic and aromatic residues. Thus, a hydrophobic pocket is identified in the vicinity of the constriction region. Moreover, the enrofloxacin molecule needs to overcome higher overall energy barriers than the ciprofloxacin molecule during translocation through the porin OmpC; therefore, a slower diffusion process is expected. To the best of our knowledge, this is the first theoretical study that demonstrates how a minor chemical modification of an antibiotic can qualitatively affect its translocation mechanism. This finding emphasizes the key role of nonpolar interactions in the permeation of large substrates across nonspecific porins. Several issues need to be addressed in future studies. First, the influence of various physiologically relevant ionic salts89,91,92 can modify permeability through the porin OmpC in a nontrivial manner because ions can strongly interact with substrates and residues involved in the affinity sites. Second, the influence of a transmembrane potential might be relevant in the diffusion process, although the inclusion of the electric field into the present protocol might be challenging. Third, a consideration of LPS in the outer leaflet of the OM will be required in the future to simulate the OM channels in a more realistic environment, as the presence of LPS might affect the dynamics of the extracellular loops of OM proteins.93−95 Finally, simulations for the translocation of different fluoroquinolones and other classes of antibiotics through OM porins would help to provide very valuable theoretical underpinnings concerning the respective structure−function relations. The present study is one of the very few theoretical studies comparing the translocation of similar but different compounds through outer-membrane channels. Because understanding the translocation of antibiotics through these nanopores constitutes a major issue in the development of new drugs,25,26 we hope that the current findings might help us to better appreciate what effects minor chemical modifications of antibiotic molecules might have on their permeation properties.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The research leading to these results was conducted as part of the Translocation consortium (www.translocation.eu) and has received support from the Innovative Medicines Joint Undertaking under Grant Agreement No. 115525; resources which are composed of a financial contribution from the European Union’s Seventh Framework Programme (FP7/2007−2013) and EFPIA companies in kind contribution.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b12568. Force-field parameters for the enrofloxacin molecule, definition of the CVs, multiple-walker WTmetaD simulation, ciprofloxacin permeation across OmpC (PDF)

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Enrofloxacin Permeation Pathways across the Porin OmpC - The

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