Substrate Access Mechanism in a Novel Membrane-Bound Phospholipase A of Pseudomonas aeruginosa Concordant with Specificity and Regioselectivity

Abstract

PlaF is a cytoplasmic membrane-bound phospholipase A 1 from Pseudomonas aeruginosa that alters the membrane glycerophospholipid (GPL) composition and fosters the virulence of this human pathogen. PlaF activity is regulated by a dimer-to-monomer transition followed by tilting of the monomer in the membrane. However, how substrates reach the active site and how the characteristics of the active site tunnels determine the activity, specificity, and regioselectivity of PlaF for natural GPL substrates have remained elusive. Here, we combined unbiased and biased all-atom molecular dynamics (MD) simulations and configurational free-energy computations to identify access pathways of GPL substrates to the catalytic center of PlaF. Our results map out a distinct tunnel through which substrates access the catalytic center. PlaF variants with bulky tryptophan residues in this tunnel revealed decreased catalysis rates due to tunnel blockage. The MD simulations suggest that GPLs preferably enter the active site with the sn-1 acyl chain first, which agrees with the experimentally demonstrated PLA 1 activity of PlaF. We propose that the acyl chain-length specificity of PlaF is determined by the structural features of the access tunnel, which results in favorable free energy of binding of medium-chain GPLs. The suggested egress route conveys fatty acid (FA) products to the dimerization interface and, thus, contributes to understanding the product feedback regulation of PlaF by FA-triggered dimerization. These findings open up opportunities for developing potential PlaF inhibitors, which may act as antibiotics against P. aeruginosa.

Rational Design Yields Molecular Insights on Leaf-Binding of Anchor Peptides

“…ASMD tackles this problem by dividing the reaction path into smaller segments called stages . Within these stages, the trajectory closest to the Jarzynski average is determined, and the final state of this trajectory is used as starting point for the consecutive stage . Thus, trajectories contributing little to the overall PMF are disregarded in the next stage, that way reducing the total number of simulations to be performed.…”

Molecular Mechanisms Underlying Medium-Chain Free Fatty Acid-regulated Activity of the Phospholipase PlaF fromPseudomonas aeruginosa

“…As a result, it has been suggested that this tilting motion underlies the activation of the monomeric PlaF by facilitating substrate access to the active site tunnel (5). In addition to the previously mentioned tunnel (T1), two other tunnels (T2 and T3) were identified, connecting the active site to the surface of PlaF (11). Unbiased MD simulations showed that myristic acid (MYR), the hydrolysis product of 1-myristoyl-2-hydroxy-sn-glycerol-3-phosphoglycerol (2LMG), a lysophospholipid, relocates from T3 and reaches the entrance of T1.…”

“…For each of the three systems, 12 replicas were performed, and binding and unbinding events of the FFA were monitored (Figure 6A). We identified an FFA as "bound" if it is within a distance < 15 Å from the T3 entrance (COM of residues K170, Q234, Y236) and has an RMSD < 1.5 Å to the previous pose (see Supplementary results) (11). Density maps of FFAs around t-PlaF show that FFAs tend to gather in T3, which connects the catalytic center to the periplasmic space (Figure 6B).…”

“…Density maps of FFAs around t-PlaF show that FFAs tend to gather in T3, which connects the catalytic center to the periplasmic space (Figure 6B). T3 has previously been suggested to play a role in relocating substrates within the catalytic center, which is necessary for the PLA1 activity of PlaF (11). Furthermore, it has been hypothesized that FFA may leave PlaF via T3 after lipid cleavage (11).…”

“…Unbiased MD simulations showed that myristic acid (MYR), the hydrolysis product of 1-myristoyl-2-hydroxy-sn-glycerol-3-phosphoglycerol (2LMG), a lysophospholipid, relocates from T3 and reaches the entrance of T1. Interestingly, MYR is also found in T1 in the crystal structure of PlaF (5,11). Accordingly, the fatty acid can reach the membrane, where it might modify its composition and properties.…”