Quorum sensing is a cell to cell signaling mechanism that enables them to coordinate their behaviors in a density-dependent manner mediated by small diffusible signaling molecules, which can control the virulence and biofilm gene expression in many Gram-negative and positive bacteria. N-acyl homoserine lactone acylase PvdQ from human opportunistic pathogen Pseudomonas aeruginosa is a quorum-quenching enzyme that can hydrolyze the amide bond of the quorum signaling N-acyl homoserine lactones (AHLs) thereby degrading the signaling molecules, turning off the biofilm phenotype and resulting in a reduction of bacterial virulence. Previous studies demonstrated that PvdQ has different preferences for N-acyl substrates with different acyl chain lengths and substituents. However, the substrate binding specificity determinants of the quorum-quenching enzyme PvdQ with the different bacterial ligands are unknown and unintuitive. Further, elucidation of these determinants can lead to mutants with efficiency and broader substrate promiscuity. To investigate this question, a computational study was carried out combining multiple molecular docking methods, molecular dynamics simulations, residue interaction network analysis, and binding free energy calculations. The main findings are: firstly, the results from pKa predictions support that the pKa of the N-terminus of Serβ1 was depressed due to the surrounding residues. Multiple molecular docking studies provide useful information about the detailed binding modes and binding affinities. Secondly, 300 ns molecular dynamics simulations were carried out to analyze the overall molecular motions of substrate-bound and substrate-free PvdQ. The specific interactions between the active site of PvdQ and different ligands revealed the determinants for the preference among the ligands. A systematic comparison and analysis of the protein dynamic fingerprint of each complex demonstrated that binding of the most favorable ligand, C12-homoserine lactone (C12-HSL), reduced the global motions of the complex and maintained the correct arrangement of the catalytic site. Further, the residue interaction network analysis of each system illustrated that there are more communication contacts and pathways between the residues in the C12-HSL complex as compared to complexes with the other ligands. The binding of the C12-HSL ligand facilitates structural communication between the two knobs and the active site. While the binding of the other ligands tend to impair specific communication pathways between the two knobs and the active site, and lead to a catalytically inefficient state. Finally, simulation results from free energy landscape and binding free energy analysis revealed that the C12-HSL ligand has the lowest binding free energy and greater stability than the less favored ligands. Each of the following residues: Serβ1, Hisβ23, Pheβ24, Metβ30, Pheβ32, Leuβ50, Asnβ57, Thrβ69, Valβ70, Trpβ162, Trpβ186, Asnβ269, Argβ297 and Leuα146, play different roles in substrate binding specificity. This is the first computational study that provides molecular information for structure-dynamic-function relationships of PvdQ with different ligands and demonstrates determinants of bacterial substrate binding specificity.
