(151e) Investigation of the Proton Translocation Mechanism in Lactose Permease of E. Coli by a Hybrid QM/MM Approach

In the biological cells, molecular traffic in and out of the cell is mainly controlled by the membrane transport proteins. The membrane transport proteins play critical roles in human physiology and their malfunctions are responsible for a range of human diseases. The Major Facilitator Superfamily (MFS) is one of the largest families of membrane transport proteins whose members are found in almost all types of organisms and are very diverse in terms of substrate transport. We are studying Lactose Permease (LacY) of E.coli as a structural and functional model for the MFS proteins. LacY, a symporter, couples the energetically downhill transport of protons with the energetically uphill transport of sugar molecules but the mechanism of this energy coupling is unknown. A hybrid quantum mechanics/molecular mechanics (QM/MM) protocol is used to study the mechanism of proton translocation. Self-consistent-charge-density-functional-tight-binding (SCC-DFTB) is used for the QM calculations and the CHARMM MM force field is used to describe the MM region, while the generalized solvent boundary potential (GSBP) method is used to describe electrostatic interactions far away from the active site. The transition between the QM and MM regions is treated using the generalized hybrid orbital (GHO) approach.

It has been established that protonation of LacY is a precursor to sugar binding but the details of the proton binding site are unclear. An extensive mutational analysis indicated that residues Tyr236, Asp240, Arg302, Lys319, His322, and Glu325, which form a tight hydrogen bond/salt bridge network at the center of the molecule, are involved in proton binding but no single amino acid could be identified as an individual proton acceptor (Smirnova et al., Biochemistry, 2009). Therefore it was postulated that a water molecule coordinated within this tight network acts as a proton acceptor. We are investigating the role of structural water molecule/s in proton binding. Multiple independent simulations are carried out with only the structural water in the QM region to determine the stability of protonated water and its form. To study whether any of the residues in the surrounding hydrogen bond network can act as a proton acceptor, several independent simulations are carried out with each of these residues included in the QM region in addition to the protonated water. The energetics of proton transfer from the protonated water to each residue are calculated using the potential of mean force (PMF) approach. The results from these proton binding studies will be used to further probe the transport of proton from its binding site to Glu269, which is an irreplaceable residue believed to be involved in both sugar binding as well as proton translocation. The results from this study will help not only in understanding the overall mechanism of LacY but also in gaining useful insights into the proton translocation by other MFS proteins such as a multidrug efflux protein EmrD.