Pleotropic potential of quorum sensing mediated N-acyl homoserine lactones (AHLs) at the LasR and RhlR receptors of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen, having complicated quorum sensing (QS) system utilizing multiple signals and receptors to coordinate virulence and pathogenicity. N-acyl-homoserine-lactones (AHLs) are the most common autoinducers responsible for regulation of QS-mediated virulence gene expression. There are four QS systems in P. aeruginosa among which the LasI/R and RhlI/R systems are regulated by 3-oxo-C12-HSL and C4-HSL respectively. They play a major role in host-associated pathogenesis. The LasR and RhlR binding specificity to cognate or non-cognate HSLs influences the QS-mediated responses. Here, we used computational approaches to consolidate the interaction of different types of HSLs produced by P. aeruginosa with LasR and RhlR receptors. To explore the binding affinity, fourteen different AHLs were subjected for molecular docking analysis with LasR and RhlR receptors. The RhlR was modelled using MMseqs2 in ColabFold: Alpha fold 2. Further, to validate the stability and interaction mechanism, molecular dynamic simulations was performed with the top docked six HSLs for 100 ns. In docking results, apart from 3-oxo-C12-HSL and C4-HSL, other HSLs such as C16-HSL and C6-HSL showed better binding affinity towards LasR and RhlR proteins, respectively. Further validation by molecular dynamic simulations showed that 3-oxo-C10-HSL and 3-oxo-C6-HSL formed stable complex with LasR and RhlR, respectively. Our comprehensive in silico study results may provide promising targets for development of anti-QS drugs against Las/Rhl QS systems.


Introduction
Pseudomonas aeruginosa is an opportunistic gram-negative bacterium causing infections in compromised immune systems [1]. It is the most commonly isolated nosocomial pathogen, associated with urinary tract infections, chronic wounds, septicaemia and respiratory infections such as cystic fibrosis and chronic obstructive pulmonary disease [2][3][4][5][6]. This bacterium induces pathogenicity through the production of virulence dominated by quorum sensing (QS) signaling mechanism [7]. The QS allows bacteria to monitor their population density and coordinate changes in their behaviour at a threshold concentration of autoinducers [8]. It facilitates the bacterial colonization and establishment of infection using multiple autoinducers like N-acylhomoserine lactones (AHL), Pseudomonas quinolone signal (PQS), integrating quorum sensing (IQS) and autoinducer-2 (AI-2) signaling systems for intra-and interspecies 1 3 communication [9,10]. In the microbial community, the functional autoinducers are prone to exploitation by the non-producing cheaters. For example, the mutants that lack the LasI synthase responds to 3-oxo-C 12 -homoserine lactone produced by the wild type of P. aeruginosa outcompeting the wild-type population [11]. In host system, QS by one species can be influenced by another species in the mixed communities which modulate the host immune responses [12] where the autoinducers play a key role in competitive interactions with the receptors.
The AHLs play a crucial role in regulating virulence factor gene expression, biofilm formation and antibiotic resistance efflux pumps [7,13]. In P. aeruginosa, AHL-mediated QS system is controlled by two major LuxR/I homologues (LasR/I and RhlR/I) where transcriptional regulators LasR and RhlR are activated through N- (3-oxo-dodecanoyl) homoserine lactone (3-oxo-C 12 -HSL) and N-butanoyl homoserine lactone (C 4 -HSL), respectively. These are generated by the LasI and RhlI synthases, respectively [14]. The substitution of the homoserine lactone (HSL) ring in the N-acylated β-and γ-sites with a fatty acyl group at the α-position is the typical characteristic of AHLs. The acyl chain differs with varying carbons from C 2 to C 20 in length, saturation level and oxidation state [15]. Even though LasI primarily synthesizes 3-oxo-C 12 -HSL, at considerably lower concentrations, the analogues 3-oxo-C 10 -HSL and 3-oxo-C 14 -HSL are also produced [16]. The Las and Rhl QS systems are coordinated in a hierarchy where LasR/3oxo-C 12 -HSL regulates the expression of lasI, constituting a positive feedback loop regulating the expression of rhlR and rhlI. The LasI/R-mediated QS system controls virulence factors associated with host cell damage and acute infection through production of exotoxins, elastases and protease during infection progression [17,18]. Similarly, RhlR produces siderophores, rhamnolipids, pyocyanin, lectins and hydrogen cyanide and represses those responsible for the type III secretion system (T3SS) [19,20]. The 3-oxo-C 12 -HSL in P. aeruginosa acts on intracellular nuclear peroxisome proliferator-activated receptors (PPAR) transcriptional regulators using Nuclear factor kappa B (NF-κB) signaling [21]. Several studies reported the role of 3-oxo-C 12 -HSL related analogues (i.e., 3-oxo-C 10 -HSL and 3-oxo-C 14 -HSL) in lipid bilayer insertion in T-lymphocyte membrane [22]. Since LasR-3-oxo-C 12 -HSL stimulates both PQS and Rhl QS machinery, these systems represent a hierarchical activation cascade that is triggered by attainment of a threshold cell density. On the other hand, a few studies have reported the presence of other AHLs in P. aeruginosa cultures including C 16 -HSL, C 6 -HSL, C 8 -HSL, 3-oxo-C 10 -HSL and C 18 -HSL, at low concentrations in the AHL profile [23]. Furthermore, studies have reported the detection of C 6 -HSL, C 8 -HSL, C 10 -HSL, 3-oxo-C 6 -HSL and 3-oxo-C 10 -HSL from sputum samples colonised by P. aeruginosa of cystic fibrosis patients [24,25]. Since the AHLs have analogous structure, the concern is how bacteria discriminate among them and compete for the binding site on receptor and regulate the virulence phenotypes.
The presence of these multiple AHLs in the P. aeruginosa's environment and associated pathogenesis during the QS process is still unclear. There is increased interest in studies using the molecular docking approaches to evaluate the molecular interaction of QS receptor and autoinducers, where LasR and RhlR interactions with 3-oxo-C 12 -HSL and C 4 -HSL, respectively, are often explored in P. aeruginosa [26,27]. The crystallography structure analyses of the factors driving ligand accommodation by LasR are enhanced when LasR ligand binding domain is bound to its cognate HSL, noncognate HSLs, and other agonists [28,29]. To gain a clear insight into the dynamic patterns of AHLs at the LasR and RhlR active sites and to confirm stability, molecular dynamic (MD) simulation was carried out for the scrutinised AHLs, based on the docking score, binding free energy and nature of interacting amino acid residues using Schrödinger suite. This study results will comprehend how other AHLs present in the environment interact with receptors and with host factors to regulate virulence. This can contribute to identify molecules with similar chemical behaviour to that of AHLs in designing and development of rational anti-QS agents.

LasR and RhlR protein preparation, refinement and validation
The three-dimensional crystallographic structure of LasR in association with P. aeruginosa N- (3-oxo-dodecanoyl) homoserine lactone was downloaded from protein data bank (PDB) under the accession number PDBID: 4NG2. The retrieved structure was prepared using Schrodinger suite's "Protein Preparation Wizard" (Schrödinger, LLC, New York, NY, 2019-1). In brief, the 4NG2 was preprocessed to add hydrogen bonds and refine missing side chains and loops. The water molecules in hetero groups greater than 5 Å were eliminated, and hetero states were created using EpiK at pH 7 ± 2.0. The generated heterostates with the lowest state penalty scores were chosen for further refining. The resultant LasR protein structure was further improved using PROPKA at pH 7 ± 2.0 and the OPLS-3e force field. Finally, the prepared complex was validated using a Ramachandran plot of the phi/psi ratio.

Homology modelling for RhlR
The protein sequence of the regulatory protein RhlR, was acquired from Uniprot (UniProtKB -P54292) and modelled using MMseqs2 in ColabFold: Alpha fold 2 [30]. The RhlR protein modelling was carried out in auto (paired + unpaired) mode with three recycles. The PDB-formatted structures were scored using the average pLLDT method, and the topranked modelled structures of RhlR were submitted to the "Protein preparation Wizard" as described previously for LasR. The Ramachandran plot was used to validate the RhlR structure.

Molecular docking
The docking process was validated prior to docking by redocking the prepared LasR with its native ligand, and the RMSD was calculated by superimposing its substructures (RMSD < 1). The receptor grid for LasR was built for molecular docking by choosing its native ligand at its binding site. Furthermore, Van der Waals radius scaling was maintained with a scaling factor of one and a partial cutoff of 0.25. The ligand size was kept comparable to the size of the native ligand during docking. Rotatable groups in active site residues, such as hydroxyls and thiols, were permitted to rotate during the docking procedure.
The receptor grid for RhlR was created by selecting active site residues. As LasR and RhlR are homologues, the RhlR active site residues were determined using sequence alignment in NCBI Protein alignment blast between LasR and RhlR. LasR's major active site residues (TYR56, TRP60, ASP73 and SER129) were linked with RhlR's (TYR64, TRP68, ASP81 and SER135). Docking ligands for RhlR were restricted to an enclosed box with RhlR active site residues that matched. Finally, the receptor grid for docking for RhlR was generated in the same way as it was for LasR. GLIDE V7.7 (Glide, Schrödinger, LLC, New York, NY, 2019-1) was used to execute flexible docking in the extra precision mode for HSLs using LasR and RhlR posing docking threshold constraint of 0.50 kcal/mol. The docked HSLs were examined and ranked based on binding affinity and interaction type.

Calculation of binding free energy (Prime MM-GBSA)
The Prime MM-GBSA for docked complexes of LasR and RhlR with HSLs were performed using the Schrodinger suite's Prime module V 3.5 (Prime, Schrödinger, LLC, New York, NY, 2019-1). In brief, the docked complexes were separated as ligand and protein and used as the input structure for calculating binding free energy (ΔG bind). The solvation model form-GBSA was VSGB, and the forcefield was kept as OPLS-3e. The following equation was used to compute the binding free energy.
where ΔEMM is the variance between the minimized energies of the protein-ligand complex and sum of minimized energies of protein and its inhibitor; ΔGSolv denotes the difference between the GBSA solvation energies of proteininhibitor complex and sum of GBSA solvation energies of protein and its inhibitor; and ΔGSA denotes the difference between the surface area energies of protein-inhibitor complex and sum of surface area of protein and its inhibitor.

Molecular dynamics (MD) simulation
The binding stability, conformation and interaction mechanisms of the chosen AHLs with LasR and RhlR binding sites were determined using molecular dynamics simulation. GROMACS 2019.2 (http:// www. groma cs. org) software was used to simulate molecular dynamics. In brief, for molecular dynamic simulation, the vacuum was reduced for 5000 steps using the steepest descent algorithm. A simple point charge (SPC) water model was used to solve the complex structure in a cubic periodic box of 0.5 nm. The complex system was then maintained at an adequate salt concentration of 0.15 M by adding an appropriate quantity of Na + and Cl counter ions. Each complex was given a simulation time of 100 ns for the final run from the NPT (Isothermal-Isobaric, constant number of particles, pressure and temperature) equilibration. The GROMACS simulation package was used to perform trajectory analysis using the online server "WebGRO for Macromolecular Simulations" (https:// simlab. uams. edu/).

Results and discussion
The opportunistic pathogen P. aeruginosa is a leading cause of nosocomial infections worldwide frequently accompanied by clinical treatment difficulties [31]. P.

ΔGbind = ΔEMM + ΔGSolv + ΔGSA
aeruginosa uses a hierarchically organised and linked QS regulatory systems to produce a slew of virulence factors such as elastase, phenazines and rhamnolipids by transcription of AHL-receptor complex [32,33]. The QS transcription factor lasR is at the top of this hierarchy, where it promotes the transcription of multiple genes, including the one encoding the QS regulator RhlR [10]. Paradoxically, inactivating lasR mutations are commonly reported in isolates from individuals with persistent P. aeruginosa infections [34]. The I mutations, on the other hand, are uncommon. It was recently found that RhlR operates independently of LasR [35]. As a result, understanding the molecular basis of LasR and RhlR interaction with AHLs is important to study host-pathogen interactions.

LasR active site in P. aeruginosa
To gain insight into the LasR and HSLs binding mechanism in P. aeruginosa QS system, the refined LasR protein structure was extensively examined using the Schrodinger module's "Sitemap" feature. The sitemap analysis revealed the presence of two active sites in LasR, each with a DScore (druggability score) of 1.193 and 0.730. The active site (site 1) of LasR is depicted in Fig. 1A, which corresponds to the co-crystallized structure of 3-oxo-C 12 -HSL with LasR. The AHLs are an assembly of amphipathic small molecules comprised of a hydrophilic homoserine lactone ring and a hydrophobic acyl side chain, produced using S-adenosylmethionine and acyl-acyl carrier protein (acyl-ACP) as the bases for HSL Fig. 1 A) The active site of LasR in complex with its co-crystallized ligand 3-oxo-C 12 -HSL; B) the homology-modelled RhlR protein; C) the predicted active site of RhlR in complex with C 4 -HSL; and D) the structural alignment of LasR (green) and RhlR (red) and their active site localization. Yellow pocket of the active site represents hydrophobic core, red zone represents H-bond acceptor region, and blue zone represent H-bond donor region ring moiety and acyl side chain, respectively [17]. When the concentration of AHLs in the environment increases, at a threshold population density, the intracellular levels of AHLs are sufficient to maximally induce the activation of LuxR, which contains a C-terminal DNA-binding domain and an N-terminal acyl-HSL-binding domain [36]. The active site of LasR is formed by the amino acid residues 36 The hydrophobic region of the active site is represented by the yellow pocket, which is occupied by the dodecanamide group. In contrast, the donor region (blue pocket) is occupied by the polar hydrogen of the amide nitrogen in dodecanamide, which mediates H-bond interaction with the negatively charged ASP73 amino acid residue. Carbonyl groups from the ligand occupy the active site's H-bond acceptor region (red pocket), facilitating interactions with TYR56, TRP60, and SER129 amino acid residues. It can be deduced that the hydrophobic side chains of the AHLs are involved in the ligand's structural stability, while the donors and acceptor groups are involved in the ligand's intrinsic activity toward LasR.

Homology modelling of RhlR and its active site
The RhlR protein was modelled using CoFold Alpha Fold 2.0, as indicated in Fig. 1B. The prepared and refined RhlR structure was validated by "PROCHEK" analysis by inspecting the phi/psi distribution of the Ramachandran Plot (Supplementary Fig. 1). The sitemap analysis performed for RhlR revealed the presence of two active sites, with D scores of 1.102 and 0.646 for site 1 and site 2, respectively. Since LasR and RhlR shares comparable homology, we superimposed their substructures for their active site localisation and the results showed that both proteins have a similar active site region localisation (Fig. 1D) with an RMSD of 2.1 Å. Furthermore, the active site of RhlR contains a hydrophobic pocket, an H-bond acceptor zone, and an H-bond donor zone (Fig. 1C).

Docking and molecular mechanics-generalized born surface area (MM-GBSA) calculation
In this study, the prepared AHLs were docked flexibly to the active sites of LasR and RhlR in the extra precision mode in Schrodinger's Glide module. The docking and binding free energies of AHLs for LasR and RhlR are shown in Tables 1  and 2 and Fig. 2, respectively. Interestingly, 3-oxo-C 12 HSL ranked second with a docking score of − 9.430 kcal/mol, whereas C 16 -HSL rated first with a binding affinity of − 10.89 kcal/mol. The 3-oxo-C 10 -HSL and C 12 -HSL had a docking score of − 9.327 and − 9.235 kcal/mol, respectively. According to the docking analysis, C 16 -HSL has the most significant binding affinity towards LasR, followed by 3-oxo-C 12 -HSL, 3-oxo-C 10 -HSL and C 12 -HSL. Based on the binding free energy (BFE), the subsequent analysis revealed that 3-oxo-C 12 HSL has shown a ΔG score of − 93.05, followed by C 16 -HSL and 3-oxo-C 10 -HSL with scores of − 83.14 and − 79.25, respectively. Overall comprehensive analysis results indicate that the activity of 3-oxo-C 12 -HSL is substantially more significant due to the binding free energy, although it has a lower binding affinity than C 16 -HSL and 3-oxo-C 10 -HSL. The detection of HSL physiological QS concentration is complicated due to their susceptibility to alkaline or enzymatic hydrolysis. Since the detectable QS concentration is 600 µM in patient samples, the role of other AHLs is underestimated due to low detection limit [16]. Considering the prevalence of these AHLs in the samples, we have explored their pleotropic role in LasR and RhlR mediated interactions using a molecular approach. McCready et al. (2019) investigated whether P. aeruginosa at high cell density is likely to compete with other AHLs than 3-oxo-C 12 -HSL through molecular studies and found that a flexible loop is present in LasR which has the ability to bind to multiple AHLs [35].
Based on this, we then explored the interaction patterns of the top-ranked AHLs based on the nature of the interaction. The C 16 -HSL mediated H-bond acceptor interaction with amino acid residues TRP60, TYR56 and SER129 and H-bond donor interaction with ASP73. These interactions were comparable to those seen with 3-oxo-C 12 -HSL and 3-oxo-C 6 -HSL. However, 3-oxo-C 12 -HSL and 3-oxo-C 10 -HSL facilitated an extra water molecule mediated H-bond interaction with ARG61. Based on C 16 -HSL occupancy at the active site of LasR, it was observed that the C-16 side chain filled the majority of the hydrophobic core, which may mediate the stability of the AHL for consistent interaction at the active site. The occupancy of the carbon side chain at the hydrophobic location was restricted to 3-oxo-C 12 -HSL and 3-oxo-C 10 -HSL (Fig. 3A). This could explain the comparable low binding affinity of AHLs for LasR.
Docking and binding free energy calculations for AHLs towards RhlR indicated an intriguing result, with the extensively studied AHL: C 4 -HSL being the least preferred AHL towards RhlR with a binding affinity of − 6.930 kcal/mol when compared to C 6 -HSL, 3-oxo-C 6 -HSL, C 8 -HSL and 3-OH-C 8 -HSL with docking scores of − 8.167, − 8.121, − 7.264 and − 7.247 kcal/mol, respectively. Further study based on binding free energy inspection revealed that 3-oxo-C 6 -HSL ranked top with a ΔG score of − 53.87, followed by C 6 -HSL (− 47.41) and C 4 -HSL (− 43.16). Furthermore, the nature of C 6 -HSL, 3-oxo-C 6 -HSL and C 4 -HSL interactions demonstrated H-bond acceptor interactions at TPR68 and TYR64 amino acid residues and H-bond donor interactions at ASP81 amino acid residue. C 4 -HSL, on the other hand, has an extra H-bond donor interaction with SER135 of RhlR. Overall, it is reasonable to infer that the hydrophobicity of the AHLs and secondary carbonyl group interactions are important in influencing binding affinity towards RhlR. Active site occupancy of 3-oxo-C 6 -HSL and C 4 -HSL indicated that the 3-oxo-C 6 -HSL skeleton occupies a larger hydrophobic zone than C 4 -HSL (Fig. 3B). An earlier study results suggest that LasR preferentially detects long chain AHLs in a mixed environment of autoinducers, with high binding efficiency towards 3-oxo-C 12 -HSL, closely followed by 3-oxo-C 14 -HSL, where SER129 ligand binding domain plays a crucial role in LasR [29].

Molecular dynamic simulation AHLs/ LasR complexes
To gain a better understanding of the dynamic patterns of AHLs at the LasR and RhlR active sites, molecular dynamic simulation was carried out for the scrutinised AHLs, based on the docking score, binding free energy and nature of interacting amino acid residues. We have chosen 3-oxo-C 12 HSL (extensively studied) as the benchmark for comparison with 3-oxo-C 10 -HSL and C 12 -HSL for LasR. For RhlR, C 4 -HSL was chosen as the standard for comparison with C 6 -HSL and 3-oxo-C 6 -HSL. A dynamic simulation was performed for 100 ns, for each of the six complexes. The root mean square deviation (RMSD), root mean square fluctuation (RMSF), H-bond interaction and solvent accessible surface area (SASA) were computed and compared.
Since the RMSD is an important component of complex equilibration and stability during the simulation run, the stability of the structural complexes was determined by plotting the backbone RMSD acquired during the production run. The computed RMSD profile for 3-oxo-C 12 -HSL/ LasR showed an average RMSD of 0.240 nm. Similarly, the average RMSD for C 16 -HSL/LasR and 3-oxo-C 10 -HSL/ LasR were 0.243 nm and 0.193 nm, respectively (Table 3). The 3-oxo-C 10 -HSL/LasR complex resulted in a significant reduction in RMSD throughout the simulation run (Fig. 4A). Thus, the stability of the regular complex formation is demonstrated. RMSD analysis indicated that, compared to the extensively studied 3-oxo-C 12 -HSL complex, the 3-oxo-C 10 -HSL complex displayed a steady RMSD and  16 -HSL binds to LasR in elastase biosynthesis regulation involved in functional analysis of genes required for the biosynthesis of pyocyanin and phenazine-1-carboxamide in P. aeruginosa PAO1 [37]. The molecular dynamic simulations depicted that 3-oxo-C 10 -HSL formed a stable complex with LasR compared to 3-oxo-C 12 -HSL by establishing strong hydrogen and hydrophobic interactions. Ortori et al. (2011) reported that 3-oxo-C 10 -HSL was identified in P. aeruginosa culture in LC MS/MS analysis [23]. The docking analysis and molecular simulations of 3-oxo-C 10 -HSL for LasR and 3-oxo-C 6 -HSL for RhlR demonstrated that it has a pleotropic role in QS mechanism. Subsequent examination of the backbone RMSF indicated that the 3-oxo-C 12 -HSL/LasR complex had an average RMSF of 0.116 nm, whereas C 16 -HSL/LasR and 3-oxo-C 10 -HSL/LasR demonstrated an average RMSF of 0.143 and 0.107 nm, respectively (Table 3) (Fig. 4B). From the RMSF graph, it is observed that complexes 3-oxo-C 12 -HSL and C 16 -HSL exhibited greater fluctuation across the complex than 3-oxo-C 10 -HSL. Additionally, the RMSF of active site residues in respective complex systems were compared with the RMSF of the active site residues in 3-oxo-C 12 -HSL/ LasR (standard). The ASP73 residues of 3-oxo-C 10 -HSL/ LasR substantially decreased fluctuation than the standard and C 16 -HSL complexes. Similarly, decrease in fluctuation was observed with amino acid residue SER129 of 3-oxo-C 10 -HSL/LasR compared to the other two complexes. Other active sites retained a similar fluctuation across the run for all the complexes (Table S1). The overall study results suggest that interaction between LasR's (ASP73, and SER129) with HSLs could stabilise the potential complex formation.
The binding free energy (ΔG bind) of all three complexes was calculated using MM/PBSA method. The BFE values for 3-oxo-C 12 -HSL, C 16 -HSL and 3-oxo-C 10 -HSL were − 130.19 ± 11.721, − 216.54 ± 17.043 and − 180.655 ± 17.165, respectively (Table S2). From these values, it can be concluded that C 16 -HSL and 3-oxo-C 10 -HSL possess strong stabilization potential in the complex formation. In addition, pre-residue energy decomposition analysis of the MD simulation derived equilibrated trajectory of three structural complexes was also estimated using MM/PBSA. Our findings indicate that LasR residues TYR56, TRP60, ASP73 and SER129 are the most critical residues for complex formation (Table S3). From the MM/ PBSA analysis, it was perceived that TYR56 and TRP60 amino acid residues were majorly contributed to the binding complexes.  Additionally, SASA analysis was performed for all the complexes. SASA is a criterion for determining the extent of protein exposure to the surrounding solvent molecules during simulation. In general, ligand binding can cause structural changes in the receptor, altering the area in contact with the solvents. To estimate the change in surface area, the SASA values of the three complexes were plotted against the time. From the SASA graph, it was observed that 3-oxo-C 10 -HSL exhibited a considerable decrease in the SASA, followed by C 16 -HSL and 3-oxo-C 12 -HSL complexes (Fig. 4C). The overall analysis revealed that proteins in all complexes were shrunken during the entire simulation.
The binding mode of these three complexes was analysed by selecting a representative pose from the final 5 ns of a simulation run. The more stable 3-oxo-C 10 -HSL complex established strong molecular interactions characterised by hydrogen and hydrophobic interactions. The TRP60, TYR56 and ARG 61 contribute to H-bond donor interaction and ILE52, LEU36, TYR47 and VAL76 contribute to hydrophobic interaction (Fig. 4D). The aforementioned interactions suggest that 3-oxo-C 10 -HSL has shown a strong binding to LasR.

Molecular dynamic simulation HSLs/RhlR complexes
A molecular dynamic simulation with C 4 -HSL, C 6 -HSL and 3-oxo-C 6 -HSL complexes of RhlR was performed. The average backbone RMSD of these complexes was 0.438, 0.381 and 0.328 nm, respectively (Table 3) (Fig. 5A). The 3-oxo-C 6 -HSL/RhlR complex resulted in a significant reduction in RMSD over the run compared to the other two complexes, indicating that a stable complex formation by 3-oxo-C 6 -HSL with RhlR. Subsequent analysis of the backbone RMSF indicated an average RMSF of 0.182, 0.199 and 0.206 for C 4 -HSL, C 6 -HSL and 3-oxo-C 6 -HSL complexes, respectively (Table 3) (Fig. 5B). From the RMSF graph, it was observed that all the three complexes had minor fluctuations over the course of run. However, the C 4 -HSL complex exhibited a relatively modest fluctuation compared to other two complexes. The RMSF data shows that TYR64 residue of the C 6 -HSL/RhlR complex rendered a substantial decrease in mean fluctuation compared to fluctuation at the other two complexes. In addition, TRP68, ASP81 and SER135 fluctuate relatively less in C 6 -HSL than in other two complexes (Table S1). Our results indicate that the amino acid residue TYR64 is crucial in stabilising complex formation.
Further, BFE analysis exhibited a score of − 56.08 ± 9.268 , − 60.988 ± 16.66 and − 69.25 ± 15.15 for C 4 -HSL, C 6 -HSL and 3-oxo-C 6 -HSL, respectively (Table S2). From these values, it can be concluded that 3-oxo-C 6 -HSL possesses strong stabilization in the complex formation, followed by Close-up view of intermolecular interaction between 3-oxo-C 6 -HSL and RhlR. Interacting residues are represented in stick form. Green and pink lines denote hydrogen and hydrophobic interactions, respectively C 6 -HSL and C 4 -HSL. Moreover, RhlR residues TYR64, TRP68, ASP81 and SER135 are critical for complex formation and activity. From pre-residues energy decomposition analysis, it was perceived that TYR64 and TRP68 contribute majorly to the binding of HSLs to RhlR (Table S3). Additional analysis of SASA revealed that all complexes were shrunken during the entire simulation run. It was also observed that C 4 -HSL exhibited a considerable decrease in SASA followed by 3-oxo-C 6 -HSL and C 6 -HSL (Fig. 5C). Furthermore, the binding mode of relative more stable complex 3-oxo-C 6 -HSL established a strong interaction for H-bond and hydrophobic interaction. The amino acid residues TYR64, TRP68 and SER135 contribute to H-bond interactions, and the amino acid residues ALA83, PHE101, LAI11 and TRP108 contribute to hydrophobic interactions. The above-mentioned interaction suggests a strong binding of 3-oxo-C 6 -HSL at the RhlR binding site.

Pleotropic role of AHLs at LASR and RhlR binding sites
The initial scrutiny of 3-oxo-C 10 -HSL for LasR and 3-oxo-C 6 -HSL for RhlR from molecular docking and dynamics studies helped to assess the pleiotropic effect of top-ranked AHLs towards both LasR and RhlR. Venn diagram was plotted for commonalities in AHLs towards both LasR and RhlR binding sites. The cut-off for the selection of AHLs towards both the receptors was kept at − 5 as the docking score and AHLs having negative binding free energy.
Based on the aforementioned criteria, thirteen AHLs were selected for LasR, and five AHLs were selected for RhlR ( Table 4). The commonality analysis shows that eight unique AHLs possess activity towards LasR. The 3-oxo-C 10 -HSL is shown that its one among the unique ligand for LasR. Further analysis exhibited five common AHLs (3-oxo-C 6 -HSL, 3-OH-C 8 -HSL, C 8 -HSL, C 6 -HSL and C 4 -HSL), to possess the activity for both LasR and RhlR (Fig. 6). Overall analysis suggests the pleotropic role of specific AHLs towards LasR and RhlR. The crystallography studies by McCready et al. (2021) show that LasR can accommodate long chain AHLs in their binding site, which is likely due to hydrogen bonding between ketone moiety and TRP-60 [35].
Despite the availability of the LasR crystalline structure, the 3D structural complex of RhlR with HSLs has yet to be understood. The development of new anti-QS agents will aid in the effective disruption of these regulatory proteins. Only a few clinical studies using azithromycin (NCT00610623) and BB536 (NCT01201577) have been done and yet to examine their QS inhibitory characteristics; hence, the development of improved probes to block the interaction of AHLs with LasR and RhlR for future implications is urgently required. This is the first study reported, by using in silico approach, the molecular basis for interactions of different AHLs with the  16 -HSL 3-oxo-C 6 -HSL 2 3-oxo-C 12 -HSL 3-oxo-C 6 -HSL 3-oxo-C 12 -HSL 3-OH-C 8 -HSL 3 3-oxo-C 10 -HSL C 8 -HSL 3-oxo-C 10 -HSL C 8 -HSL 4 C 12 -HSL 3-OH-C 8 -HSL C 12 -HSL C 6 -HSL 5 3-OH-C 12 -HSL C 4 -HSL 3-OH-C 12 -HSL C 4 -HSL 6 3-OH-C 10 -HSL 3-OH-C 10 -HSL 7 3-oxo-C 6 -HSL C 14 -HSL 8 C 14 -HSL C 10 -HSL 9 3-OH-C 8 -HSL 10 C 10 -HSL 11 C 8 -HSL 12 C 6 -HSL 13 C 4 -HSL Fig. 6 Venn diagram illustrating the activity of AHLs and demonstrating the overlapped and distinct AHLs for LasR and RhlR regulatory proteins LasR and RhlR in P. aeruginosa. Since these AHLs form stable complexes and are predominantly present in infectious samples, they need to be further validated using in vitro assays and exploited for their role in virulence gene expression. Our study results will pave the way for target specific examination of QS regulatory mechanisms for P. aeruginosa that is ubiquitous in distribution. Overall, our results show that LasR and RhlR can detect and bind to the noncognate long chain AHLs and can activate QS responses. The versatility of the AHL-receptor complexes may provide survival advantages for the bacteria in the complex environmental conditions.

Conclusion
The structural modification of AHLs to target the LasR/ RhlR receptor can be made for generating small molecule inhibitors that can impair the process of signal transmission and QS gene activation and regulation. Our findings support that 3-oxo-C 10 -HSL forms a stable complex with LasR and 3-oxo-C 6 -HSL forms a stable complex with RhlR, which will assist in the development of inhibitors that preferentially target the LasR/RhlR receptors.