Article
Conversion of OprO into an OprP-like Channel by Exchanging Key Residues in the Channel Constriction Sonalli Ganguly,1,* Anusha Kesireddy,2 Iva´n Ba´rcena-Uribarri,1 Ulrich Kleinekatho¨fer,2 and Roland Benz1 1
Department of Life Sciences and Chemistry and 2Department of Physics and Earth Sciences, Jacobs University Bremen, Bremen, Germany
ABSTRACT Under phosphate-limiting conditions, the channels OprP and OprO are induced and expressed in the outer membrane of Pseudomonas aeruginosa. Despite their large homology, the phosphate-specific OprP and the diphosphate-specific OprO pores show structural differences in their binding sites situated in the constriction region. Previously, it was shown that the mutation of amino acids in OprP (Y62F and Y114D) led to an exchange in substrate specificity similar to OprO. To support the role of these key amino acids in the substrate sorting of these specific channels, the reverse mutants for OprO (F62Y, D114Y, and F62Y/D114Y) were created in this study. The phosphate and diphosphate binding of the generated channels was studied in planar lipid bilayers. Our results show that mutations of key residues indeed reverse the substrate specificity of OprO to OprP and support the view that just a few strategically positioned amino acids are mainly responsible for its substrate specificity.
INTRODUCTION Gram-negative bacterial outer membrane (OM) channels are major access points for most antibiotics currently present in the market. These channels are strategically positioned in the outer membrane of Gram-negative bacteria to allow the exchange of substrates for its survival. The uptake is generally by passive diffusion driven by the respective concentration gradients across the membrane. General diffusion porins such as OmpF and OmpC nonspecifically allow the passage of substrates based on the size extrusion principle, whereas substrate-specific membrane channels allow particular types of substrates to pass through, as their name implies (1,2). Examples of such channels include the nucleoside-specific Tsx (3), the sugar-specific LamB and ScrY (4,5), and the phosphate-specific OprO and OprP channels (6). This specificity is guided by the presence of favorable substrate affinity sites inside the channels and the specificity is especially effective when the external substrate concentrations are minimal. This kind of nutrient-deficient environment is usually present in the case of pathogenic Pseudomonas bacteria, which make them rely
Submitted August 12, 2016, and accepted for publication July 10, 2017. *Correspondence: [email protected] Sonalli Ganguly and Anusha Kesireddy contributed equally to this work. Editor: Emad Tajkhorshid. http://dx.doi.org/10.1016/j.bpj.2017.07.004
exclusively on substrate-specific channels. This restricted uptake mechanism renders Pseudomonas aeruginosa resistant to many antibiotics (7). Exploiting the idea of specificity by gaining deeper understanding of the OM channel transport properties is an active area of research that provides useful insights to combat antibiotic resistance (8,9). OprP and OprO are phosphate-specific channels expressed in P. aeruginosa under phosphate starvation conditions (10,11). The crystal structures of both channels show trimeric arrangements, where each monomer consists of 16 antiparallel b-strands connected by long extracellular loops and short periplasmic turns. A closer look into the crystal structures of these channels show how phosphate molecules can pass from the extracellular to periplasmic side of the cell gliding along the electropositive arginine and lysine ladder of the channel (12). Despite having a high homology at the amino acid level (74%), both channels differ in their conductance and substrate-binding properties (13,14). The pore-forming properties of OprP and OprO have been characterized previously using planar lipid bilayers. OprP has a single channel conductance of 260 pS in 1 M KCl, whereas the conductance of OprO is 440 pS, caused by a wider constriction zone. With respect to substrate specificity, it has been shown that OprP features a higher affinity for monophosphate whereas OprO is specialized in uptake of polyphosphate molecules such as
Ó 2017 Biophysical Society.
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diphosphate and triphosphate. Only a very few amino acids situated in the constriction zone seem to play a major role in the different single channel conductance and specificities for monophosphate and diphosphate. The previously reported exchange of the amino acids Y62F and Y114D in OprP led to a conversion of its pore properties to those of OprO. The OprP double mutant (Y62F/Y114D) resulted in an increase of single channel conductance and its binding affinity for diphosphate reaching values similar to those found for OprO (13). Therefore, we envisioned that the mutation F62Y/D114Y in OprO alters its specificity favoring the transport of monophosphate molecules over the diphosphate molecules like in OprP. In this study, we explored the role of the F62 and D114 residues in the substrate specificity of OprO (Fig. 1). To this end, we performed single channel conductance and binding affinity measurements of monophosphate and diphosphate substrates using electrophysiology. A similar approach has been used in the previous studies of bacterial OM channels to shed light on the molecular details of substrate specificity (13,15,16). Moreover, molecular dynamics (MD) simulations presented here offer atomistic insights into the KCl conductance properties of OprO and its mutant channels. The results show that two residues, F62 and D114, in the constriction region of the OprO channel determine the substrate specificity of the channel. The study further confirms that the two residues in the constriction region play a major role in OprO and OprP transport properties across the OM of P. aeruginosa. MATERIALS AND METHODS Bacterial strains and growth conditions Bacterial Escherichia coli cells CE1248 (11) and XL-1 blue containing OprO and OprO mutant genes incorporated in pTZ19R plasmid were grown in 1000 mL baffled Erlenmeyer flasks containing 250 mL of LB medium and DYT medium at 37 C with shaking at 220 rpm. Agar plates and liquid media were supplemented with 100 mg/mL ampicillin antibiotic (Carl Roth, Karlsruhe, Germany). The pTZ19R vector (Thermo Fisher Scientific, Schwerte, Germany) was used for cloning and overexpression of the
oprO gene. It is a phagemid vector, 2862 basepairs in length, derived from the pUC19 vector by inserting the DNA of the phage intergenic region (IG) as well as the T7 promoter sequence near the multiple cloning sites of pUC19. The pTZ19R plasmid contains the pMB1 replicon rep (responsible for the replication of phagemid), a bla gene (resistance to ampicillin), the f1 intergenic region (for initiation and termination of phage f1 DNA synthesis), a T7 promoter, a lac containing CAP protein binding site, the promoter Plac, a lac repressor binding site, and the 50 -terminal part of the lacZ gene encoding the N-terminal fragment of b-galactosidase.
Site-directed mutagenesis in oprO gene using mismatch nucleotides The wild-type oprO gene in the plasmid pTZ19R was subjected to mutation using the QuikChange Lightning site-directed mutagenesis kit (Thermo Fisher Scientific) where a phenylalanine present in the oprO gene is replaced by a tyrosine at amino acid position 62, and an aspartic acid at position 114 is mutated into a tyrosine as well. The primers as given in Table S1 were constructed using the company guidelines. These plasmids were checked for correctness using sequencing by GATC Biotech (Ko¨ln, Germany).
Expression and purification of OprO and OprO mutant recombinant proteins Briefly, the pTZ19R OprO plasmid and the pTZ19R OprO D114Y E. coli CE1248 were transformed into XL-1 blue E. coli cells, respectively. After successful transformation, the positive colonies were chosen and grown overnight at 37 C in LB and DYT media. After overnight incubation, the cells were pelleted and resuspended in a buffer containing 40% glucose containing 50 mg/mL DNase. The mixture was subjected to sonication and ultracentrifugation. The resultant pellet containing outer membrane components was further extracted using increasing concentrations of octyl-polyoxyethylene. The final fraction containing purified OprO protein was loaded into SDS-polyacrylamide gels and electrophoreses. These proteins were then run through a Mono-Q column (GE Healthcare Life Sciences, Marlborough, MA) and various fractions of the column were collected and analyzed via SDS-gels.
Electrophysiology The black lipid membrane assay has been performed as explained elsewhere (17). In brief, a membrane was formed from a 1% (w/v) solution of diphytanoylphoshatidylcholine (DiPhPC; Avanti Polar Lipids, Alabaster, AL) in n-decane (Fluka, Steinheim, Germany) over a 0.4 mm2 aperture,
FIGURE 1 Top-view cartoon of (a) OprO WT and (b) OprO DM monomers. The key residues F62 and D114 together with D94 are shown in blue. Colored in red are oxygen molecules. The asterisk symbols represent the respective conducting pathway. To see this figure in color, go online.
830 Biophysical Journal 113, 829–834, August 22, 2017
Conversion of OprO to OprP between two aqueous compartments in a Teflon (https://www.chemours. com/) cell. Two Ag/AgCl electrodes with salt bridges were inserted on both sides of the Teflon cell. One of the electrodes was connected via a voltage source to ground and the other electrode was connected to a current amplifier (Keithley 427; Tektronix, Beaverton, OR) to measure the current passing through membrane and electrodes. Titration experiments were carried out to measure the inhibition of chloride conductance by phosphate binding to the binding sites, as previously described in detail with OprP wild-type and its mutants (13). More details are given in the Supporting Material.
ions (24,25). The long-range electrostatic interactions were calculated us˚ . Moreover, the van ing the PME method with a short-range cutoff of 12 A ˚ whereas a der Waals interactions were considered up to a distance of 10 A switch function was used to turn off the interactions smoothly to reach ˚ . The final unbiased simulations were performed in a NPT zero at 12 A ensemble, achieved by semi-isotropic coupling to a Parrinello and Rahman (26) barostat at 1 bar with a coupling constant of 5 ps, whereas the appliedfield simulations were performed in a NVT ensemble. The temperature was set to 303.15 K using a Nose-Hoover thermostat (27,28) with a coupling constant of 1 ps. Moreover, the simulations were carried out with 2-fs time step by applying constraints on hydrogen atom bonds using the LINCS algorithm (29).
MD simulations The starting coordinates of the OprO wild-type (WT) and OprO double mutant (DM) (F62Y, D114Y) channels were obtained from the PDB database (PDB: 4RJW and 4RJX, respectively (13)). In addition, two variants of OprO were prepared by either replacing F62 by a tyrosine or D114 by a tyrosine. All four proteins were aligned to the membrane normal, also termed the z axis, with the extracellular loops toward the negative z values. Subsequently, the protein structures in their trimeric form were inserted into a 1-palmitoyl-2-oleoyl-sn-glycero-3 phosphoethanolamine lipid bilayer ˚ of modified TIP3P water on both sides and 0.15 M KCl solution with 40 A of the membrane. Likewise, systems of OprO WT and OprO DM in 1 M KCl salt concentrations were prepared. All six systems were equilibrated in six steps by reducing the positional restraints on the heavy atoms of proteins and lipids for a total of 20 ns. The systems were built and equilibrated using the protocol proposed in the CHARMM GUI Membrane Builder (18,19). Four equilibrated systems, i.e., OprO WT, OprO-F62Y, OprOD114Y, and OprO DM in the 0.15 M KCl salt solution were simulated in NPT ensemble for 100 ns each without any applied biasing force. Furthermore, we performed applied-field simulations (3 100 ns) for the OprO WT and OprO DM in 1 M KCl salt solutions by applying a constant electric field E along the z direction of the channel. The applied field strength E is proportional to the voltage V, E ¼ V/Lz, where Lz represents the length of the system in the z direction (20–22). The applied-field simulations were performed in a NVT ensemble with the electric field pointing from the extracellular to the periplasmic space corresponding to transmembrane potential of 500 mV. The flux of K and Cl ions passing through the channels in the presence of applied field can be estimated using (20)
IðtÞ ¼
N X 1 qi z i ; Lz Dt i ¼ 1
where qi denotes the charge of the atom i and Dzi is the displacement of atom i during the time step Dt. The MD simulations were performed with the software GROMACS 4.6.5 (23) using the CHARMM36 force field for proteins, lipids, and
RESULTS AND DISCUSSION Our initial goal was to investigate if the channel properties of OprO can be converted to those of OprP by modifying the key residues in the constriction region. Two single mutants, F62Y, and D114Y, and a DM F62Y/D114Y of OprO were generated and reconstituted into the lipid bilayer. The single channel conductance of these channels was measured at þ50 mV applied voltage at room temperature (RT) in 0.1 M and 1 M KCl solutions supplemented with 10 mM (2-(N-morpholino) ethanesulfonic acid) (MES) and adjusted to pH 6. Fig. 2 shows typical single channel recordings in a step-like manner caused by insertion of OprO WT, OprO mutants, and OprP WT into lipid bilayers. DM shows an increase in single channel conductance As expected, based on previous studies, the OprO protein exhibits higher conductance than the OprP (see Table 1). At the level of single amino acid mutations, the exchange of phenylalanine by tyrosine in OprO, F62Y, produced a slight drop in the conductance of the channel. On the other hand, replacement of the aspartic acid with more bulky tyrosine in OprO, D114Y, did not show any visible effect on the KCl conductance. The introduction of both mutations simultaneously, i.e., OprO F62Y/D114Y, surprisingly exhibits a conductance higher than that of OprO WT. Thus, the OprO DM mutant channel did not yield a conductance value
FIGURE 2 Pore-forming events of OprO WT, OprO F62Y, OprO D114Y, OprO DM, and OprP WT. Single-channel conductance as observed for OprO WT, OprO mutants, and OprP WT in a DiPhPC membrane bathed in 0.1 M KCl, 10 mM MES-KOH, pH 6, at þ50 mV.
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Ganguly et al. TABLE 1 The Experiments were Carried Out with DiPhPC Membranes and an Applied Voltage of D50 mV at RT
ation of chloride ions compared to the native channel, and this leads to higher conductance in OprO DM than OprO WT.
Average Single Channel Conductance Experiments
MD Simulations
Proteins
0.1 M KCl
1 M KCl
1 M KCl
OprO WT OprO (F62Y) OprO (D114Y) OprO DM OprP WT
240 5 15 200 5 37 240 5 28 400 5 43 160 5 9
440 5 33 400 5 70 440 5 67 600 5 76 260 5 18
532 5 43 — — 625 5 89 —
At least 100 single events were used to calculate the average conductance while simulations were only performed for two of the channels.
similar to that of OprP. On the contrary, in the previous study we have shown that the OprP DM (Y62F/Y114D) channel yielded conductance values similar to those of OprO WT. To understand the rise in conductance of the OprO DM channel, we performed all-atom MD simulations and calculated the conductance values of the OprO WT and OprO DM channels in 1 M KCl solution in the presence of a 500-mV applied field pointing from the extracellular to the periplasmic side. Table 1 summarizes the average conductance values from three independent simulations of 100-ns length each. Consistent with the experiments, the OprO DM has shown a higher conductance compared to the WT. The increased single channel conductance of the OprO DM might be due to the change in the dimensions of the channel and the electrostatics inside the channel. The radius profiles clearly indicate that OprO DM forms a wider channel than the OprO WT with a twofold increase of the cross-section area in the eyelet region (Fig. 3). Moreover, the electrostatic network inside the pore is altered by the replacement of phenylalanine by the less hydrophobic tyrosine and the negatively charged aspartic acid by the neutral tyrosine. These modifications can result in a smaller barrier for the perme-
FIGURE 3 Plot showing the average radius of the OprO WT as well as OprO single and double mutant channels along with the corresponding SD derived from unbiased MD simulations. The pore radii have been determined from 100-ns-long equilibrated trajectories of the respective channels using the HOLE program (30). ‘‘EC’’ and ‘‘PP’’ denote the extracellular and the periplasmic sides of the channel, respectively. To see this figure in color, go online.
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DM channel of OprO has OprP-like substrate specificity Previous titration experiments have shown that the OprO and OprP pores have specific binding affinities for diphosphate and monophosphate, respectively. Mutations of two important amino acid residues present in the vicinity of the affinity site in OprP reversed its specificity from monophosphate to diphosphate (13). To understand the role of these two residues i.e., F62 and D114, in substrate binding of OprO and to see if mutations can alter the specificity of OprO, the inhibition of channel conductance by adding mono- and diphosphate solution on both sides of membrane was measured as a function of concentration. The experiments were carried out with DiPhPC membranes and an applied voltage of þ50 mV at RT. At least 100 single events were used to calculate the average conductance values. The half-saturation constants for inhibition of channel conductance by mono- and diphosphate were obtained from titration experiments using Lineweaver-Burk plots are shown in Fig. 4. Table 2 summarizes the results of the titration experiments of OprO WT and mutant proteins with monophosphate and diphosphate. The single and double mutations reduce the binding affinity of the OprO channel for diphosphate and increases the affinity toward monophosphate ions. A considerable difference can be observed in the stability constants of mono- and diphosphate binding to OprO WT and to OprO DM. In case of the OprO DM, the current through the membrane decreases almost to 0 at monophosphate concentration of 6.36 mM, which indicates that for the OprO DM channels, the KCl conductance is blocked due to the binding of monophosphate inside the channel lumen. The stability constant for monophosphate binding to OprO DM (K ¼ 720 1/M) is increased by a factor of 3 compared to that of OprO WT (220 1/M). It is noteworthy that the replacement of F62/D114 from OprO with bulkier tyrosine amino acids led to a substantial increase of the binding affinity of monophosphate ions with similar binding kinetics as that of OprP. Table 2 shows the data from similar experiment where a membrane containing the OprO DM was titrated with diphosphate. The OprO DM conductance was blocked at a higher diphosphate concentration of 48.70 mM. This finding is demonstrated in Fig. 3 (red straight line), which shows a Lineweaver-Burk plot of the data from the titration experiment for the OprO DM. The straight line in the LineweaverBurk plot corresponds to a stability constant of 343 1/M, which indicates that the ability of diphosphate to block the ion conductance in OprO DM is decreased by approximately a factor of 4 as compared to OprO WT. The binding affinity of the OprO channel for diphosphate ions was reduced when key amino acid residues (F62 and D114) are mutated with tyrosine. The results suggest that the replacement of
Conversion of OprO to OprP
FIGURE 4 Lineweaver-Burk plots for OprO WT and its mutants for phosphate- or diphosphate-mediated inhibition of membrane conductance. The straight lines correspond to stability constants K given by the Eq. 1 (fraction of closed channels) ¼ K∙c/(K∙c þ 1). (a) Lineweaver-Burk plots for diphosphate: OprO WT shows the highest stability constant for diphosphate K ¼1450 1/M followed by an intermediate stability constant for OprO D114Y of K ¼ 630 1/M and for OprO F62Y of stability constant K ¼ 490 1/M; OprP WT has the smallest stability constant K ¼ 310 1/M for diphosphate and OprO DM shows a similar stability constant as that of OprP WT with K ¼ 343 1/M. (b) Lineweaver-Burk plots for phosphate: OprP WT shows the highest stability constant K ¼ 770 1/M followed by OprO DM with a stability constant of K ¼ 720 1/M. OprO WT shows the smallest stability constant K ¼ 220 1/M and intermediate stability constants were found for D114Y with a stability constant K ¼ 500 1/M and for OprO F62Y with K ¼ 690 1/M. To see this figure in color, go online.
OprO F62/D114 by tyrosine residues reverses the binding affinity from diphosphate to monophosphate. CONCLUSION The OMs of Gram-negative bacteria contain several specific channels that are involved in the uptake of specific substrates across the bacterial cell. It has been demonstrated previously that a few amino acids in the affinity site of these TABLE 2 Monophosphate- and Diphosphate-Mediated Inhibition of Chloride Conductance of OprO WT, OprO Mutants, and OprP WT in 0.1 M KCl, 10 mM MES, pH 6, at an Applied Voltage of D50 mV at RT Diphosphate Proteins OprO WT OprO (F62Y) OprO(D114Y) OprO DM OprP WT
Phosphate
1/K (mM)
K (1/M)
1/K (mM)
K (1/M)
0.6 2 1.5 2.9 3.1
1450 5 120 490 5 110 630 5 90 343 5 20 310 5 35
4.5 1.4 2 1.4 1.3
220 5 50 690 5 140 500 5 70 720 5 90 770 5 150
channels play a major role in sorting substrate. Electrophysiology experiments revealed that OprP and OprO have specific affinities toward monophosphate and diphosphate, respectively. In a previous study, we reported that the Y62F/Y114D mutations successfully swapped the OprP pore properties to OprO-like (13). In this study, we probed the role of two key residues, F62, and D114Y, in the ion and substrate transport properties of the OprO channel. The F62Y OprO channel displayed a slight decrease in conductance, whereas the D114 OprO mutation does not show any change in conductance. Surprisingly, the OprO DM (F62Y/D114Y) displayed a higher conductance than the expected OprP-like behavior. Molecular level analysis using MD simulations helped us to investigate the reason for this rise in conductance. The OprO DM has a wider lumen than that of the OprO and OprP WT channels and this increase in the pore dimension along with the altered electrostatics made the passage of chloride ions easier, leading to an increased conductance in OprO DM. Binding affinity studies of OprO and its mutants revealed the importance of two key amino acid residues, F62 and D114 (present in the constriction region) for their substrate specificity, and any change of these amino acids can influence the functionality of this pore. The most substantial reversal of substrate specificity was observed when both residues, F62 and D114, were replaced by tyrosine residues, followed by F62Y and D114Y single mutant OprO channels. Our results suggest that the engineered OprO DM channels have an OprP-like substrate specificity. The two key residues seem to render the unlike phosphate specificities of the OprO and OprP channels. The results presented here and in previous studies (12,13,16) of OprO and OprP channels indicate that only a few residues in the constriction region of OM channels play a key role in sorting the substrates. Future investigations of transport of phosphonic antibiotics, e.g., fosfomycin and fosmidomycin, across these channels will improve our understanding of antibiotic influx across the OM of P. aeruginosa. SUPPORTING MATERIAL Supporting Materials and Methods and one table are available at http:// www.biophysj.org/biophysj/supplemental/S0006-3495(17)30755-5.
AUTHOR CONTRIBUTIONS R.B., S.G., and U.K. designed research. S.G., I.B.-U., A.K., U.K., and R.B. performed and analyzed electrophysiology and MD simulations, analyzed data, and wrote the article.
ACKNOWLEDGMENTS We thank Prof. R.E.W. Hancock, University of British Columbia, Vancouver, Canada, for providing basic materials for our experiments. We would like to thank Niraj Modi for his help with the initial simulations.
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Ganguly et al. This research has received support from the Innovative Medicines Initiatives Joint Undertaking under grant agreement No. 115525, resources that are composed of financial contributions from the European Union’s Seventh Framework Program (FP7/2007-2013) and EFPIA. In addition, this work has been supported by the grants BE 865/16-2 and KL 1299/9-2 of the Deutsche Forschungsgemeinschaft (DFG).
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Conversion of OprO into an OprP-like Channel by Exchanging Key Residues in the Channel Constriction Sonalli Ganguly,1,* Anusha Kesireddy,2 Iva´n Ba´rcena-Uribarri,1 Ulrich Kleinekatho¨fer,2 and Roland Benz1 1
Department of Life Sciences and Chemistry and 2Department of Physics and Earth Sciences, Jacobs University Bremen, Bremen, Germany
ABSTRACT Under phosphate-limiting conditions, the channels OprP and OprO are induced and expressed in the outer membrane of Pseudomonas aeruginosa. Despite their large homology, the phosphate-specific OprP and the diphosphate-specific OprO pores show structural differences in their binding sites situated in the constriction region. Previously, it was shown that the mutation of amino acids in OprP (Y62F and Y114D) led to an exchange in substrate specificity similar to OprO. To support the role of these key amino acids in the substrate sorting of these specific channels, the reverse mutants for OprO (F62Y, D114Y, and F62Y/D114Y) were created in this study. The phosphate and diphosphate binding of the generated channels was studied in planar lipid bilayers. Our results show that mutations of key residues indeed reverse the substrate specificity of OprO to OprP and support the view that just a few strategically positioned amino acids are mainly responsible for its substrate specificity.
INTRODUCTION Gram-negative bacterial outer membrane (OM) channels are major access points for most antibiotics currently present in the market. These channels are strategically positioned in the outer membrane of Gram-negative bacteria to allow the exchange of substrates for its survival. The uptake is generally by passive diffusion driven by the respective concentration gradients across the membrane. General diffusion porins such as OmpF and OmpC nonspecifically allow the passage of substrates based on the size extrusion principle, whereas substrate-specific membrane channels allow particular types of substrates to pass through, as their name implies (1,2). Examples of such channels include the nucleoside-specific Tsx (3), the sugar-specific LamB and ScrY (4,5), and the phosphate-specific OprO and OprP channels (6). This specificity is guided by the presence of favorable substrate affinity sites inside the channels and the specificity is especially effective when the external substrate concentrations are minimal. This kind of nutrient-deficient environment is usually present in the case of pathogenic Pseudomonas bacteria, which make them rely
Submitted August 12, 2016, and accepted for publication July 10, 2017. *Correspondence: [email protected] Sonalli Ganguly and Anusha Kesireddy contributed equally to this work. Editor: Emad Tajkhorshid. http://dx.doi.org/10.1016/j.bpj.2017.07.004
exclusively on substrate-specific channels. This restricted uptake mechanism renders Pseudomonas aeruginosa resistant to many antibiotics (7). Exploiting the idea of specificity by gaining deeper understanding of the OM channel transport properties is an active area of research that provides useful insights to combat antibiotic resistance (8,9). OprP and OprO are phosphate-specific channels expressed in P. aeruginosa under phosphate starvation conditions (10,11). The crystal structures of both channels show trimeric arrangements, where each monomer consists of 16 antiparallel b-strands connected by long extracellular loops and short periplasmic turns. A closer look into the crystal structures of these channels show how phosphate molecules can pass from the extracellular to periplasmic side of the cell gliding along the electropositive arginine and lysine ladder of the channel (12). Despite having a high homology at the amino acid level (74%), both channels differ in their conductance and substrate-binding properties (13,14). The pore-forming properties of OprP and OprO have been characterized previously using planar lipid bilayers. OprP has a single channel conductance of 260 pS in 1 M KCl, whereas the conductance of OprO is 440 pS, caused by a wider constriction zone. With respect to substrate specificity, it has been shown that OprP features a higher affinity for monophosphate whereas OprO is specialized in uptake of polyphosphate molecules such as
Ó 2017 Biophysical Society.
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diphosphate and triphosphate. Only a very few amino acids situated in the constriction zone seem to play a major role in the different single channel conductance and specificities for monophosphate and diphosphate. The previously reported exchange of the amino acids Y62F and Y114D in OprP led to a conversion of its pore properties to those of OprO. The OprP double mutant (Y62F/Y114D) resulted in an increase of single channel conductance and its binding affinity for diphosphate reaching values similar to those found for OprO (13). Therefore, we envisioned that the mutation F62Y/D114Y in OprO alters its specificity favoring the transport of monophosphate molecules over the diphosphate molecules like in OprP. In this study, we explored the role of the F62 and D114 residues in the substrate specificity of OprO (Fig. 1). To this end, we performed single channel conductance and binding affinity measurements of monophosphate and diphosphate substrates using electrophysiology. A similar approach has been used in the previous studies of bacterial OM channels to shed light on the molecular details of substrate specificity (13,15,16). Moreover, molecular dynamics (MD) simulations presented here offer atomistic insights into the KCl conductance properties of OprO and its mutant channels. The results show that two residues, F62 and D114, in the constriction region of the OprO channel determine the substrate specificity of the channel. The study further confirms that the two residues in the constriction region play a major role in OprO and OprP transport properties across the OM of P. aeruginosa. MATERIALS AND METHODS Bacterial strains and growth conditions Bacterial Escherichia coli cells CE1248 (11) and XL-1 blue containing OprO and OprO mutant genes incorporated in pTZ19R plasmid were grown in 1000 mL baffled Erlenmeyer flasks containing 250 mL of LB medium and DYT medium at 37 C with shaking at 220 rpm. Agar plates and liquid media were supplemented with 100 mg/mL ampicillin antibiotic (Carl Roth, Karlsruhe, Germany). The pTZ19R vector (Thermo Fisher Scientific, Schwerte, Germany) was used for cloning and overexpression of the
oprO gene. It is a phagemid vector, 2862 basepairs in length, derived from the pUC19 vector by inserting the DNA of the phage intergenic region (IG) as well as the T7 promoter sequence near the multiple cloning sites of pUC19. The pTZ19R plasmid contains the pMB1 replicon rep (responsible for the replication of phagemid), a bla gene (resistance to ampicillin), the f1 intergenic region (for initiation and termination of phage f1 DNA synthesis), a T7 promoter, a lac containing CAP protein binding site, the promoter Plac, a lac repressor binding site, and the 50 -terminal part of the lacZ gene encoding the N-terminal fragment of b-galactosidase.
Site-directed mutagenesis in oprO gene using mismatch nucleotides The wild-type oprO gene in the plasmid pTZ19R was subjected to mutation using the QuikChange Lightning site-directed mutagenesis kit (Thermo Fisher Scientific) where a phenylalanine present in the oprO gene is replaced by a tyrosine at amino acid position 62, and an aspartic acid at position 114 is mutated into a tyrosine as well. The primers as given in Table S1 were constructed using the company guidelines. These plasmids were checked for correctness using sequencing by GATC Biotech (Ko¨ln, Germany).
Expression and purification of OprO and OprO mutant recombinant proteins Briefly, the pTZ19R OprO plasmid and the pTZ19R OprO D114Y E. coli CE1248 were transformed into XL-1 blue E. coli cells, respectively. After successful transformation, the positive colonies were chosen and grown overnight at 37 C in LB and DYT media. After overnight incubation, the cells were pelleted and resuspended in a buffer containing 40% glucose containing 50 mg/mL DNase. The mixture was subjected to sonication and ultracentrifugation. The resultant pellet containing outer membrane components was further extracted using increasing concentrations of octyl-polyoxyethylene. The final fraction containing purified OprO protein was loaded into SDS-polyacrylamide gels and electrophoreses. These proteins were then run through a Mono-Q column (GE Healthcare Life Sciences, Marlborough, MA) and various fractions of the column were collected and analyzed via SDS-gels.
Electrophysiology The black lipid membrane assay has been performed as explained elsewhere (17). In brief, a membrane was formed from a 1% (w/v) solution of diphytanoylphoshatidylcholine (DiPhPC; Avanti Polar Lipids, Alabaster, AL) in n-decane (Fluka, Steinheim, Germany) over a 0.4 mm2 aperture,
FIGURE 1 Top-view cartoon of (a) OprO WT and (b) OprO DM monomers. The key residues F62 and D114 together with D94 are shown in blue. Colored in red are oxygen molecules. The asterisk symbols represent the respective conducting pathway. To see this figure in color, go online.
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Conversion of OprO to OprP between two aqueous compartments in a Teflon (https://www.chemours. com/) cell. Two Ag/AgCl electrodes with salt bridges were inserted on both sides of the Teflon cell. One of the electrodes was connected via a voltage source to ground and the other electrode was connected to a current amplifier (Keithley 427; Tektronix, Beaverton, OR) to measure the current passing through membrane and electrodes. Titration experiments were carried out to measure the inhibition of chloride conductance by phosphate binding to the binding sites, as previously described in detail with OprP wild-type and its mutants (13). More details are given in the Supporting Material.
ions (24,25). The long-range electrostatic interactions were calculated us˚ . Moreover, the van ing the PME method with a short-range cutoff of 12 A ˚ whereas a der Waals interactions were considered up to a distance of 10 A switch function was used to turn off the interactions smoothly to reach ˚ . The final unbiased simulations were performed in a NPT zero at 12 A ensemble, achieved by semi-isotropic coupling to a Parrinello and Rahman (26) barostat at 1 bar with a coupling constant of 5 ps, whereas the appliedfield simulations were performed in a NVT ensemble. The temperature was set to 303.15 K using a Nose-Hoover thermostat (27,28) with a coupling constant of 1 ps. Moreover, the simulations were carried out with 2-fs time step by applying constraints on hydrogen atom bonds using the LINCS algorithm (29).
MD simulations The starting coordinates of the OprO wild-type (WT) and OprO double mutant (DM) (F62Y, D114Y) channels were obtained from the PDB database (PDB: 4RJW and 4RJX, respectively (13)). In addition, two variants of OprO were prepared by either replacing F62 by a tyrosine or D114 by a tyrosine. All four proteins were aligned to the membrane normal, also termed the z axis, with the extracellular loops toward the negative z values. Subsequently, the protein structures in their trimeric form were inserted into a 1-palmitoyl-2-oleoyl-sn-glycero-3 phosphoethanolamine lipid bilayer ˚ of modified TIP3P water on both sides and 0.15 M KCl solution with 40 A of the membrane. Likewise, systems of OprO WT and OprO DM in 1 M KCl salt concentrations were prepared. All six systems were equilibrated in six steps by reducing the positional restraints on the heavy atoms of proteins and lipids for a total of 20 ns. The systems were built and equilibrated using the protocol proposed in the CHARMM GUI Membrane Builder (18,19). Four equilibrated systems, i.e., OprO WT, OprO-F62Y, OprOD114Y, and OprO DM in the 0.15 M KCl salt solution were simulated in NPT ensemble for 100 ns each without any applied biasing force. Furthermore, we performed applied-field simulations (3 100 ns) for the OprO WT and OprO DM in 1 M KCl salt solutions by applying a constant electric field E along the z direction of the channel. The applied field strength E is proportional to the voltage V, E ¼ V/Lz, where Lz represents the length of the system in the z direction (20–22). The applied-field simulations were performed in a NVT ensemble with the electric field pointing from the extracellular to the periplasmic space corresponding to transmembrane potential of 500 mV. The flux of K and Cl ions passing through the channels in the presence of applied field can be estimated using (20)
IðtÞ ¼
N X 1 qi z i ; Lz Dt i ¼ 1
where qi denotes the charge of the atom i and Dzi is the displacement of atom i during the time step Dt. The MD simulations were performed with the software GROMACS 4.6.5 (23) using the CHARMM36 force field for proteins, lipids, and
RESULTS AND DISCUSSION Our initial goal was to investigate if the channel properties of OprO can be converted to those of OprP by modifying the key residues in the constriction region. Two single mutants, F62Y, and D114Y, and a DM F62Y/D114Y of OprO were generated and reconstituted into the lipid bilayer. The single channel conductance of these channels was measured at þ50 mV applied voltage at room temperature (RT) in 0.1 M and 1 M KCl solutions supplemented with 10 mM (2-(N-morpholino) ethanesulfonic acid) (MES) and adjusted to pH 6. Fig. 2 shows typical single channel recordings in a step-like manner caused by insertion of OprO WT, OprO mutants, and OprP WT into lipid bilayers. DM shows an increase in single channel conductance As expected, based on previous studies, the OprO protein exhibits higher conductance than the OprP (see Table 1). At the level of single amino acid mutations, the exchange of phenylalanine by tyrosine in OprO, F62Y, produced a slight drop in the conductance of the channel. On the other hand, replacement of the aspartic acid with more bulky tyrosine in OprO, D114Y, did not show any visible effect on the KCl conductance. The introduction of both mutations simultaneously, i.e., OprO F62Y/D114Y, surprisingly exhibits a conductance higher than that of OprO WT. Thus, the OprO DM mutant channel did not yield a conductance value
FIGURE 2 Pore-forming events of OprO WT, OprO F62Y, OprO D114Y, OprO DM, and OprP WT. Single-channel conductance as observed for OprO WT, OprO mutants, and OprP WT in a DiPhPC membrane bathed in 0.1 M KCl, 10 mM MES-KOH, pH 6, at þ50 mV.
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Ganguly et al. TABLE 1 The Experiments were Carried Out with DiPhPC Membranes and an Applied Voltage of D50 mV at RT
ation of chloride ions compared to the native channel, and this leads to higher conductance in OprO DM than OprO WT.
Average Single Channel Conductance Experiments
MD Simulations
Proteins
0.1 M KCl
1 M KCl
1 M KCl
OprO WT OprO (F62Y) OprO (D114Y) OprO DM OprP WT
240 5 15 200 5 37 240 5 28 400 5 43 160 5 9
440 5 33 400 5 70 440 5 67 600 5 76 260 5 18
532 5 43 — — 625 5 89 —
At least 100 single events were used to calculate the average conductance while simulations were only performed for two of the channels.
similar to that of OprP. On the contrary, in the previous study we have shown that the OprP DM (Y62F/Y114D) channel yielded conductance values similar to those of OprO WT. To understand the rise in conductance of the OprO DM channel, we performed all-atom MD simulations and calculated the conductance values of the OprO WT and OprO DM channels in 1 M KCl solution in the presence of a 500-mV applied field pointing from the extracellular to the periplasmic side. Table 1 summarizes the average conductance values from three independent simulations of 100-ns length each. Consistent with the experiments, the OprO DM has shown a higher conductance compared to the WT. The increased single channel conductance of the OprO DM might be due to the change in the dimensions of the channel and the electrostatics inside the channel. The radius profiles clearly indicate that OprO DM forms a wider channel than the OprO WT with a twofold increase of the cross-section area in the eyelet region (Fig. 3). Moreover, the electrostatic network inside the pore is altered by the replacement of phenylalanine by the less hydrophobic tyrosine and the negatively charged aspartic acid by the neutral tyrosine. These modifications can result in a smaller barrier for the perme-
FIGURE 3 Plot showing the average radius of the OprO WT as well as OprO single and double mutant channels along with the corresponding SD derived from unbiased MD simulations. The pore radii have been determined from 100-ns-long equilibrated trajectories of the respective channels using the HOLE program (30). ‘‘EC’’ and ‘‘PP’’ denote the extracellular and the periplasmic sides of the channel, respectively. To see this figure in color, go online.
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DM channel of OprO has OprP-like substrate specificity Previous titration experiments have shown that the OprO and OprP pores have specific binding affinities for diphosphate and monophosphate, respectively. Mutations of two important amino acid residues present in the vicinity of the affinity site in OprP reversed its specificity from monophosphate to diphosphate (13). To understand the role of these two residues i.e., F62 and D114, in substrate binding of OprO and to see if mutations can alter the specificity of OprO, the inhibition of channel conductance by adding mono- and diphosphate solution on both sides of membrane was measured as a function of concentration. The experiments were carried out with DiPhPC membranes and an applied voltage of þ50 mV at RT. At least 100 single events were used to calculate the average conductance values. The half-saturation constants for inhibition of channel conductance by mono- and diphosphate were obtained from titration experiments using Lineweaver-Burk plots are shown in Fig. 4. Table 2 summarizes the results of the titration experiments of OprO WT and mutant proteins with monophosphate and diphosphate. The single and double mutations reduce the binding affinity of the OprO channel for diphosphate and increases the affinity toward monophosphate ions. A considerable difference can be observed in the stability constants of mono- and diphosphate binding to OprO WT and to OprO DM. In case of the OprO DM, the current through the membrane decreases almost to 0 at monophosphate concentration of 6.36 mM, which indicates that for the OprO DM channels, the KCl conductance is blocked due to the binding of monophosphate inside the channel lumen. The stability constant for monophosphate binding to OprO DM (K ¼ 720 1/M) is increased by a factor of 3 compared to that of OprO WT (220 1/M). It is noteworthy that the replacement of F62/D114 from OprO with bulkier tyrosine amino acids led to a substantial increase of the binding affinity of monophosphate ions with similar binding kinetics as that of OprP. Table 2 shows the data from similar experiment where a membrane containing the OprO DM was titrated with diphosphate. The OprO DM conductance was blocked at a higher diphosphate concentration of 48.70 mM. This finding is demonstrated in Fig. 3 (red straight line), which shows a Lineweaver-Burk plot of the data from the titration experiment for the OprO DM. The straight line in the LineweaverBurk plot corresponds to a stability constant of 343 1/M, which indicates that the ability of diphosphate to block the ion conductance in OprO DM is decreased by approximately a factor of 4 as compared to OprO WT. The binding affinity of the OprO channel for diphosphate ions was reduced when key amino acid residues (F62 and D114) are mutated with tyrosine. The results suggest that the replacement of
Conversion of OprO to OprP
FIGURE 4 Lineweaver-Burk plots for OprO WT and its mutants for phosphate- or diphosphate-mediated inhibition of membrane conductance. The straight lines correspond to stability constants K given by the Eq. 1 (fraction of closed channels) ¼ K∙c/(K∙c þ 1). (a) Lineweaver-Burk plots for diphosphate: OprO WT shows the highest stability constant for diphosphate K ¼1450 1/M followed by an intermediate stability constant for OprO D114Y of K ¼ 630 1/M and for OprO F62Y of stability constant K ¼ 490 1/M; OprP WT has the smallest stability constant K ¼ 310 1/M for diphosphate and OprO DM shows a similar stability constant as that of OprP WT with K ¼ 343 1/M. (b) Lineweaver-Burk plots for phosphate: OprP WT shows the highest stability constant K ¼ 770 1/M followed by OprO DM with a stability constant of K ¼ 720 1/M. OprO WT shows the smallest stability constant K ¼ 220 1/M and intermediate stability constants were found for D114Y with a stability constant K ¼ 500 1/M and for OprO F62Y with K ¼ 690 1/M. To see this figure in color, go online.
OprO F62/D114 by tyrosine residues reverses the binding affinity from diphosphate to monophosphate. CONCLUSION The OMs of Gram-negative bacteria contain several specific channels that are involved in the uptake of specific substrates across the bacterial cell. It has been demonstrated previously that a few amino acids in the affinity site of these TABLE 2 Monophosphate- and Diphosphate-Mediated Inhibition of Chloride Conductance of OprO WT, OprO Mutants, and OprP WT in 0.1 M KCl, 10 mM MES, pH 6, at an Applied Voltage of D50 mV at RT Diphosphate Proteins OprO WT OprO (F62Y) OprO(D114Y) OprO DM OprP WT
Phosphate
1/K (mM)
K (1/M)
1/K (mM)
K (1/M)
0.6 2 1.5 2.9 3.1
1450 5 120 490 5 110 630 5 90 343 5 20 310 5 35
4.5 1.4 2 1.4 1.3
220 5 50 690 5 140 500 5 70 720 5 90 770 5 150
channels play a major role in sorting substrate. Electrophysiology experiments revealed that OprP and OprO have specific affinities toward monophosphate and diphosphate, respectively. In a previous study, we reported that the Y62F/Y114D mutations successfully swapped the OprP pore properties to OprO-like (13). In this study, we probed the role of two key residues, F62, and D114Y, in the ion and substrate transport properties of the OprO channel. The F62Y OprO channel displayed a slight decrease in conductance, whereas the D114 OprO mutation does not show any change in conductance. Surprisingly, the OprO DM (F62Y/D114Y) displayed a higher conductance than the expected OprP-like behavior. Molecular level analysis using MD simulations helped us to investigate the reason for this rise in conductance. The OprO DM has a wider lumen than that of the OprO and OprP WT channels and this increase in the pore dimension along with the altered electrostatics made the passage of chloride ions easier, leading to an increased conductance in OprO DM. Binding affinity studies of OprO and its mutants revealed the importance of two key amino acid residues, F62 and D114 (present in the constriction region) for their substrate specificity, and any change of these amino acids can influence the functionality of this pore. The most substantial reversal of substrate specificity was observed when both residues, F62 and D114, were replaced by tyrosine residues, followed by F62Y and D114Y single mutant OprO channels. Our results suggest that the engineered OprO DM channels have an OprP-like substrate specificity. The two key residues seem to render the unlike phosphate specificities of the OprO and OprP channels. The results presented here and in previous studies (12,13,16) of OprO and OprP channels indicate that only a few residues in the constriction region of OM channels play a key role in sorting the substrates. Future investigations of transport of phosphonic antibiotics, e.g., fosfomycin and fosmidomycin, across these channels will improve our understanding of antibiotic influx across the OM of P. aeruginosa. SUPPORTING MATERIAL Supporting Materials and Methods and one table are available at http:// www.biophysj.org/biophysj/supplemental/S0006-3495(17)30755-5.
AUTHOR CONTRIBUTIONS R.B., S.G., and U.K. designed research. S.G., I.B.-U., A.K., U.K., and R.B. performed and analyzed electrophysiology and MD simulations, analyzed data, and wrote the article.
ACKNOWLEDGMENTS We thank Prof. R.E.W. Hancock, University of British Columbia, Vancouver, Canada, for providing basic materials for our experiments. We would like to thank Niraj Modi for his help with the initial simulations.
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Ganguly et al. This research has received support from the Innovative Medicines Initiatives Joint Undertaking under grant agreement No. 115525, resources that are composed of financial contributions from the European Union’s Seventh Framework Program (FP7/2007-2013) and EFPIA. In addition, this work has been supported by the grants BE 865/16-2 and KL 1299/9-2 of the Deutsche Forschungsgemeinschaft (DFG).
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