Unlocking the mystery behind the activation phenomenon of T1 lipase: a molecular dynamics simulations approach

Abstract

The activation of lipases has been postulated to proceed by interfacial activation, temperature switch activation, or aqueous activation. Recently, based on molecular dynamics (MD) simulation experiments, the T1 lipase activation mechanism was proposed to involve aqueous activation in addition to a double-flap mechanism. Because the open conformation structure is still unavailable, it is difficult to validate the proposed theory unambiguously to understand the behavior of the enzyme. In this study, we try to validate the previous reports and uncover the mystery behind the activation process using structural analysis and MD simulations. To investigate the effects of temperature and environmental conditions on the activation process, MD simulations in different solvent environments (water and water-octane interface) and temperatures (20, 50, 70, 80, and 100°C) were performed. Based on the structural analysis of the lipases in the same family of T1 lipase (I.5 lipase family), we proposed that the lid domain comprises α6 and α7 helices connected by a loop, thus forming a helix-loop-helix motif involved in interfacial activation. Throughout the MD simulations experiments, lid displacements were only observed in the water-octane interface, not in the aqueous environment with respect to the temperature effect, suggesting that the activation process is governed by interfacial activation coupled with temperature switch activation. Examining the activation process in detail revealed that the large structural rearrangement of the lid domain was caused by the interaction between the hydrophobic residues of the lid with octane, a nonpolar solvent, and this conformation was found to be thermodynamically favorable.

Figure 1

Structure superposition of T1 lipase with BTL2 lipase. RMSD value of 0.545 Å was obtained from the superimposition. The helices that formed the lid for BTL2 are colored in green (α6 helix) and yellow (α7 helix), whereas the proposed helices that formed the lid for T1 are colored in blue (α6 helix) and brown (α7 helix). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 2

Quantitative analysis of conformational changes for closed T1 lipase during MD simulations in different environments and temperatures. A: RMSD of T1 lipase Cα-backbone atoms as a function of time during simulations of 10 ns in aqueous environment at 20°C (red), 50°C (yellow), 70°C (blue), 80°C (green) and 100°C (purple); B: RMSD of T1 lipase Cα-backbone atoms as a function of time during simulations of 10 ns in water-octane interface at 20°C (red), 50°C (yellow), 70°C (blue), 80°C (green), and 100°C (purple); C: Radius of gyration as a function of time during simulations of 10 ns of closed T1 lipase in aqueous environment at 20°C (red), 50°C (yellow), 70°C (blue), 80°C (green), and 100°C (purple); D: Radius of gyration as a function of time during simulations of 10 ns of closed T1 lipase in water-octane interface at 20°C (red), 50°C (yellow), 70°C (blue), 80°C (green), and 100°C (purple). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 3

Time variation of the solvent accessible surface area of closed T1 lipase in distinct environments and temperatures. A: T1 lipase in aqueous environment at 20°C (red), 50°C (yellow), 70°C (blue), 80°C (green), and 100°C (purple) during simulations of 10 ns; B: T1 lipase in water-octane interface at 20°C (red), 50°C (yellow), 70°C (blue), 80°C (green), and 100°C (purple) during simulations of 10 ns. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 4

Flexibility and lid movement of closed T1 lipase in terms of B-factor. A: Calculated B-factors of T1 lipase residues from MD simulations carried out in aqueous environment at 20°C (red), 50°C (yellow), 70°C (blue), 80°C (green), and 100°C (purple); B: Calculated B-factors of T1 lipase residues from MD simulations carried out in water-octane interface at 20°C (red), 50°C (yellow), 70°C (blue), 80°C (green), and 100°C (purple). The secondary structure of T1 lipase is shown on the graph for reference. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 5

The lid domain of T1 lipase. The lid of T1 lipase consists of α6 and α7 helices connected by a loop and forming a helix-loop-helix motif. The hydrophobic residues of the lid domain are colored in red. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 6

Conformation of closed T1 lipase before and after MD simulations in the presence of water-octane interface at 70°C. A: Surface view of initial structure of closed T1 lipase before MD simulations; B: Surface view of final structure of open T1 lipase after MD simulations. The lid domain is colored in green, whereas, the active site is colored in red. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 7

The distance between the catalytic residues (Asp317-His358-Ser113) of T1 lipase at two different temperatures in water-octane interface. A: MD simulation at 70°C; B: MD simulation at 80°C. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 8

Conformational transition of the lid during simulations of 10 ns in different environments at 70°C. A: Conformation of closed T1 lipase in water; B: Conformation of closed T1 lipase in water-octane interface. The lid gradually opens in water-octane interface but remains closed in water. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 9

Superposition of the final T1 lipase structure after the MD simulations with the initial structure. A: Superposed images of T1 lipase after MD simulations in water at 20, 50, 70, 80, and 100°C; B: Superposed structures of T1 lipase after MD simulations in water-octane interface at 20, 50, 70, 80, and 100°C. The α6 and α7 helices of the initial structure are colored in cyan and yellow, whereas in final structure those helices are colored in blue and brown. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 10

Lid opening structure stabilization by salt bridges formation. After the lid was displaced, two salt bridges between Asp205-Arg214 and Asp209-Arg214 were formed. The salt bridges within the lid region stabilized the opening structure of the lid. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 11

Schematic illustration of T1 lipase activation. The unidirectional lid (yellow) movements occurred in the presence of octane micelles and were enhanced with increases in temperature. At lower temperature and with the presence of octane micelles, the lid only opened slightly. In the absence of octane micelles, the lid remained closed. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 12

Quantitative analysis of conformational changes for lid-closed simulation of T1 lipase in aqueous environment at 70°C. A: RMSD of T1 Cα-backbone atoms as a function of time during simulations of 10 ns; B: Time variation of the solvent accessible surface area during simulations of 10 ns; C: Radius of gyration as a function of time during simulations of 10 ns; D: Calculated B-factors of T1 lipase residues during simulations of 10 ns. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 13

Conformational transition of the lid during lid-closed simulations of 10 ns in aqueous environment at 70°C. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]