(650g) Free Energy Landscape of C Rugosa Lid Closing by Well-Tempered Metadynamics

Lipase family enzymes are common components of most organisms’ lipid metabolism pathway. In humans and many yeasts and bacteria, lipases hydrolyze triglycerides. Their importance in this role explains why human lipases have been implicated in a variety of health conditions, including heart disease and diabetes. Industrial utilization of lipases, which primarily come from yeasts, has predominantly been for producing food additives and in detergents, yet in the past decade for the production of biodiesel and other sustainable fuels from plant-derived oils, including waste oils from the food industry.

Despite their importance in both technological application and human health, little is known about the dynamics of lipases at the atomic level or the atomic scale features of key molecular events in the action of lipases.  Previous experimental investigations have shown a common structural motif among lipases is a lid-like domain that protects the active site from the solvent until in the enzyme contacts a solvent-lipid interface. The C. rugosa lipase (CRL) structure has been resolved for both the lid-open and lid-closed states. The two states have nearly identical structure, except for the 26 amino-acid lid, which differs by 17 Å between the open and closed states. Given the importance of this conformational change in regulating lipase activity, the molecular details of the lid mechanism are essential knowledge for engineering improved lipases.  However, direct observation of this process is difficult or impossible via experiment alone. Therefore, we have used simulation to investigate the behavior of the CRL lid in explicit water and a number of different solvents that show varying levels of lipase activity. The major challenge of using classical MD in investigating conformational rearrangement and rare events (e.g., lid opening/closing) lies in sufficient sampling.  As we have previously demonstrated, even microsecond-long simulations of the all-atom (AA) system cannot provide enough sampling to calculate the equilibrium probability distribution of this process. Building off of our previous AA simulation of CRL behavior in water, ionic liquid and organic solvents, we have applied coarse-graining and other multiscale techniques toward investigating the lid behavior for CRL. We use the AA simulation trajectories in addition to coarse-grained (CG) models to develop a set of collective variables that describe the lid’s low energy states. Finally, we use the metadynamics [1] algorithm to calculate the free-energy landscape along these collective variables and identify key structural transitions in the opening and closing process.

 [1] A. Laio and M. Parrinello (2002). "Escaping free-energy minima." PNAS 99(20): 12562-12566.