A conserved salt bridge network stabilizes the hepatic organic anion transporters OATP1B1 and OATP1B3.

The organic anion transporting polypeptide (OATP)-1B1 and -1B3 are liver-specific transporters that govern the uptake of numerous endogenous molecules and drugs before their metabolism and excretion by the hepatocytes. Structurally, these two transporters are members of the major facilitator superfamily, operating by the alternating access mechanism that facilitates the movement of solutes between extracellular and intracellular compartments. Given their dynamic nature, salt bridges often modulate the conformations of transporters and participate in the orchestration of conformational changes. In this study, we identified and characterized a network of salt bridges within the internal cavities of OATP1B1 and OATP1B3 by cell-based uptake assays, uptake kinetics, and molecular dynamics simulations. These experiments revealed that a salt bridge network centered around E185 is crucial for uptake activities in these two proteins, as it stabilizes the inward cavity of the proteins and bridges the N- and C- bundles of the protein. Interestingly, this salt bridge network changes as a function of conformation. Furthermore, the residues studied do not participate in ligand coordination in the published structures nor in our simulations. These findings advance our understanding of the elaborate network of ionic interactions that govern the structure and dynamics of OATP1B1, OATP1B3, and other MFS transporters.