(597e) Quantification of Sugar Binding Affinity and Study of Proton Translocation In Lactose Permease of Escherichia 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, a secondary active transporter that transports various sugar molecules across the plasma membrane by proton symport mechanism, is studied as a model for the MFS proteins.

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). Only a subtle change in the substrate stereochemistry results in a significant change in its binding affinity to LacY. To investigate this anomeric binding phenomenon, free energies of binding of αα-, αβ-, and ββ-(Galp)₂ to LacY are calculated using the alchemical free energy perturbation (FEP) method (Mobley et al., JCP, 2006). Since free energy is a state function, the double decoupling scheme was used to construct an alchemical thermodynamic cycle between a sugar molecule bound to LacY and a solvated sugar molecule. For FEP calculations, sampling windows are generated from independent molecular dynamics (MD) trajectories for all the alchemical transformation steps using the CHARMM simulation package. The sampling data is processed with the weighted histogram analysis method (WHAM) to get the ΔG values. Since the sugar molecules bind to LacY in multiple binding conformations, multiple independent MD simulations were performed to get all the binding modes. The initial protein conformations were obtained from the x-ray crystal structure (1PV7) and our model for the outward-facing state (Pendse et al., JMB, 2010) to get the conformations in the inward- and outward-facing states of LacY, respectively. To validate our methods, ΔG_bind for p-nitrophenyl α-D-galactopyranoside (NPG) from our simulations was compared and agrees favorably with the values obtained from the isothermal calorimetry experiments (Nie et al., JBC, 2006). The binding of the α-anomer of a sugar was determined to be more favorable compared to the β-anomer. The dissimilarity in binding affinities was found to be a result of differences in the binding conformations for different anomeric states.

It has been established that protonation of LacY, in other words binding of a proton, is a necessary precursor to sugar binding. LacY then couples translocation of protons with the transport of sugar molecules but the mechanism of this coupling is unclear. The hybrid quantum mechanics/ molecular mechanics (QM/MM) approach is used to study the proton translocation. The self consistent charge- density functional tight binding (SCC-DFTB) method is used for the QM calculations. The generalized hybrid orbital (GHO) method is used to treat the transition between the QM and MM regions across the covalent bonds. The QM/MM calculations are used to understand the key protein residues involved in proton translocation and the translocation pathway.