Solvent-Dependent Gating Motions of an Extremophilic Lipase from Pseudomonas aeruginosa

Abstract

Understanding how organic solvent-stable proteins can function in anhydrous and often complex solutions is essential for the study of the interaction of protein and molecular immiscible interfaces and the design of efficient industrial enzymes in nonaqueous solvents. Using an extremophilic lipase from Pseudomonas aeruginosa as an example, we investigated the conformational dynamics of an organic solvent-tolerant enzyme in complex solvent milieux. Four 100-ns molecular dynamics simulations of the lipase were performed in solvent systems: water, hexane, and two mixtures of hexane and water, 5% and 95% (w/w) hexane. Our results show a solvent-dependent structural change of the protein, especially in the region that regulates the admission of the substrate. We observed that the lipase is much less flexible in hexane than in aqueous solution or at the immiscible interface. Quantified by the size of the accessible channel, the lipase in water has a closed-gate conformation and no access to the active site, while in the hexane-containing systems, the lipase is at various degrees of open-gate state, with the immiscible interface setup being in the widely open conformation ensembles. The composition of explicit solvents in the access channel showed a significant influence on the conformational dynamics of the protein. Interestingly, the slowest step (bottleneck) of the hexane-induced conformational switch seems to be correlated with the slow dehydration dynamics of the channel.

Pressure‐induced conformational switch of an interfacial protein

“…Also, PAO1 lipase shares the same fold as the lipase from C. antarctica (PDB:1TCA). Simulation data for the PAO1 lipase at room pressure was obtained from a previous work which examined lipase gating motions at an immiscible solvent interface . The initial structure of the protein was obtained from crystal structure PDB:1EX9 (with ligand removed) .…”

“…In addition, a tightly bound calcium ion was reported be important to the structural integrity of this lipase. However, the metallic bonding properties of the calcium can be quite challenging to treat efficiently; therefore, we opted to fix the position of the calcium atom and the four coordinated oxygens as previously described . All simulations performed under different pressures have the same starting configuration.…”

“…Long‐range interactions were treated using the particle mesh Ewald method, all bonds involving hydrogen atoms were constrained using the SHAKE algorithm, and pressure control was achieved using the Berendsen method . Other pertinent simulation details are similar to those reported previously . Each simulation was conducted with a 2 fs time step for 150 ns, and snapshots were generated at 1 ps intervals, producing 150,000 snapshots for each system besides the trajectories obtained previously at room pressure …”

“…It has been suggested that movement of the lid is essential for ligand admission and, thus, enzyme activity . Previous simulation studies have revealed the molecular details of the conformational switch from a closed‐gate state in aqueous solution to an open‐gate state at water–oil interfaces …”

Lid closure dynamics of porcine pancreatic lipase in aqueous solution

“…Further, microbial lipases, which are structurally similar to the catalytic N-terminal domain of pancreatic lipases, show similarity in the lid conformation-dependent accessibility of the active site region. The studies on these proteins suggest that lid closer movement in aqueous environment is driven by constricting the hydrophobic face of the amphiphilic helix of the lid towards inner core of the protein [63][64][65]. Though in some lipases the process can be considered as rigid body movement as secondary structural changes are minimal (for instance, Burkholderia cepacia lipase [65,66]), other microbial lipases show major secondary structural changes during the lid closure (for instance, Candida rugosa lipase [67]) as observed in PPL.…”