Simultaneous molecular docking of different ligands to His₆-tagged organophosphorus hydrolase as an effective tool for assessing their effect on the enzyme

Introduction

To date, bacterial diseases are among the most common and hazardous to humans. While some bacteria play a vital role in ecology, others can become sources of dangerous human diseases, including fatal ones. Today, a number of antimicrobial agents are used to fight bacterial pathogenicity, the most important of which are various antibiotics (Von Döhren, 2004). The wide dissemination of pathogenic microorganisms has recently led to an uncontrolled increase in the amount of antibiotics consumed, which, in turn, has caused the development of bacterial strains resistant to the action of antimicrobial agents. The development of antibiotic-resistant pathogens has resulted in the loss of the action effectiveness of some antibiotics applied in practice and the subsequent need to increase their usage doses (Fischbach & Walsh, 2009). As a result, antibiotic resistance has become one of the major problems of our time, requiring the development of effective approaches to address the issue (Ventola, 2015).

The resistance mechanisms of most bacterial species are well studied today (Mah & O’toole, 2001). The Quorum Sensing (QS), mechanism describes the ability of bacterial cells to interact with each other within the same population to develop highly resistant antibiotic associations (Ng & Bassler, 2009). It is known that the majority of pathogenic gram-negative bacteria use signal molecules, acyl homoserine lactones (AHLs), as QS inducers. The synthesis of different AHLs is typical for various bacteria and the acyl chain length of AHLs usually ranges from C4 to C18 (Whitehead et al., 2001; Miller & Bassler, 2001). For example, common bacterial pathogens such as Pseudomonas aeruginosa, Burkholderia cepacia, and Yersinia enterocholitica secrete C12-, C8-, and C6- containing AHLs as QS signal molecules, respectively (Parsek & Greenberg, 2000).

It is supposed that one of the effective methods to overcome bacterial resistance may be the enzymatic degradation of the QS signal molecules of gram-negative bacteria. There are two basic groups among the known enzymes capable of hydrolyzing AHLs: (1) lactonases which eliminate ester bond in lactones that results in the lactone ring opening, and (2) acylases which hydrolyze an amide bond between the lactone ring and the acyl chain, that results in the production of homoserine lactone and fatty acids (Chen et al., 2013). The lactonases catalyzing the lactone ring cleavage in AHLs are of particular interest. Hexahistidine-tagged organophosphorus hydrolase (His₆-OPH) is one such enzyme. It was previously shown that His₆-OPH, in addition to its high catalytic activity in the process of hydrolysis of a number of organophosphorous compounds, also has lactonase activity against a number of AHLs (Maslova et al., 2017a; Maslova et al., 2017b). It has been established that combinations of His₆-OPH with β-lactam antibiotics provide an increase in the efficiency of action of both the enzyme and antibiotics (Maslova et al., 2017a; Maslova et al., 2017b; Aslanli, Lyagin & Efremenko, 2018). These results reinforce the interest in studying the possibility of combining His₆-OPH with other antibiotics characterized by different chemical properties and a spectrum of bacterial targets (Azzam & Algranati, 1973; Salmon, Watts & Yancey Jr, 1996). Recently, it was shown that in the presence of the enzyme, there is an observed decrease in the minimum inhibitory concentrations of the puromycin and ceftiofur against Pseudomonas sp. cells simultaneously producing different AHLs (Maslova et al., 2018). Therefore, it is of high scientific interest to determine the effect of these antibiotics on the catalytic activity of His₆-OPH.

The previous study (Aslanli, Lyagin & Efremenko, 2018) demonstrated and experimentally confirmed the possible application of molecular docking as a method for the simulation of models of the concurrently binded ligands to the single active site of the His₆-OPH enzyme and developing its combined preparations with β-lactam antibiotics. However, there is no available information about the dynamics between the protein and ligands in such models of combined biocatalytic systems. So, the originality of the present study is the use of computational methods to better understand the simultaneous interactions between His₆-OPH and different ligands being substrates or stabilizers. In this regard, the methods of molecular docking and molecular dynamics were applied in this work to simulate models of His₆-OPH in combination with antibiotics or/and AHLs to estimate the possible expanding the spectrum of antibiotics suitable for combining with the enzyme and evaluate its lactonase activity. These studies were carried out by using antibiotics such as puromycin and ceftiofur and different AHLs-like enzyme substrates (N-butyryl-DL-homoserine lactone (C4-HSL), N-(3-oxooctanoyl)-L-homoserine lactone (C8-HSL) and N-(3-oxododecanoyl)-L-homoserine lactone (C12-HSL)) naturally synthesized by various bacteria under real conditions (Fig. 1).

Materials & Methods

Results

Docking simulations

The interactions of C4-HSL, C8-HSL and C12-HSL with the active sites of enzyme dimer molecules were simulated in the presence of puromycin and ceftiofur docked to the surface of His₆-OPH at pH 7.5 (which corresponds to the physiological conditions of the antibiotics’ use) and 10.5 (which ensures the maximum catalytic activity of His₆-OPH) (Votchitseva et al., 2006) (Figs. 2 and 3).

Small, individual differences were shown in the His₆-OPH active sites when the relative position of the molecules of each of the antibiotics and the studied AHLs were taken in different combinations. The possibility of the intersection of AHLs with both antibiotic molecules was revealed when entering the enzyme’s active site. Moreover, the increase of AHLs acyl chain length was accompanied by an increase in the probability of its intersection with the antibiotics.

The binding energies of various ligands (AHLs and antibiotics) with the His₆-OPH dimer surface were calculated (Table 1).

Table 1:

Binding energy of ligands (antibiotics and AHLs) with His₆-OPH at different pH.

Scoring function named affinity (Trott & Olson, 2010) is an estimated minimum of potential energy during electrostatic and hydrophobic interactions, and hydrogen bonding between ligand (antibiotic or AHL) and receptor (enzyme).

Ligand	pH	Affinity, (kJx mol−1)				p value
		Mean	Median	Range
Puromycin	7.5	−29.9	−30.1  ± 0.8	−29.3	−30.5	0.317
	10.5	−30.3	−30.1  ± 1.0	−29.3	−31.0
Ceftiofur	7.5	−29.7	−29.3  ± 1.2	−29.0	−30.1	0.837
	10.5	−29.8	−29.5  ± 1.3	−29.0	−30.7
C4-HSL	7.5	−21.8	−21.9  ± 1.8	−23.1	−20.7	0.170
	10.5	−22.5	−22.7  ± 0.8	−22.7	−22.0
C8-HSL	7.5	−23.2	−23.2  ± 2.3	−23.9	−22.5	0.206
	10.5	−23.9	−23.4  ± 1.3	−24.5	−22.8
C12-HSL	7.5	−23.8	−23.7 ± 1.2	−24.4	−23.2	0.360
	10.5	−23.5	−23.0  ± 1.1	−24.3	−22.7

DOI: 10.7717/peerj.7684/table-1

Statistically significant differences (according to one-way ANOVA, p = 0.003) were observed between the values obtained for C4-HSL and C12-HSL, whereas there were no statistically significant differences between the values of the other groups (p ≥ 0.100). It has also been shown that the binding energies of AHLs with His₆-OPH were slightly higher in comparison with the binding energies of antibiotics with the enzyme. Therefore, the binding of antibiotics to the enzyme’s active site seems to be an energetically more beneficial process, which means that when competing with AHLs, they must displace the latter from the His₆-OPH dimer active sites.

Binding properties

To estimate how does the antibiotics binding to the surface of His₆-OPH affect the behavior of enzyme-substrate interactions, the root-mean-square-deviation (RMSD) and the root-mean-square-fluctation (RMSF) analysis were performed (Figs. 4 and 5). Same parameters established for His₆-OPH in enzyme-substrate complex with paraoxon being pesticide and the regular substrate of the enzyme (Votchitseva et al., 2006; Lyagin & Efremenko, 2019) were used as a reference in this analysis.

It is known that the lower the RMSD value, the better ligands position is fixed in the protein-ligand complex. According to Fig. 4 the steadiness of substrate position in the complex of His₆-OPH with AHL increases from C4-HSL to C12-HSL. In all cases, the combination of His₆-OPH with antibiotic led to an increase in the steadiness of AHLs position in enzyme-substrate complexes.

Significant fluctuations were observed for AHL atoms in enzyme-substrate complexes in the absence of antibiotics, except C8-HSL (Fig. 5). Evidently, combination of antibiotics with the His₆-OPH provides the fixation of the position of substrate molecule in the His₆-OPH-AHL complexes.

To quantify the strength of the interaction between AHLs and His₆-OPH in the absence and presence of antibiotics the interaction energies between enzyme and AHLs were calculated (Table 2).

Table 2:

Effect of His₆-OPH combination with antibiotics on the interaction energy of AHLs as substrates with the enzyme.

Enzyme or combinations	Interaction energy, kJx mol−1
	S1*	S2	S3
His6-OPH	−25.05  ± 7.38	−57.51  ± 12.08	−153.62  ± 8.60
His6-OPH/A1**	−72.27  ± 11.97	−227.60  ± 15.01	−138.44  ± 7.58
His6-OPH/A2	−86.60  ± 3.06	−109.52  ± 12.18	−129.67  ± 6.52

DOI: 10.7717/peerj.7684/table-2

Notes:

*S₁, S₂, S₃ are C4-, C8- and C12-HSLs, respectively.

**A₁, A₂ are puromycin and ceftiofur, respectively.

As a result, the highest interaction strength was observed for His₆-OPH-C8-HSL complex in the presence of pyromycin. The interaction strength of C12-HSL with all three tested enzymatic samples were high enough, whereas C4-HSL showed relatively low interaction strength.

To understand whether reaction of AHLs as substrates with His₆-OPH in the presence of antibiotics affects the interactions between enzyme and antibiotics, the interaction energies of antibiotics with His₆-OPH were calculated for His₆-OPH/antibiotic-AHL systems (Table 3).

Table 3:

Effect of enzyme-substrate binding on the interactions of His₆-OPH with antibiotics.

Substrate	Interaction energy, kJx mol−1
	His6-OPH/A1**	His6-OPH/A2
w/o substrate	−76.22  ± 5.34	−81.32  ± 7.44
S1*	−94.54 ± 5.52	−130.52 ± 28.32
S2	−11.73  ± 6.86	−139.09 ± 11.08
S3	−55.70  ± 6.38	−61.79  ± 11.83

DOI: 10.7717/peerj.7684/table-3

Notes:

*S₁, S₂, S₃ are C4-, C8- and C12-HSLs, respectively.

**A₁, A₂ are puromycin and ceftiofur, respectively.

The results presented in the Table 3 showed that ceftiofur binds with His₆-OPH stronger than puromycin in all cases. Formation of the His₆-OPH-C4-HSL complex in the presence of antibiotics led to increased interaction strength between enzyme and antibiotic, while complex with C12-HSL resulted in its decrease. In case of enzyme reaction with C8-HSL the interaction strength inside the combination His₆-OPH/ceftofur was increased and significantly decreased in His₆-OPH/puromycin.

Discussion

It is known that the closer and more accessible the lactone ring of the hydrolyzable AHLs is oriented to the active site and to the Co²⁺ ions that participate in the catalytic act of the enzyme, the higher the probability of the occurrence of effective enzymatic catalysis. In addition, the acyl chain of AHLs should be localized outside of the active site.

The docking of AHLs to the His₆-OPH dimer surface in the presence of some antibiotic (Figs. 2 and 3) showed that the latter can directly influence the orientation of the AHLs in the enzymatic active site and that the longer the acyl chain of AHLs, the greater the observed effect. When the molecules of both ligands are adjacent to the enzymatic active site, the antibiotics competes with the AHLs for localization in the same place, thereby affecting the efficiency of AHLs hydrolysis.

RMSD and RMSF were used to evaluate the interactions of the His₆-OPH with substrates (various AHLs) in the presence and absence of different antibiotics. The results of RMSD and RMSF analysis showed that the combination of His₆-OPH with both puromycin and ceftiofur leads to a significant fixation of the AHLs position in coordination with the active center of His₆-OPH in enzyme-substrate complexes. Moreover, the longer the acyl chain of AHLs, the more stable it appears (Fig. 4).

It is known that His₆-OPH exhibits the highest catalytic activity against paraoxon among the other organophosphorus substrates for the action of this enzyme (Lyagin & Efremenko, 2019). Based on this, the enzyme-substrate complex of His₆-OPH with paraoxon was used as a “reference standard” when comparing the results of RMSD and RMSF analysis obtained for His₆-OPH-AHL complexes. It has been established, that the combination of His₆-OPH with antibiotics leads to the same efficient hydrolysis of AHLs as paraoxon.

To assess how the presence of puromycin or ceftiofur affects the behavior of the enzyme-substrate interactions, the values of interaction energy between AHLs and His₆-OPH in the absence and presence of antibiotics were calculated. As a result, it was revealed that from a thermodynamic point of view, the longer the acyl chain of AHLs, the more favorable and, as a result, the more effective is a formation of the enzyme-substrate complex. The use of His₆-OPH/antibiotic combinations has proven thermodynamically to be more efficient for C4-HSL and C8-HSL hydrolysis as compared to C12-HSL, for which the most effective interaction was observed with native His₆-OPH. By that, the results of MD simulations confirmed the assumptions made on the basis of molecular docking: the combination of His₆-OPH with antibiotics leads to a direct influence on the enzyme-substrate binding and, therefore, on the efficiency of the enzymatic reaction. Moreover, the longer the acyl chain of AHLs, the pronounced is the affect of the antibiotic on the catalytic reaction, and the less effective is the interaction between the enzyme and the AHLs.

Conclusions

The results obtained in this study indicate that puromycin and ceftiofur, being combined with His₆-OPH, increase the steadiness of AHLs position in the enzyme-substrate complexes, but at the same time tested antibiotics reduce the effectiveness of enzymatic hydrolysis of long-chain AHLs as compared to the native enzyme. Therefore, this should be taken into account when creating a possible therapeutic composition based on combining antibiotics with His₆-OPH.

Based on the data obtained, it can be concluded that the computational methods such as molecular docking and molecular dynamic simulations can be further utilized for effective selection of possible partners among differently used antibiotics for enzymes with lactonase activity, like His₆-OPH, in order to create maximally efficient preparations with improved action against antibiotic-resistant bacterial cells.

Supplemental Information