Article pubs.acs.org/biochemistry
Insight into the Mechanism of Hydrolysis of Meropenem by OXA-23 Serine-β-lactamase Gained by Quantum Mechanics/Molecular Mechanics Calculations Jacopo Sgrignani,†,§ Giovanni Grazioso,*,‡ and Marco De Amici‡ †
Istituto di Chimica del Riconoscimento Molecolare, CNR, Via Mario Bianco 9, 20131 Milan, Italy Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Via L. Mangiagalli 25, 20133 Milan, Italy
‡
S Supporting Information *
ABSTRACT: The fast and constant development of drug resistant bacteria represents a serious medical emergency. To overcome this problem, the development of drugs with new structures and modes of action is urgently needed. In this work, we investigated, at the atomistic level, the mechanisms of hydrolysis of Meropenem by OXA-23, a class D β-lactamase, combining unbiased classical molecular dynamics and umbrella sampling simulations with classical force field-based and quantum mechanics/molecular mechanics potentials. Our calculations provide a detailed structural and dynamic picture of the molecular steps leading to the formation of the Meropenem−OXA-23 covalent adduct, the subsequent hydrolysis, and the final release of the inactive antibiotic. In this mechanistic framework, the predicted activation energy is in good agreement with experimental kinetic measurements, validating the expected reaction path.
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and, for this reason, are also known as oxacillinases (OXAs).10 Depending on the specific substrate that is preferentially hydrolyzed, this family of enzymes has been additionally classified into groups of penicillinases (e.g., OXA-1), cephalosporinases (e.g., OXA-10, OXA-14, and OXA-17), and carbapenemases (e.g., OXA-23, OXA-40, previously known as OXA-24, and OXA-48). Numerous crystal structures shed light on the structural features that make this class of enzymes atypical. For example, Docquier et al. in 2009,11 determining the apo structure of OXA-48, revealed that the large binding site permits the carbamylation of a lysine residue. This peculiar derivatization was recognized as being essential to trigger the catalytic activity of the enzyme, thus activating the serine residue that attacks the β-lactam ring of substrates.12 In addition, the presence of an oxyanion hole, occupied by a water molecule, and of pivotal residues involved in the catalytic cycle was put in evidence. Among class D BLs, OXA-23 is the major carbapenemase produced by Acinobacter baumannii, an opportunistic pathogen found worldwide.13,14 The structure of this enzyme was determined by X-ray diffraction in 2013,13 and like other BLs of the same class, OXA-23 showed the N-carboxylated Lys82, which is fully active only at neutral pH. Conversely, at pH 4.0,
he repeated occurrence of drug resistant bacteria constitutes a real emergency for human and animal health.1 One of the most relevant mechanisms of resistance in Gram-negative pathogens is the expression of β-lactamases (BLs), specific enzymes that can hydrolyze the most commonly used antibiotics, such as penicillins, cephalosporins, monobactams, and carbapenems. The consequence of this hydrolytic reaction is the complete inactivation of the drugs.2−4 Over the years, four classes of BLs (A, B, C, and D) have been globally adopted. While in classes A, C, and D a serine residue directly participates in the hydrolytic reaction,3 in enzymes belonging to class B metal cofactors, in particular zinc ions, play a pivotal role in accelerating the reaction.5 A common therapeutic strategy for overcoming bacterial resistance envisages the co-administration of one β-lactam drug and one BL inhibitor. However, the continuous use of these drug combinations has generated an evolutionary pressure resulting in the accelerated emergence of resistant bacterial strains.2 At present, several BL inhibitors, such as clavulanic acid, tazobactam, sulbactam, and avibactam, are in clinical use while other promising compounds such as diazabicyclo[3.2.1]octanone derivatives and boronic acids are being added to the therapeutic toolbox.6,7 Only a few novel inhibitors of metalloBLs have recently been reported,8,9 and no effective inhibitors are currently used in clinical practice. Similarly, class D BLs are not efficiently inhibited by some BL inhibitors, and at variance with BLs belonging to other classes, they hydrolyze oxacillin © 2016 American Chemical Society
Received: May 11, 2016 Revised: August 10, 2016 Published: August 18, 2016 5191
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associated with this important class of BLs, for which novel and potent inhibitors are still lacking.
decarboxylation of this lysine residue severely impairs the enzymatic activity.13 Moreover, X-ray studies suggested that the OXA-23 Ser79 residue covalently bound Meropenem (Mer), a carbapenem antibiotic (Figure 1)13 whose interaction is additionally stabilized by several hydrogen bonds and hydrophobic contacts with the enzyme.
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COMPUTATIONAL METHODS Model System and MD Setup. The OXA-23−Mer complex model was built taking into account the crystal structures acquired at different pH values (Protein Data Bank entries 4JF4 and 4JF6).13,31 One includes Mer covalently bound to the enzyme, but because of the acidic crystallographic conditions (pH 4.1), it did not contain the N-carboxylated form of Lys82 (Kcx82), essential to the enzyme’s activation and the subsequent substrate hydrolysis. Conversely, the structure of 4JF6 displayed Kcx82, but Mer was missing. Thus, to create the starting Michaelis complex model, we merged both structures, restoring manually the hydrolyzed β-lactam ring of Mer found in the 4JF4 structure. The final enzyme protonation state was determined at pH 7 by the H++ Web server.32 Furthermore, the OXA-23−Mer complex was prepared for the simulations through the removal of the chloride ions included in the X-ray structure and the assignment of Kcx82 and Mer partial atomic charges by the RESP33 method, starting from an electrostatic potential calculated by Gaussian0934 and considering a HF/6-31g(d) level of theory. Then, fully solvating the complex considering a box with a minimum distance between its surface and the nearest protein atom of 10 Å, we built a system with ∼20000 solvent atoms and a box volume of 234756 Å3. The TIP3P35 model was employed to describe the solvent molecules’ effect, and ff12SB and GAFF36 force fields were applied to the enzyme and the ligand, respectively. The neutrality of the system was ensured by adding two sodium ions. After the calculation of grid charges, tleap (AMBER1237,38) inserted ions on the protein surface where the energies were the lowest.19,39 MM energy minimization and MD simulations optimized the resulting complex, initially by the sander module and then by the pmemd.cuda module of the AMBER1237,38 package, in the production MD run. During a first minimization step, only water molecules, keeping the protein atoms frozen, were optimized. Then, the whole system was geometrically refined by setting a convergence criterion on the gradient of 10−4 kcal mol−1 Å−1. Prior to the start of the production run of MD simulations, the system was equilibrated for 40 ps at 300 K under isocore conditions (NVT). Subsequently, 50 ns MD simulations in an isothermal−isobaric ensemble (NPT) were performed at 300 K, with a 2 fs time step. All the simulations were performed under periodic boundary conditions (PBC). van der Waals and short-range electrostatic interactions were estimated within a 10 Å cutoff, whereas long-range electrostatic interactions were assessed using the particle mesh Ewald method.40 The SHAKE41 algorithm was applied to all bonds involving hydrogen atoms. Once the complex model had reached equilibration and structural stability [root-mean-square deviation (rmsd) of protein Cα atoms in the range of 1.5−2 Å], the trajectories were further examined by visual inspection with VMD,42 thus ensuring that the thermalization did not cause any structural distortion. Finally, snapshots (334) extracted from the last 10 ns of MD simulations were analyzed by the MM-GBSA energy decomposition approach implemented in Amber14,43 to improve the characterization of the molecular determinants of the initial Mer binding in the Michaelis complex (additional details are available as Supporting Information).
Figure 1. (A) Full representation of the OXA-23/Mer model used for simulations. Water molecules have been omitted for the sake of clarity. The surface is colored according to the atom electrostatic charges (blue for positive and red for negative), suitably calculated by Pymol tools. (B) Chemical structure of Meropenem, used in this study.
However, because crystal structures show the static image of the mutual interaction, the mechanism by which substrates are hydrolyzed can be hypothesized only by taking into account the position of some key residues in the Michaelis complex and the similarity with other serine-BLs already investigated by computational methods.15,16 In fact, it is widely accepted that carbapenem hydrolysis by serine-BLs involves a nucleophilic attack by a serine residue, previously activated by a nucleophile. For class A BLs, the general base is represented by the side chain of Glu166,16 whereas in the oxacillinases, this role is played by the carbamylated Lys82 (Kcx82).12 In the subsequent step, the activated serine attacks the carbonyl group of the βlactam ring, in which the nitrogen atom is hydrogen-bound to the side chain of an adjacent serine residue. The following βlactam ring opening and the formation of the covalent complex require a negative charge neutralization step, assisted by the side chain of a Lys residue. The final acyl−enzyme hydrolysis is controlled by the presence of a water molecule and supported by the starting base, i.e., Glu166 for class A or Kcx82 for class D BLs. This step can be bypassed through inactivation of the water molecule by the 6α-1R-hydroxyethyl group of the substrate17 or the tautomerization of carbapenems from the Δ2-pyrroline to the less reactive Δ1-pyrroline form.13 However, despite these mechanistic hypotheses, the precise role of each residue and/or the specific interaction with the water molecules is still not entirely clarified and/or could be different across BLs.18 In this study, as in our previous investigations,19 we used a multiscale20 computational approach, based on the application of the classic molecular mechanics (MM) molecular dynamics (MD) simulations to hybrid quantum mechanics/molecular mechanics (QM/MM),21−29 to investigate at the atomistic level the Mer hydrolysis reaction by OXA-23. In particular, we estimated the free energy profiles of all the steps necessary for complete substrate hydrolysis, using an enhanced sampling method, i.e., umbrella sampling (US) simulations.30 Our computational results, in line with known experimental parameters, highlight the importance of Lys82, Ser216, and Lys216 in the hydrolysis mechanism catalyzed by OXA-23. Likewise, this study shed some light on the antibiotic resistance 5192
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Biochemistry Table 1. Collective Variables Considered in the US Simulations US1 CV = A − B QM residues: Mer, Ser79, Kcx82, Trp165, Ser126, Lys216
A is the distance between the oxygen atom of Kcx82 and the hydrogen atom belonging to the Ser79 side chain B is the distance between the oxygen and the hydrogen atoms of the Ser79 OH group
US2 CV = A − B QM residues: Mer, Ser79, Kcx82, Trp165, Ser126, Lys216
A is the distance between the oxygen atom of Ser79 and the C7 atom belonging to the β-lactam ring of Mer B is the distance between the C7 and N1 atoms, internal to the Mer β-lactam ring
US3 CV = A − B QM residues: Mer, Ser79, Kcx82, Lys216, Wat
A is the distance between the hydrogen atom of Wat and the N1 atom belonging to the β-lactam ring of Mer B is the distance between the oxygen and hydrogen atoms of the Wat molecule
US4 CV = A − B QM residues: Mer, Ser79, Kcx82, Lys216, Wat
A is the distance between the oxygen atom of Wat and the C7 atom belonging to the β-lactam ring of Mer B is the distance between the C7 and oxygen atoms belonging to Ser79 side chain
QM/MM Calculations. The study of an enzymatic reaction mechanism requires computational methods that can take into account the formation and cleavage of covalent bonds in solvated ligand−protein complexes.21,44−46 Despite recent hardware and software advancements, quantum mechanics (QM) calculations capable of treating a fully solvated biological system at a high level of theory are still too expensive. A common solution to this limitation is the application of mixed QM/MM potentials, in which only the residues and solvent molecules involved in the reaction are simulated at the QM level, whereas the residual part of the system is simulated at the MM level.22,44,47,48 All the QM/MM calculations were performed by means of Amber12 3 7 , 3 8 patched by PLUMED1.3.49 In this study, the last frame of the MM MD simulations was used as the starting point for the QM/MM MD calculations. The QM region of the system was formed by the substrate (Mer), by OXA-23 residues Ser79, Kcx82, Ser126, Trp165, and Lys216, and by a water molecule closely bound to the last residue (Table 1 for details). Semiempirical Hamiltonians have some limitations in describing the electronic structure of a given molecular system; however, they can produce satisfactory results when specifically parametrized and tested.50,51 In this case, the choice of a semiempirical Hamiltonian is justified by previous benchmarks and permits an extensive sampling of the system that would be barely affordable considering a computationally more expensive level of theory such as the density functional theory (DFT) of post-Hartree−Fock calculations.21,28,44 In particular, the QM zone was simulated using the PM3-PDDG52 Hamiltonian that, as reported by Pierdominici-Sottile and Roitberg,53 permitted the estimation of the protonation energy in Trypanosoma cruzi trans-sialidase and in our previous study of the avibactam hydrolysis by TEM-1.19 The PM3-PDDG52 Hamiltonian was proposed by Jorgensen and co-workers in 2002, leading to a significant improvement in the evaluation of both formation heat and isomerization energies with respect to that achieved with the simpler PM3 methods.54 In this case, because we additionally aimed to verify the suitability of this specific level of theory for investigating BL reactions, once the reaction mechanism was fully characterized we calculated the free energy profile of the reaction also using the AM155 Hamiltonian (Figure S3) and achieved similar results.
Importantly, the use of this specific Hamiltonian allowed us to simulate the entire system for more than 1.5 ns, a simulation time that, considering a higher level of theory, is still inaccessible even using the most powerful supercomputers. The MM part of the system was simulated using the same parameters employed during the MM MD simulations (see previous section), and the link atom approach implemented in Amber1237,38 was employed to obtain the correct theoretical description of the bonds crossing the boundaries between the QM and MM portions. The system was equilibrated for 30 ps considering the new potential, with a time step of 0.05 fs in a NVT ensemble. The temperature was set to 300 K using a Langevin thermostat with a collision frequency of 5.0 ps−1.56 Short- and long-range electrostatic interactions were estimated in the same manner as classical MD.38,40 Umbrella Sampling Simulations. The activation energy of the steps necessary for the formation of the Mer−OXA-23 covalent complex and the final release of the hydrolyzed antibiotic were investigated using US simulations. In the first reaction step, the last frame obtained by MM MD simulations was utilized as the starting coordinates, and then the other simulations were started from the last configuration of the previous reaction step. Different numbers of windows were used for the various reaction steps, depending on the range values of the considered CV (Table 1). In particular, 24, 28, 33, and 31 windows of CV values for steps 1−4, respectively, were explored in the US simulations, constraining the system on specific CV values by harmonic constants ranging from 100 to 500 kcal mol−1 Å−2 (Tables S1−S4). Each US window ran for 7.5 ps, reaching a total simulation time of 1.02 ns. Finally, the free energy profile was estimated by the weighted histogram analysis method, as implemented in the WHAM program, developed by the Grossfield laboratory.57 The statistical uncertainty of the free energy profiles was estimated by Montecarlo Boostrap error analysis.
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RESULTS AND DISCUSSION MD Simulations of the OXA-23−Mer Complex. A structural inspection of the 50 ns classical MD simulations of the OXA-23−Mer complex did not highlight any significant changes in the active site geometry or in the secondary structures. The average rmsd for the Cα atoms of OXA-23, calculated with respect to the starting structure during the 5193
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Reaction Path Identification by QM/MM MTD and US Calculations. US is an enhanced sampling technique associated with a QM/MM scheme, which has been successfully applied in several studies.50,59 We used this method to acquire atomistic details about the mechanism by which Mer is hydrolyzed by OXA-23. US simulations additionally can be used to explore the potential of mean force (PMF) associated with a variation of a properly defined CV. CV definition is a critical step in all the US studies, and frequently, it is a trial and error process in which the availability of experimental data on the reaction under study should be complemented by the knowledge of the general principles of bioorganic chemistry. Thus, to validate the CVs involved in the reaction coordinate of the Mer hydrolysis, we accurately estimated the free energy profile of all reaction steps, and the calculated energy barriers separating all steps were compared with the experimentally derived kcat value. From kcat, a ΔF# value of 19.1 kcal/mol13 was estimated by applying the Eyring equation, as implemented in the applet available at http://www-jmg.ch.cam.ac.uk/tools/ magnus/eyring.html and described in ref 60. By taking into account the knowledge of the OXA-23 reaction mechanism,13 we postulated a reaction path (Figure 4) in which Ser79 is activated by Kcx82 (N-carboxylated Lys82, step 1), becoming able to attack the C6 carbonyl atom of Mer (step 2). These two steps lead to the formation of an intermediate (2), in which the negatively charged Mer is covalently bound to OXA-23 by Ser79. A proton is then transferred to the Mer negatively charged atom (step 3) by a water molecule, which is hydrogen-bound to the side chain of Lys216. The resulting hydroxide ion performs the direct nucleophilic attack on the carbonyl group of Mer (step 4), leading to the final release of the hydrolyzed substrate. The proton transfer from Kcx82 to Ser79, which re-establishes the initial protonation state of the enzyme, marks the end of the catalytic cycle. Ser79 Deprotonation (step 1) and Mer β-Lactam Ring Opening (step 2). The Mer hydrolysis reaction catalyzed by OXA-23 is triggered by the Ser79 deprotonation [US1, step 1 (Figure 4)] and the subsequent nucleophilic attack on the βlactam carbonyl group of Mer [US2, step 2 (Figure 4)]. In US1, the considered CV defined the Ser79 protonation state in the presence of the negatively charged Kcx82 (Table 1 and Figure 5A). The attained potential of mean force (PMF) profile showed a transition state (TS1) at CV values of −0.2 Å and an energy minimum (B, Figure 5A) at CV values close to −0.5 Å. Here, the Ser79 side chain hydrogen atom was abstracted by Kcx82, remaining at a distance compatible with the formation of a hydrogen bond with the resulting serinate (minimum B in Figure 5A). In terms of energy values, the estimated activation free energy (ΔF#) from the starting geometry (A) to energy minimum B was 20.5 kcal/mol, while the energy difference (ΔF) between A and B minima was 18.6 kcal/mol. Conversely, minimum B was slightly more energy favored than TS1, because of the estimated low energy difference with TS1 (1.9 kcal/mol). This suggests that the nucleophilic attack of Ser79 on Mer could start as TS1 is formed. We investigated this step by US2, in which the difference between (i) the distance from the Ser79 side chain oxygen atom to the C6 atom of Mer and (ii) the distance between O7 and N1 of Mer was taken as the CV. The free energy profile resulting from US2 simulations again showed two energy minima (Figure 5B): the first corresponding to the starting
entire simulation, was 1.25 Å (Figure S1). Mer occupied the catalytic site for the entire simulation time, conserving the same binding mode observed in the starting complex. In this respect, the carboxylic group of Mer was found to bind to OXA-23 by a salt bridge involving Arg259 of OXA-23.13 Furthermore, this same group of Mer was stabilized by two hydrogen bonds with the side chains of the enzyme, Ser126 and Thr217. Also, the oxygen atom of the β-lactam ring [O8 (Figure 1B)] created two hydrogen bonds, the first with the backbone of Trp219 and the second with the side chain of Ser79 (Figure 2). The side chain of Kcx82 stably interacted with the indole ring of Trp165 through hydrogen bonding, as observed in the X-ray structures.
Figure 2. Representative configuration of the Mer binding geometry extracted from the last frame of the 50 ns MD simulations. For the sake of clarity, the protein is represented as a ribbon, while Mer (cyan) and some key residues are represented as sticks. The hydrogen bonds made by Mer and OXA-23 are represented by dashed yellow lines.
A more thorough estimation of the molecular determinants of the Mer−OXA-23 recognition process was achieved by analyzing the MD simulation outputs by the MM-GBSA energy decomposition method,58 which allowed the decomposition of the total ligand−protein interaction energy into the sum of pairwise residue−ligand energetic contributions. The results of these calculations (Figure 3) confirmed that binding of the Mer carboxylic group with the positively charged region defined by Arg259 and Lys216 represents the most relevant interaction. Other significant contributions are given by the hydrogen bond interactions between the same carboxylic group of Mer and the OXA-23 Thr217 and Ser126 residues.
Figure 3. MM-GBSA energy decomposition. The contribution of the most relevant residues to the Mer−OXA-23 interaction energy is reported. Standard deviations are shown as error bars. 5194
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Figure 4. Proposed mechanism for the covalent binding and hydrolysis of Meropenem by OXA-23.
Figure 5. PMF obtained from (A) US1 and (B) US2. We verified that the statistical uncertainty of the energy profile never exceeds 0.2 or 0.5 kcal/ mol for US1 or US2, respectively.
was found to be exothermic, with an estimated ΔF# of 11.3 kcal/mol and a ΔF of −33.6 kcal/mol. In addition, it is important to stress that the formation of 2 is irreversible, because the inverse reaction is prevented by an energy barrier close to 45 kcal/mol. Transfer of a Proton from a Water Molecule to Mer with the Assistance of Lys216 (step 3). The step following formation of the covalent adduct was the neutralization of the
geometry (A in Figure 5B) and the second on which the covalent OXA-23−Mer adduct was formed (B in Figure 5B). Here, the β-lactam ring was cleaved, and the negative charge on the Mer N1 atom was stabilized by a hydrogen bond network involving the side chains of Ser126 and Lys216. It is plausible to hypothesize that these interactions are central in assisting the βlactam ring hydrolysis, weakening the C7−N1 bond during the nucleophilic attack on serinate. Also, this latter reaction step 5195
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Figure 6. Potential of mean force obtained by umbrella sampling for step 3. We verified that the statistical uncertainty of the energy profile never exceeds 0.2 kcal/mol.
Figure 7. Potential of mean force obtained by umbrella sampling for step 4. We verified that the statistical uncertainty of the energy profile never exceeds 0.5 kcal/mol.
Lys216 and the water oxygen atom to values of 10 kcal/mol. General Overview of the Hydrolysis Mechanism. Through US calculations, we computationally determined the most plausible mechanism of hydrolysis of Mer by OXA-23. Our data indicate that the catalytic cycle proceeds in four stages: (1) the deprotonation of Ser79 by Kcx82, (2) the nucleophilic attack on the Mer β-lactam ring by the previously generated serinate, (3) the neutralization of the Mer negative charge by a Lys216-stabilized water molecule, and (4) the nucleophilic attack on the Mer−Ser79 ester group by the hydroxide ion from the previous step, followed by release of the hydrolyzed form of carbapenem. As a final remark, the role of both Kcx82 and Lys216 should be further stressed. The first residue acts as a general base and is involved in the activation of Ser79 as well as in assisting the final hydrolysis of the Mer−Ser79 covalent bond by interacting with a water molecule. On the other hand, Lys216 supports the β-lactam ring breakage during the nucleophilic attack by Ser79, creating a hydrogen bond with the side chain of Ser126. Additionally, Lys216 is also crucial in the Mer negative charge neutralization cooperating with a water molecule. Our simulations allowed prediction of the energy balance for each reaction step. The estimated reaction free energy profile (Figure 8) indicates that the entire catalytic cycle is favored by an overall energy gain of approximately 11 kcal/mol. Intermediate 2, representing the anionic covalent complex, was found to be more stable than the neutral acyl−enzyme 3. The electrostatic contribution to the free energy might be greatly influenced by the hydrogen bond network involving Ser126 and, mostly, the positively charged Lys216. In fact, the latter residue could act like the zinc ion in metallo-BLs, in which the catalytic cation significantly stabilizes the anionic acyl−enzyme intermediate.68 From a kinetic point of view, the available data imply that the formation of the acyl−enzyme intermediate is rather fast (kcat/ Km ≥ 6.8 × 104), whereas the reaction rate, from the formation of the Michaelis complex to the final deacylation step, is quite slow (kcat = 0.068 s−1). Our computations suggest that two steps, requiring the highest activation energy, could each represent the rate-determining step, i.e., (i) the Ser79 deprotonation (20.5 kcal/mol) and (ii) the final deacylation
atom. Although such a conversion could be observed with an increase in the simulation time, the formation of the less reactive tautomer is more likely a consequence of the conditions used for acyl−enzyme crystallization (i.e., the nonneutral environment). Therefore, given the lack of experimental evidence of Mer tautomerization at the end of the enzymatic reaction coupled with the difficulty of investigating the pH effect during MD simulations, the effect of this presumed tautomerization step on the overall energy profile is still a matter of debate. Final Hydrolysis of the Acyl−Enzyme Complex (step 4). We hypothesized that the hydrolysis mechanism would proceed with retention of the more hydrolysis-prone Δ2pyrroline tautomer. Next, a nucleophilic attack by the hydroxide ion, formed in the previous step, on the carbonyl atom of Mer leads to the hydrolysis of the Mer−Ser79 bond (Figure 7) with the release of the inactive form of the antibiotic. For this reaction step, to estimate only the energy profile associated with the hydroxide nucleophilic attack on Mer, we did not take into account the unproductive hydroxide ion neutralization by Kcx82. To such an end, we constrained the distance difference between (i) the O and H atoms of Kcx82 and (ii) the H atoms of Kcx82 and the water oxygen atom to values of
