Project summary The polymorphic transporter OATP1B1 plays significant roles in the hepatic uptake and disposition of endogenous molecules and drugs. Further, OATP1B1 is involved in numerous drug-drug interactions (DDIs) due to its multispecificity. Despite decades of research, many factors related to the determinants of ligand binding, transport mechanism and energetics, as well as the structure of this important transporter are unresolved. The long-term goal is to characterize hepatic transport at the microscopic and macroscopic levels. Due to the functional importance of OATP1B1 in liver clearance, this is the first target studied. The central hypothesis is that structures of human OATP1B1 and functional assays performed on isolated proteins will unequivocally define the mechanism of ligand binding, transport, and inhibition. The rationale that underlies this research is that protein structures coupled with unambiguous, reproducible activity assays are absolutely required to fully characterize the mechanism of transport and resolve long-standing conflicting data reported in the literature. Establishing robust expression and purification protocols for human OATP1B1 facilitates pursuing the two Specific Aims: 1) Resolve structures of OATP1B1 by Cryo-EM and analyze their motions by MD simulations. 2) Functionally characterize the transport mechanism of OATP1B1. For the 1st Aim, the optimal conditions that result in the highest image quality for Cryo-EM of OATP1B1 would be screened, and data would be acquired in the presence and absence of ligands. The Cryo-EM studies will be augmented by MD simulations and docking studies that would reveal details that are not accessible by mere structural analysis. The different biochemical conditions (apo and holo) increase the likelihood of A) solving the structural determinants of the binding of various ligands (with distinct chemistries) to the two ligand-binding sites of OATP1B1. B) solving the structures of multiple conformations that are part of the transport cycle. For the 2nd Aim, we will use purified protein reconstituted into proteoliposomes to characterize the inherent properties of OATP1B1 such as the energizing ion for transport, the influence of the membrane voltage, and pH. Those results would be compared with cellular assays that include intact cellular machinery, and other plasma membrane transporters that might directly or indirectly affect OATP1B1. The research proposed in this application is innovative, in the applicant’s opinion because it introduces two methodologies that have never been explored for any OATP isoform. These could uncover a plethora of novel insights into the structure/function relationship of OATP1B1. The proposed research is significant since OATP1B1 is involved in DDIs of several commonly prescribed drugs, sometimes resulting in life-threatening situations. This study would unravel the exact determinants of ligand binding to OATP1B1 and transport. Ultimately, the structu...