(602b) Binding Free Energy Calculations to Understand the Mechanism of Sugar Binding to Lactose Permease of E. Coli

In biological cells, molecular traffic in and out of the cell is mainly controlled by membrane transport proteins. The Major Facilitator Superfamily (MFS) is an important class of membrane transporters whose members are found in almost all types of organisms and are very diverse in terms of substrate transport. Lactose Permease (LacY) of E.coli is studied as a model for the MFS proteins. LacY, a secondary active transporter, transports various sugar molecules across the plasma membrane by the proton symport mechanism. Although LacY binds to different anomeric states of a disaccharide with the same specificity, binding affinity of the α-anomer is greater than β (Sahin-Toth et al., Biochem, 2000). Molecular simulation studies on sugar-protein interactions have indicated that αβ-(Galp)₂ and ββ-(Galp)₂ bind to LacY differently and that the binding of αβ-(Galp)₂ is enthalpically more favorable (Klauda & Brooks, JMB, 2007). The aim of this research is to quantify the binding affinities of the different anomeric states of (Galp)₂ by calculating the binding free energy.

Alchemical free energy perturbation (FEP) method is used to calculate the change in free energy between the state in which ligand is bound to the protein and the state in which ligand is fully solvated (Mobley et al., JCP, 2006). The Hamiltonian is gradually perturbed by eliminating the interactions between the ligand and its surroundings in a series of alchemical steps. The interactions of the ligand with its surroundings are then gradually restored in water to complete the thermodynamic cycle. The CHARMM simulation package is used to carry out all molecular dynamics (MD) simulations. The CHARMM family of force fields is used to describe interatomic interactions for the protein and sugars and the TIP3P model is used to represent water. For FEP calculations, sampling windows are generated from independent MD trajectories for all the alchemical transformation steps. The sampling data is processed with the weighted histogram analysis method (WHAM) to get the ΔG values.

To test the accuracy of our methods, free energy change for binding of p-nitrophenyl α-D-galactopyranoside (NPG) to LacY was calculated and compared with values obtained from the isothermal titration calorimetry experiments (Nie et al., JBC, 2006). Six independent MD trajectories for 25-30 ns were run starting with different orientations of NPG near the binding site to determine the binding conformations of NPG in LacY. Only conformations that were stable for at least 1ns were s elected for FEP calculations. The contributions to the binding free energy from electrostatic and non-polar interactions between the ligand and protein were calculated separately. The non-polar contribution was further divided into dispersive and repulsive contributions using the Weeks, Chandler and Andersen (WCA) separation of Lennard-Jones potential. A similar strategy was used to obtain the binding free energies for αβ-(Galp)₂ and ββ-(Galp)₂. The FEP calculations for αβ-(Galp)₂ and ββ-(Galp)₂ reveal a detailed description of LacY's anomeric binding phenomenon which is crucial in understanding of the overall transport mechanism. The method will also be extended to different anomers of other disaccharides, such as melibiose and lactose, to throw more light on anomeric binding.