Bacterial Outer Membrane Porins as Electrostatic Nanosieves: Exploring Transport Rules of Small Polar Molecules

Transcription

1 Bacterial Outer Membrane Porins as Electrostatic Nanosieves: Exploring Transport Rules of Small Polar Molecules Harsha Bajaj, Silvia Acosta Gutiérrez, Igor Bodrenko, Giuliano Malloci, Mariano Andrea Scorciapino, Mathias Winterhalter and Matteo Ceccarelli

2 Supplementary information (SI) SI-Figure 1: Norfloxacin structure of the three different charged forms with their respective dipoles a. cationic form, b. zwitterionic form and c. anionic form; d. norfloxacin isoelectric point plot. At ph 7, majority of the norfloxacin tautomer distribution is zwitterionic which has the highest dipole as shown in figure. The zwitterionic molecule has a very high dipole since the two charges are at the opposite ends of the molecule.

3 SI-Figure 2: Single channel ion current trace of OmpF WT channel a., b. without antibiotic (Blank) and c., d. with 100 µm norfloxacin on cis side (corresponding dwell time in inset) and e., f. trans side at ± 100 mv in 200 mm KCl, 5mM PO 4 ph 7

4 SI-Figure 3: Dissociation rate of two kinds of events above -125mV represented as τ 1 and τ 2 for norfloxacin addition on cis side of OmpF WT at 200mM KCl, 5mM PO 4 ph 7, at room temperature (22 C- 26 C). The above figures shows that at high voltage like -150mV two kinds of events are observed (analysis is based on single channel analysis, this is also confirmed using power spectral method 1 3 ). This two kinds of events with residence time τ 1 and τ 2 are observed for room temperature ( 25 C).

5 SI-Figure 4: Single channel ion current trace of OmpF channel without antibiotic (Blank) and with 100 µm norfloxacin on cis side (corresponding dwell time in inset) at ± 25mV and -150mV; Blank and 100 µm norfloxacin on trans side at 150 mv in 200 mm KCl, 5mM PO 4 ph 7. The association and dissociation rates of norfloxacin were calculated using both single channel analysis and power spectral analysis methods. 1 3 For high voltages (>150mV) where the protein

6 without any antibiotic shows intrinsic gating as shown in SI-Figure 4 Blank -150mV, power spectral method analysis (where the power spectrum is fit by subtracting the blank from substrate addition) in such cases might be more reliable. However, both methods gave comparable results for the kinetic rate constant. SI-Figure 4 shows the ion current trace at low voltage ± 25mV and -150mV, zoomed in ion current trace shows single events clearly and the dwell time histogram inset which is fit using single exponential for ± 25mV and 2 term exponential fit for -150mV. SI-Figure 4 histogram in inset at -150mV norfloxacin addition on cis side, at room temperature, which is fit using two term exponential function.

7 SI-Figure 5: Voltage dependence of a. association rate constant for norfloxacin/ompf cis side addition and b. dissociation rate constant for cis addition for different temperature from 10 C to 35 C in 200mM KCl, 5mM PO 4 ph 7. Temperature dependence of c. the association rate constant and d. the dissociation rate constant at the selected applied voltages; the trend well fits the exponential law. e. The activation enthalpy for the association and for the dissociation; the same values are shown in a smaller voltage range corresponding the linear response regime.

8 SI-Figure 6: Power spectrum (subtracted from blank) of norfloxacin cis side addition to OmpF WT at a. 35 C -199mV and -100mV, the spectrum are fit using a single Lorentzian and b. 35 C -199mV the spectrum is fit using both a single and two Lorentzians. At - 100mV the spectrum is fit using a single Lorentzian at 200mM KCl, 5mM PO 4 ph7. This two kinds of events with residence time τ 1 and τ 2 are observed for room temperature ( 25 C) and lower temperature at voltage higher than -125mV (as seen in SI-Figure 3). At 35 C, though only one kind of event is observed even at high voltage like -199mV, SI-Figure 6a shows the subtracted power spectrum of norfloxacin addition on cis side at -199mV or -100mV which can be fit using one term lorentzian. On the other hand, at lower temperature like 10 C, high voltage like -199mV could not be fit using a single lorentzian function but two term lorentzian fit the curve as shown in SI-Figure 6b, relatively lower voltage like -100mV could be fit using single term lorentzian (SI-Figure 6b). SI-Figure 6c shows the dissociation rate vs voltage at temperature 25 C which shows such two kinds of events at higher voltages. In general, the second kind of event τ 2 might not be voltage dependent and is 3-10 times higher (i.e residence time is very low about 30-60µs) than τ 1 for their respective voltages (SI-Figure 6c).

9 SI-Figure 7: Single channel ion current trace of OmpF R167E and R168E without antibiotic (Blank) at a. -100mV, d. 100mV and with 100 µm Norfloxacin on cis side b. at - 100mV, e. at 100mV and trans side addition for 200mM KCl, 5mM PO 4 ph 7. The ion current trace of mutant (R167E/R168E- OmpF EE mutant) in absence of antibiotics does not show significant fluctuations (SI- Figure 7a and d). Norfloxacin addition to the extracellular side of the channel causes blockages of residence time 35µs at -100mV (SI- Figure 7b). Therefore, with this particular mutation the residence time of the antibiotic has been reduced almost 40 times compared to OmpF WT at the same voltage indicating that the two arginines mutated that define the positive selectivity filter of OmpF WT might be involved in the binding site of norfloxacin (ES1).

10 SI- Figure 8: Single channel ion current trace of OmpC WT without antibiotic (Blank) at a. -100mV, c. 100mV and with 100 µm Norfloxacin on cis side b. at -100mV, Norfloxacin on trans side addition d. at 100 mv and 200mM KCl, 5mM PO 4 ph 7; Voltage dependence of a. Association rate on cis and trans side b. Dissociation rate at cis and trans for 200mM KCl, 5mM PO 4 ph 7. In OmpC WT, interaction with norfloxacin shows a similar exponential trend in k on (SI- Figure 8e and 7f) but for the k off such effect is not observed. This effect is similar to that observed for R167/R168E mutant where we observed that the electric field calculated for this mutant is similar to that of OmpC (SI- Figure 13b).

11 SI- Figure 9: Single channel ion current trace of OmpF D113N without antibiotic (Blank) at a. -100mV, d. 100mV and with 100 µm Norfloxacin on cis side b. at -100mV, e. at 100mV and trans side addition c., f. at ± 100 mv ; Voltage dependence of g. Association rate on cis and trans side h. Dissociation rate at cis and trans for 200mM KCl, 5mM PO 4 ph 7.

12 SI- Figure 10: Voltage dependence of Association rate and dissociation rate on cis and trans side for a, b OmpF D121A and c, d OmpF E117Q; comparison among WT, D121A and E117Q of e. association rate on cis side f. dissociation rate from cis side. 200mM KCl, 5mM PO4 ph 7.

13 SI- Figure 11: Single channel ion current trace of OmpF WT without antibiotic (Blank) at a. -100mV, c. 100mV and with 100 µm Ciprofloxacin on cis side b. at -100mV, Ciprofloxacin on trans side addition d. at 100 mv; Voltage dependence of a. Association rate on cis and trans side b. Dissociation rate at cis and trans for 200mM KCl, 5mM PO 4 ph 7 Other antibiotic like ciprofloxacin which has majority zwitterionic in nature at ph 7 were also measured with OmpF WT to generalize the model we have hypothesized. a-d shows the ion current trace, where in this case also the binding for ciprofloxacin is asymmetric from cis and trans side. Strong binding with high residence time (lower dissociation rate is observed from extracellular side) and low binding from the periplasmic side. The high affinity binding site from extracellular side mostly corresponds to the dipole alignment with the transverse electric field in the region with R167, R168 and D121 in OmpF WT channel as seen for norfloxacin. The association and dissociation rates are both exponentially dependent as seen for norfloxacin in the main text (SI- Figure 11e and f). Two kinds of events with residence time τ 1 and τ 2 are observed

14 for cis side addition of ciprofloxacin, although this can be observed already at or above -100mV (SI- Figure 11f). Our model for norfloxacin in this case should also be valid for ciprofloxacin. SI- Figure 12: (from left to right) Free energy for Norfloxacin interactions through OmpF in absence of salt and a. 0mV, b. -100mV and c. -200mV transmembrane voltage. To note how the double path for translocation at 0mV (50 and 130 degrees) disappears progressively with external voltage applied.

15 SI- Figure 13: Transversal (purple) and longitudinal (cyan) components (respect to the axis of diffusion of the channel) of the intrinsic electric field of OmpF WT, OmpC WT, OmpF R167E/R168E double mutant, OmpF D113N mutant. The pre-orientation region (PR) and the constriction region (CR) have been highlighted respectively in orange and red.

16 SI-Figure 14. (a) MD-ES1 stability, the Z-coordinate of the center of mass (COM) of Norfloxacin with respect to the center of mass of the channel, is depicted for 6 different replicas of 100ns each at 1M KCl with an applied voltage of 100mV. (b) Accumulated charge over voltage (100mV) versus time at 1M KCl. In red for the empty OmpF trimer, from green to black for the six replicas of the MD-ES1 conformation. SI- Table 1: OmpF-WT trimer conductance in 1M KCl solution at applied voltage of 100mV, with and without norfloxacin, calculated from MD simulations. System Simulation length(ns) Conductance (ns) OmpF-empty-100mV MD-ES1-100mV 6x ± 0.2

17 SI- Table 2: Ion selectivity of OmpF WT, mutant OmpF and OmpC WT channel using KCl concentration gradients on both sides of the DPhPC membranes. Porin 1M(cis)/0.1M (trans) Transmembrane potential pk + /pcl - ph (mv) OmpF WT OmpF-EE mutant OmpC WT OmpF WT

18 In our previous study, we had reported the flux of norfloxacin through OmpF channel at zero potential in liposome based assay and (close to zero) in electrophysiology measurement 4 where the flux in both techniques correlated. However, in this study we report that most of the events are not translocation. Another possibility is that high affinity events (from ES1 site) from trans side are not observed at the concentration range used (up to 1mM due to solubility), in case where a much higher concentrations (around 10mM) used likely some events might correspond to translocation. MD methods Starting from the final configuration of the two OmpF simulations described above, the drug was placed inside the lumen of the first monomer. The difference between the z-coordinate of the center of mass (com) of the antibiotic two-ring system and the z-coordinate of the c.o.m of the protein monomer for each simulation was used to follow the diffusion of antibiotic inside the pore. A thousand steps of energy minimization were performed. The equilibration stage followed for 1 ns in the NVT ensemble at 300 K as described hereinbefore. For all the cases presented, well-tempered metadynamics 8,9 simulation were performed with the ACEMD code, 10 until the first effective translocation through the protein constriction region (CR) was observed. Then, four configurations were randomly selected, two with the antibiotic located in the extracellular vestibule (EV), two in the periplasmic vestibule (PV). Correspondingly, four multiple-walkers 11 were set to extend the metadynamics reconstruction of the free-energy surface (FES). Two biased collective variables were used, namely, the antibiotic position and the projection of the dipole moment of the molecule onto the axis of diffusion. The z was defined as the difference of the z-coordinate between the com of the antibiotic two-ring system and that of the porin first monomer. Norfloxacin was placed at z=19 Å for the three cases and the first effective translocation took place in 400ns for OmpF-WT without applied voltage and 170ns when - 500mV were applied. The initial parameters chosen for well-tempered metadynamics were: initial Gaussian height of 1kcal/mol, bias factor of 15.67, and σ CV1 = 0.3 Å and σ CV2 = 0.4 Degrees. The initial sigma chosen for the cv2 was too small. In order to speed up calculations and arrive to convergence in a later stage, well-tempered metadynamics the already deposited bias was given to a new well-tempered metadynamics simulations as static energy bias 12, and σ CV2 was updated to σ CV2 =5.0 Degrees. In this way we avoid re-visiting the states that were

19 sampled in the previous run, and we avoid starting from scratch. References 1. Raj Singh, P.; Ceccarelli, M.; Lovelle, M.; Winterhalter, M.; Mahendran, K. R. Antibiotic Permeation across the OmpF Channel: Modulation of the Affinity Site in the Presence of Magnesium. J. Phys. Chem. B 2012, 116, Hilty, C; Winterhalter, M. Facilitated Substrate Transport through Membrane Proteins. Phys. Rev. Lett. 2001, 86, Bodrenko, I.; Bajaj, H.; Ruggerone, P.; Winterhalter, M; Ceccarelli, M. Analysis of Fast Channel Blockage: Revealing Substrate Binding in the Microsecond Range. Analyst 2015, 140, Cama, J.; Bajaj, H.; Pagliara, S.; Maier, T.; Braun, Y.; Winterhalter, M.; Keyser, U. Quantification of Fluoroquinolone Uptake through the Outer Membrane Channel OmpF of Escherichia coli. J. Am. Chem. Soc. 2015, 137, Hänggi, P.; Borkovec, M. Reaction-Rate Theory: Fifty Years after Kramers. Rev. Mod. Phys. 1990, 62, Schwarz, G.; Danelon, C.; Winterhalter, M. On Translocation through a Membrane Channel via an Internal Binding Site: Kinetics and Voltage Dependence. Biophys. J. 2003, 84, Truhlar, D. G.; Garrett, B. C.; Klippenstein, S. J. Current Status of Transition-State Theory. J. Phys. Chem. 1996, 100, Barducci, A.; Bussi, G.; Parrinello, M. Well-Tempered Metadynamics: a Smoothly Converging and Tunable Free-Energy Method. Phys. Rev. Lett. 2008, 100, Laio, A.; Parrinello, M. Escaping Free-Energy Minima. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, Harvey, M. J.; Giupponi, G.; De Fabritiis, G. ACEMD: Accelerating Biomolecular Dynamics in the Microsecond Time Scale. J. Chem. Theory Comput. 2009, 5, Raiteri, P.; Laio, A.; Gervasio, F. L.; Micheletti, C.; Parrinello, M. Efficient Reconstruction of Complex Free Energy Landscapes by Multiple Walkers Metadynamics. J. Phys. Chem. B 2006, 110, Barducci, A., Bonomi, M., Prakash, M. K. & Parrinello, M. Free-Energy Landscape of Protein Oligomerization from Atomistic Simulations. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, E4708-E4713.