1. Introduction
Organophosphorus hydrolase (OPH) is a bacterial enzyme that can detoxify a wide range of organophosphorus (OP) agents by hydrolyzing various phosphorus-ester bonds (P-O, P-F, P-CN, and P-S). OP compounds are toxic molecules used primarily as pesticides and nerve agents. OP compounds cause severe neurotoxic effects by covalently binding to acetylcholinesterase (AChE), an enzyme that catalyzes the breakdown of neurotransmitters such as acetylcholine. Binding by OP inhibits AChE, resulting in accumulation of neurotransmitter and rapid death in insects and humans due to lost control of respiratory muscles [
1]. Current medical countermeasures, including atropine and oxime-based reactivators [
2] that target the down-stream pathways of OP, act through reactivation of AChE, blocking acetylcholine receptor activity or simply easing the symptoms. A direct method that can hydrolyze OP agents before they enter the central nervous system is needed. Currently, OPH is the only enzyme used in organophosphate remediation [
3], but more investigation is needed to improve the use of OPH in medical therapy. The advantage of using OPH therapy over oxime-based treatment is rapid hydrolysis of OP agents in the circulatory system. Thus, nerve agents are eliminated before penetrating the blood-brain barrier and exerting effects in the nervous system.
While the hydrolytic products of OP breakdown by OPH are generally of reduced toxicity relative to their parent compounds [
3,
4], substrate binding affinity is inadequate for therapy. Many traditional protein engineering studies have shown that inducing mutations in or near the active site residues can increase the catalytic efficiency (
kcat/
KM) by increasing the catalytic rate constant (
kcat) of the enzyme [
5]. Those efforts, however, resulted in OPH variants with reduced effectiveness for hydrolysis of multiple nerve agents. In particular, the wild-type OPH and variants currently available demonstrate poor substrate binding affinity (
KM). Poor binding affinity means that the substrate concentration required for enzymatic conversion exceeds the nerve agent lethal dose. Therefore, an important topic of research is understanding how the structure of OPH correlates with substrate binding.
One hypothesis aimed at improving KM is that stabilization of substrate binding in the OPH active site could increase the probability of transition state formation for OP conversion. To test this hypothesis, we augmented traditional protein approaches with computational approaches. Using molecular dynamics simulations, we analyzed the wild-type (WT) OPH active site. The analysis led to identification of amino acid residues that stabilize the OPH active site. Experiments tested the molecular dynamics predictions.
A challenge in analyzing the OPH active site is that the native substrate remains unknown, and only structures with substrate analogs exist. While paraoxon (diethyl p-nitrophenyl phosphate) is considered a near-native substrate of OPH [
6,
7,
8] (
Figure 1), and induces neurotoxicity through the same manner as nerve agents by inhibiting AChE, it is also safe to test in research labs, which is an important consideration. It is not possible to obtain a co-crystal structure of the WT-OPH with the bound paraoxon because of the likelihood of hydrolysis. A co-crystal structure of OPH with diethyl 4-methylbenzylphosphonate (DMBP), which is a substrate analog sharing a high similarity with paraoxon, is available [
9]. Consequently, most researchers use the binding mode of DMBP as a starting structure in their studies in order to model paraoxon [
7,
10,
11]. In the absence of a bound structure, however, the validity of this analogy is uncertain. Thus, careful study of the paraoxon binding mode, and paraoxon’s potential interactions within the OPH binding site, is needed.
Before performing mutagenesis on OPH bound to paraoxon, we used docking and ligand-based methods, combined with molecular dynamics (MD) simulations, to model possible binding modes of paraoxon in the OPH binding site. Overall, we found a stable binding pose of paraoxon, which differs from the native binding mode of the substrate analog. Moreover, using the predicted binding mode, we performed MD simulations on different OPH mutants and found three different residues, D253, H254, and I255, involved in the stability of the catalytic site. Here, we report a detailed computational structural analysis on five mutant complexes that include those residues (D253E, D253E-H254R, D253E-I255G, H254S, and I255S).
Additionally, we tested the accuracy of our predictions using an experimental mutagenesis approach. When expressed experimentally, all designed single and double mutations (H254S, I255S, D253E, D253-H254R, and D253E-H254R) achieved improved substrate binding affinity. Our kinetics results showed, on the one hand, a substantial increase in substrate binding affinity of 19-, 10-, 5.2-, and 4.8- folds to D253E-H254R, D253E, H254S, D253E-I255G, respectively. The designed single mutation I255S, on the other hand, presented a modest increase of substrate binding affinity (1.6 fold) compared to the WT-OPH.
2. Results and Discussion
Despite several engineered variants, applications of OPH suffer from the enzyme’s low substrate binding efficiency, hindering its action as an OP scavenger. Here, we focused on designing OPH mutants that yield stronger substrate binding while minimizing the reduction of catalytic effectiveness. Many previous studies have been done to improve OPH substrate binding through rational design or direct evolution [
5,
12,
13]. None of the resulting designed variants, which mainly targeted active-site modifications, improved binding properties to nerve agents sufficiently to prevent lethality.
In the current work, we targeted residues surrounding the OPH active site (
Figure 2). The selected residues do not coordinate the Zn(II) cations in the active site, and are not directly involved in the hydrolysis reaction. We hypothesized that the alterations of these residues would stabilize the OPH active site by increasing the hydrogen bonding associated with the side chains of the active site residues, thus leading to a more stable substrate binding. In contrast with previously designed OPH variants focused on modification of the active site residues to increase the catalytic activity of OPH, our current findings suggest that mutations near the enzyme active site can enhance paraoxon binding affinity.
2.1. Paraoxon Binding Mode
Prior to mutagenesis of the OPH residues, we wanted to predict the correct binding mode of paraoxon in the OPH active site. As described in the Methods section, we performed docking using three pieces of docking software and selected several representative docking poses (
Figure 3).
Then, we ran short MD simulations on the selected poses to assess their stability in the OPH active site. For this purpose, we calculated the RMSD of the poses at the end of the 15 ns long MD trajectories with respect to the starting poses before MD, and found that the pose with the lowest RMSD value (1.77 Å) was generated by HYBRID (
Figure 4A). Interestingly, we found that the docking pose generated using POSIT, which best overlays the structure of diethyl 4-methylbenzylphosphonate, is not stable and scores an RMSD of 6.6 Å (
Figure 4B). Essentially, while the ligand can
dock in an overlapping pose, our simulations indicate it is not stable in that binding mode and that other binding modes are more favorable/stable. In contrast to previous assumptions, this finding suggests that the binding mode of paraoxon is different than that of 4-methylbenzylphosphonate.
2.2. The Single Mutant D253E
In wild type OPH, the carboxylate group of Asp 253 forms a hydrogen bond with the imidazole of His 230 (
Figure 5A). This hydrogen bond orients His 230 for an optimal coordination of one of the Zn(II) in the active site. We computationally mutated the aspartate residue at position 253 to a glutamate to create two hydrogen bonds with two residues in the active site (His 230 and His 55) (
Figure 5B), instead of the single hydrogen bond in the WT OPH structure.
Then, we performed MD simulations on the WT-OPH complexed with paraoxon and the D253E mutant complexed with paraoxon to evaluate whether the substitution of the aspartate’s side chain by a longer side chain would have an impact on the number and the stability of the interactions between the OPH active site residues. A structural analysis of our MD simulations showed that, in the mutant form D253E, the active site residues establish four additional stable hydrogen bonds compared to the WT OPH. As shown on
Figure A1 and
Figure A2, and based on the distance between atoms that could form potential hydrogen bonds during MD simulations, we concluded that four interactions are more stable during the simulations of the D253E mutant complex. These interactions occur between: (i) Thr 54 and His 55, (ii) His 57 and Trp 302, (iii) Thr 200 and His 201, and (iv) Leu 271 and His 254. His 55, His 57, and His 201 take part in the Zn(II) ligation while His 254 is not involved in the Zn(II) coordination. However, His 254 plays a role in the stability of the OPH active site by stacking with His 230, which guides and stabilizes the Zn(II)-His 230 interaction.
2.3. The Double Mutant D253E-H254R
After identifying D253 as a hot-spot residue, we designed the double mutant D253E-H254R and tested its effect on interactions in the OPH active site using MD simulations. We chose to mutate position 254 since His 254 influences catalysis by interacting with active site residues, particularly His 230 (
Figure 5A). Through comparing the donor-acceptor distances in the MD trajectory of the WT OPH to the donor-acceptor distances in the MD trajectory of the mutant form, we found that D253E-H254R induces three additional hydrogen bonds in the OPH active site. Out of these hydrogen bonds, two involve the side chain of Arg 254
Figure A3A,B and
Figure A4A,B, which interacts with Leu 271, and His 257. The remaining hydrogen bond mediated by the double mutant occurs between Lys 169 and Asp 100 and appears after 40 ns of MD (
Figure A3C and
Figure A4C).
2.4. The Double Mutant D253E-I255G
The role of Ile 255 in the OPH active site is not described in the literature, to our knowledge. To test whether a subtle change near the important active site residues would affect the stability of the interactions and the flexibility of the substrate binding pocket, we coupled the mutation D253E with a second mutation at position 255. Then, we investigated whether the double mutation disrupted the coupled interactions observed in the OPH active site. Our MD simulations performed on the D253E-I255G mutant in complex with paraoxon showed the same hydrogen bonding profile observed during the simulations of WT OPH. One additional hydrogen bond between Asp 100 and His 257 was found in the trajectory of the D253E-I255G mutant form (
Figure A5 and
Figure A6A). This finding suggests that position 255 has an indirect role in stabilizing interactions in the OPH active site.
2.5. The Single Mutants H254S and I255S
Since the double mutants D253E-H254R and D253E-I255G induced changes in the interactions near the OPH active site and the single mutant D253E expanded the interaction network within the binding site, we decided to explore whether positions 254 and 255 are also important for the stability of the binding site. Thus, we designed two single mutants (H254S and I255S), where we changed the properties of the basic residue at position 254 and the non-polar residue at position 255, by replacing each of them with an apolar amino acid: Serine. Next, we performed MD simulations on the two complexes: OPH:H254S and OPH:I255S. We inspected the residues’ interactions in and near the binding site during the MD simulations and compared the hydrogen bonds found to those identified during the MD simulations on the WT complex.
For OPH:H254S, the same interactions were observed compared to WT:OPH. Only one hydrogen bond, detected between the mutated residue H254S and a water molecule in the binding site, appears after 60 ns of MD simulations on the mutant complex (
Figure A7 and
Figure A6B). It is worth noting that this water molecule is one of the two crystal water molecules that are involved in the coordination of one of the Zn(II) ions in the active site. This result suggests that the induced mutation, H254S, contributes to the stabilization of the Zn(II) coordination in the active site, and could perhaps improve paraoxon binding affinity.
For OPH:I255S, the interactions established in and near the binding site are similar to those identified during the simulations of the WT complex. One stable hydrogen bond was induced by the mutation and formed between the mutated residue Ser 255 and Ser 299 (
Figure A8 and
Figure A6C). To our knowledge, S299 is not directly involved in the binding site activity, suggesting that the S255 mutation would not affect the stability of the active site and the catclytic activity.
2.6. Absolute Binding Free Energy Calculations on WT OPH and OPH Mutants
To obtain more detailed information about the affinity of paraoxon towards WT OPH and OPH mutant forms, we performed absolute binding free energy calculations in implicit solvent on these different complexes. Although binding free energy calculations in implicit solvent may not be as accurate as those in explicit solvent, we wanted to test the accuracy of this approach on our studied protein target and to assess the potential strength of OPH and OPH-mutants interactions with paraoxon within the limitations of the method.
Overall, the binding free energies appear sufficiently converged with reasonable uncertainties (
Figure A9 and
Table 1). Three designed mutants show more favorable binding free energies to WT OPH with an improvement of 0.63 kcal/mol, 0.26 kcal/mol, and 1.63 kcal/mol for D253E, D253E-H254R, and D253E-I255G, respectively. These findings confirm that the single and the double mutations we introduced here enhance the stability of the substrate in the OPH active site.
Some of our other calculations, however, show that the single mutants (H254S and I255S) we designed to alter the property of the side chains at positions 254 and 255 failed to improve the binding of paraoxon to the protein. The calculated binding free energies of the mutants I255S (−20.44 kcal/mol) and H254S (−17.61 kcal/mol) are less favorable than that of the WT complex (−20.70 kcal/mol).
2.7. Spectrophotometric Assay of WT and Mutant OPH Activity
To evaluate the accuracy of our computational analysis, we experimentally tested the predicted mutations D253E, D253E-H254R, D253E-I255G, H254S, and I255S for catalysis of P-O bond cleavage of paraoxon. Using a continuous spectrophotometric assay,
kcat and
KM values were measured and compared to WT OPH (
Table 2).
Notably, we found that the double mutant D253E-H254R exhibits a 19-fold increase in substrate binding affinity over WT. In addition, D253E and D253E-I255G enhanced the substrate binding affinity by 10- and 4.8-fold, respectively. In contrast, I255S did not improve the binding affinity of paraoxon significantly. These results exhibit a similar trend to the computational findings and suggest that hydrogen bonding within the OPH active site is important for stabilizing the OPH active site and enhancing substrate binding.
For H254S, our kinetic measurements show an improvement in substrate binding affinity by 5.2 fold compared to the WT form. This enhancement was not captured in our binding free energy predictions and might be due to the limitations of the computational method. One major limitation is the use of implicit solvent since the water structure could affect changes in protein-ligand complexes. In contrast, our MD simulations performed in explicit solvent align with the experimental results, supporting our speculations regarding the solvent. MD simulations show that H254S induces a hydrogen bond that stabilizes one of the Zn(II) coordinators, which could explain the substrate binding affinity of the enzyme. An additional limitation, of course, is that experimental measurement includes other factors, which is a function of more than just the binding free energy.
This study focused mainly on improving OPH substrate binding. While all mutants examined resulted in reduced values of kcat compared to that of WT, the overall catalytic efficiency (kcat/KM) achieved for D253E, D253E-H254R and H254S mutants were still on the order of 10 M−1 s−1, which approaches the limit of substrate diffusion (on the order of 10 to 10 M−1 s−1). Future work will address improvements to kcat while maintaining the gains to KM reported here.
4. Conclusions
Organophosphorus hydrolase is a bacterial enzyme that can detoxify a wide range of OP nerve agents. Despite thousands of engineered variants, OPH suffers from a low substrate binding affinity, hindering its action as an OP scavenger. Here, we focused on testing amino acid substitutions on residues near the enzyme active site to assess how these modulate substrate binding while minimizing the effect on enzyme catalytic efficiency. We computationally investigated single and double mutants to examine the effect of these mutations on the strong interactions occurring in and near the catalytic site. According to the modeling data, additional hydrogen bonds appear in the MD trajectories of the modified OPH enzymes. Hence, changes in the hydrogen bond network surrounding the active site may be important for the function of OPH and must be considered in the design of OPH variants. Strikingly, experimental measurements of kinetics showed that the predicted mutations improved substrate binding affinity compared to WT OPH substantially, thus confirming the computational predictions.
This work provides new insights guiding the protein design of organophosphorus hydrolase. Through coupling kinetic measurements and molecular modeling, we found hotspot residues that enhance the stability of paraoxon in the binding site and produced OPH mutants exhibiting better paraoxon binding properties. Two of the engineered mutants, D253E-H254R and D253E, enhanced the substrate binding of the enzyme significantly. Although both mutants resulted in reduced values of kcat, the overall catalytic efficiencies kcat/KM achieved for both mutants were maintained on the order of 10 M–1s–1. Additionally, we identified a stable binding mode of paraoxon that may help other researchers model OPH-substrate interactions. We expect that our structural findings highlighting the importance of the residues surrounding the active site of OPH and their strong interactions will assist future research. Combined with work on other important and extensively studied active site residues, these results could help further improve the catalytic function of OPH and its variants.