Hydrophobic Interactions Induced Activation of a Thermo-alkalophilic Lipase from Geobacillus SBS-4S by Molecular Dynamics Simulations.
Summary: Recently, we have reported the 3-dimensional structure of the lipase (LIPSBS) from Geobacillus SBS-4S in closed configuration by X-ray crystallography. In the present study, we report on activation of the close configuration of LIPSBS to its open form. The presence of metal ions or a non-ionic detergent in the crystallization buffer could not activate LipSBS. However, addition of octane molecules initiated upward movement of the lid, leaving rest of the protein intact, by molecular dynamics simulations. The hydrophobic interactions between amino acids of the lid and octane molecules resulted in shifting the inactive configuration to the active one. These results were reinforced by root-mean-sqaure deviation (rmsd) data. Our data shed light on the structural changes that take place during activation of LIPSBS.
Keywords: Lipase; Geobacillus SBS-4S; Thermo-alkalophilic; 3-dimensional structure; Molecular dynamics simulations
Lipases are hydrolytic enzymes involved in the breakdown of triglycerides to glycerol and free fatty acids. The production of lipases has been reported from mammals, yeast, fungi, bacteria and plants . Their demand in food, detergent, cosmetic and pharmaceutical industries have been increasing day by day. Therefore, recombinant DNA technologies have been utilized to fulfill the industrial requirements [2, 3].
X-ray crystallographic structure determination plays an important role in elucidation the mechanism of action of enzymes. Three dimensional structures of lipases have demonstrated the presence of various conserved domains including active site motif, metal binding pocket, a/AY hydrolase fold and regulatory elements binding domains [4, 5]. Hydrophobic lid over the active site, in most of the lipases [6, 7], prevents the interaction of active site and the substrate.
The opening of the lid shifts inactive configuration to active one due to hydrophobic interactions between the substrate and non-polar amino acids of the lid . We have previously characterized the thermo-alkalophilic lipase (LipSBS) from Geobacillus SBS-4S . Three dimensional structures of LipSBS have been resolved in close configuration at 1.6 A resolution . In the present study the three dimensional structure resolved by X-ray crystallography was utilized for in silico analysis of conformational changes during the activation of LipSBS in the presence of hydrophobic molecules.
Molecular dynamics simulations were performed using the closed configuration structure of LIPSBS (PDB Code 3AUK). Hydrophobic octane molecules were used to analyze the activation of LIPSBS. Protein Data Bank coordinates for octane molecule were generated from SMILES string specification using Coot, a molecular-graphics application . Molecular dynamics simulation studies were done using GROMACS ver. 3.3.3 utilizing the GROMOS G53a6 forcefield on a DELL DPeR900 server (HPC technologies) running on CentOS 4. LIPSBS was packed in the box (X=55 A , Y= 55 A and Z= 58 A ) with hydrophobic octane molecules surrounding the lid of LIPSBS. PACKMOL program  was used for packing the LIPSBS.
Energy minimization was performed in such a way that maximum force on any atom (Fmax) was less than 1000 kJ. Position restrained simulation to equilibrate water molecules were carried out at 37 C for 100 ps. Two sodium ions were added to neutralize the overall charge of the system. Molecular dynamics simulation was performed at 37 C with a pressure of 1 atm at neutral pH. The simulation trajectories were analyzed using several auxiliary programs provided with the GROMACS package. The root mean square deviations (rmsd) of atomic positions and atom-atom distances were computed using g_rms and g_dist utilities, respectively. The rmsd of the lid and entire protein atoms were calculated following least-square fitting of the backbone atoms of trajectories .
Results and Discussion
We have previously reported the three dimensional structure of LIPSBS in closed configuration . In order to crystallize LIPSBS in open configuration we tried Ca2+ ions as it was involved in the enhancement of LIPSBS activity  and Triton X-100 expecting that hydrophobic interactions between Triton X-100 molecules and the lid over the active site of LIPSBS might be able to shift close configuration to open one . In spite of several attempts we were unable to get LIPSBS crystals in open configuration even in the presence of both of these reagents. Therefore, molecular dynamics simulation studies were adopted as they have been previously used for the interfacial activation of various lipases .
In molecular dynamics simulations, hydrophobic octane molecules aggregated to form circular micellar structures that interacted with the hydrophobic amino acids of the lid resulting in opening of the lid (Fig. 1). The interaction of hydrophobic amino acids and octane molecules moved the lid portion while keeping the rest of the protein intact as indicated by rsmd values. A sudden increase in rmsd values of the lid portion was recorded after 7ns whereas no change was detected in the non-lid portion before and after or during the simulation (Fig. 2).
LIPSBS, a member of family 1.5, contains a single lid  similar to lipases from families 1.1 and 1.2  and in contrast to PML lipase from family 1.3 that contains two lids . When the mechanism of lid opening in LIPSBS was examined it was found that the lid helix was fixed at one end while the other end moved upward during the activation similar to PML lipase from Pseudomonas , and in contrast to T1 lipase from Geobacillus zalihae where the two fixed helices moved in the opposite direction .
A comparison of close configuration to simulated open form of LIPSBS demonstrated the upward movement of the two helices from closed (C- I and C-II) to simulated open (O-I and O-II) structures (Fig. 3). A higher level of movement was observed in helix-I as compared to helix-II. This study explored the activation mechanism of LIPSBS and demonstrated that the hydrophobic interactions between substrate and lid amino acids are involved in the activation of this lipase.
In conclusion, the inactive LIPSBS was changed to active one by molecular dynamics simulation studies. The activation involved the movement of the lid helixes which resulted in opening of the lid and providing space for the substrate to interact with the active site.
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|Publication:||Journal of the Chemical Society of Pakistan|
|Date:||Oct 31, 2015|
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