Insights into the Substrate Binding Specificity of Quorum-Quenching Acylase PVDQ

The 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 sensing signaling N-acyl homoserine lactones (AHLs) thereby degrading the signaling molecules, inhibiting the biofilm formation and reducing virulence gene expression. Previous studies demonstrated that PvdQ has different preferences for AHLs with different acyl chain lengths and substituents. However, the substrate binding specificity determinants of PvdQ with different bacterial ligands remain unknown and unintuitive. 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 (MD) simulations, residue interaction network analysis, and binding free energy calculations. The main findings are: firstly, results from pKa predictions support the observation that the pKa of the N-terminus of Serβ1 was depressed due to the surrounding residues. Multiple molecular docking studies provided information about PvdQ binding modes and binding affinities. Secondly, analysis of the protein dynamic fingerprint of each complex from MD simulations demonstrated that binding of C12-homoserine lactone (C12-HSL) ligand reduced the global motion 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 other complexes. The binding of the C12-HSL ligand facilitates structural communication between the two knobs and the active site. The binding of other ligands tends to impair these specific communication pathways, leading 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 most favorable 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 bacterial ligands and demonstrates determinants of substrate binding specificity.