Free Energy Landscape for the Entire Transport Cycle of Triose-Phosphate/Phosphate Translocator.

Secondary active transporters translocate their substrates using the electrochemical potentials of other chemicals and undergo large-scale conformational changes. Despite extensive structural studies, the atomic details of the transport mechanism still remain elusive. We performed a series of all-atom molecular dynamics simulations of the triose-phosphate/phosphate translocator (TPT), which exports organic phosphates in the chloroplast stroma in strict counter exchange with inorganic phosphate (P). Biased sampling methods, including the string method and umbrella sampling, successfully reproduced the conformational changes between the inward- and outward-facing states, along with the substrate binding. The free energy landscape of this entire TPT transition pathway demonstrated the alternating access and substrate translocation mechanisms, which revealed that P is relayed by positively charged residues along the transition pathway. Furthermore, the conserved Glu207 functions as a "molecular switch", linking the local substrate binding and the global conformational transition. Our results provide atomic-detailed insights into the substrate transport mechanism of the antiporter.