Towards Thermodynamic Characterization of Transport Cycle in Secondary Transporters using Enhanced Sampling Techniques

Secondary active transporters undergo large-scale conformational changes to couple the uphill and downhill transport of substrates. We have developed a powerful computational approach for the study of secondary active transport by combining several state-of-the-art enhanced sampling techniques primarily based on loosely coupled multiple-copy MD simulations (e.g., Bias-Exchange Umbrella Sampling and String Method with Swarms of Trajectories) within a novel, empirical, iterative sampling framework. Using this novel approach we were able to, for the first time, reconstruct an entire thermodynamic cycle associated with a secondary active transporter, namely, Glycerol-3-phosphate transporter (GlpT), based on its only available crystal structure. We have calculated the free energy profile of GlpT along a “cyclic” transition pathway connecting four distinct states of GlpT-phosphate complex including inward-facing apo, inward-facing bound, outward-facing bound, and outward-facing apo. Our results, which are in agreement with alternating access mechanism, indicate that the substrate binding lowers the free energy barrier of the transition between the inward- and ouward-facing states. When the substrate is present, the global conformational changes of the protein are coupled to the substrate translocation within the binding site. These results particularly highlight the significance of coupling between the local conformational changes of the binding site and global conformational changes of the protein. The simulations performed take advantage of tens to hundreds of loosely coupled all-atom MD simulations of GlpT in an explicit membrane/solvent environment with a total simulation time equivalent to ∼20 microseconds of single-copy MD on a system of ∼125,000 atoms. The novel approach developed here, which attempts to address the complexities associated with large-scale conformational changes of transporters and their coupling with the substrate translocation, may open opportunities for the study of other secondary transporters using enhanced sampling techniques and state-of-the-art supercomputing.