Modeling of solvent-dependent conformational transitions in Burkholderia cepacia lipase

Abstract

Background: The characteristic of most lipases is the interfacial activation at a lipid interface or in non-polar solvents. Interfacial activation is linked to a large conformational change of a lid, from a closed to an open conformation which makes the active site accessible for substrates. While for many lipases crystal structures of the closed and open conformation have been determined, the pathway of the conformational transition and possible bottlenecks are unknown. Therefore, molecular dynamics simulations of a closed homology model and an open crystal structure of Burkholderia cepacia lipase in water and toluene were performed to investigate the influence of solvents on structure, dynamics, and the conformational transition of the lid.

Results: The conformational transition of B. cepacia lipase was dependent on the solvent. In simulations of closed B. cepacia lipase in water no conformational transition was observed, while in three independent simulations of the closed lipase in toluene the lid gradually opened during the first 10-15 ns. The pathway of conformational transition was accessible and a barrier was identified, where a helix prevented the lid from opening to the completely open conformation. The open structure in toluene was stabilized by the formation of hydrogen bonds.In simulations of open lipase in water, the lid closed slowly during 30 ns nearly reaching its position in the closed crystal structure, while a further lid opening compared to the crystal structure was observed in toluene. While the helical structure of the lid was intact during opening in toluene, it partially unfolded upon closing in water. The closing of the lid in water was also observed, when with eight intermediate structures between the closed and the open conformation as derived from the simulations in toluene were taken as starting structures. A hydrophobic beta-hairpin was moving away from the lid in all simulations in water, which was not observed in simulations in toluene. The conformational transition of the lid was not correlated to the motions of the beta-hairpin structure.

Conclusion: Conformational transitions between the experimentally observed closed and open conformation of the lid were observed by multiple molecular dynamics simulations of B. cepacia lipase. Transitions in both directions occurred without applying restraints or external forces. The opening and closing were driven by the solvent and independent of a bound substrate molecule.

Figure 1

Structure of open and closed BCL. The open and closed conformation of BCL is shown in a cartoon representation. The lid of the open crystal structure of BCL [PDB: 3LIP] is colored blue and of the closed homology model green, the sphere of the Ca²⁺-ion is colored yellow. The catalytic triad consisting of Ser, His, and Asp is colored red. For analysis distances were measured between atom C_α of residue 132 from the simulated to the initial structure, respectively.

Figure 2

Flexibility of closed BCL in water. The flexibility of the closed conformation of BCL in water, indicated by calculated B-factors per residue [Ų], was calculated from the last 10 ns of simulation.

Figure 3

Structure of open BCL in toluene. The open crystal structure of BCL (green) in a cartoon representation showed a further lid opening after simulation of 30 ns in toluene (red).

Figure 4

Flexibility of open BCL in toluene. The flexibility of the open conformation of BCL in toluene, indicated by calculated B-factors per residue [Ų], was calculated from the last 10 ns of the simulation in toluene.

Figure 5

RMSD in simulations of closed BCL in toluene. Root mean squared deviations (RMSD) of backbone atoms of the core and the flexible lid as a function of time during simulation of 30 ns of closed BCL in toluene starting from the closed homology model.

Figure 6

Movement of the lid of closed BCL in toluene. The movement of the lid of closed BCL as a function of time during the simulation of 30 ns in toluene was measured by the distance C_α 138-C_α 250. The lid was opening about 18 Å from the closed to the open conformation.

Figure 7

Barrier during opening of the lid. The movement of helix α5 (residues 134 to 150) was blocked by helix α6 (residues 156 to 166) during simulation of closed BCL in toluene (B) from their starting conformation (A). The unfolding of residues 156 to 159 of helix α6 would be necessary for the complete movement of the lid (C).

Figure 8

Conformations of closed BCL during conformational transition. Conformations of closed BCL (grey) in a cartoon representation during gradually lid opening (blue) in simulation of 30 ns in toluene (A) at the beginning and (B) after 3 ns, (C) 7 ns, (D) 10 ns, (E) 17 ns and, (F) 30 ns.

Figure 9

Hydrophobic surface during conformational transition. The hydrophobic surface of BCL during the 30 ns simulation of closed conformation in toluene is represented as a function of time. The hydrophobic surface increased by 1100 Ų by lid opening.

Figure 10

Reverses simulations in toluene. (A) After simulation of open BCL the lid opening of BCL in toluene was reversed in simulations of 6 ns in water, represented as a function of time. The open crystal structure (magenta) and several conformations from the previous lid opening (red, green, blue) in the simulation of closed BCL in toluene (black) were closing again after changing the solvent to water. The lid closing of BCL was measured by the distance between atoms C_α 138 and C_α 250. (B) The frequency of the distance between atoms C_α 138 and C_α 250 in the reversed movement of the lid in water was analyzed. Frequently occurring distances are 6, 10, 14, and 16.5 Å.

Figure 11

Stabilization of Asp130 by hydrogen bonds. After the simulation of BCL in toluene Asp130 in the lid was coordinated by six hydrogen bonds to backbone and side chains of Thr132, Ser135 and Thr136. The secondary structure of BCL is colored grey, hydrogen, carbon, nitrogen and oxygen atoms are colored white, green, blue and red, respectively.