Cryo-EM structures of human organic anion transporting polypeptide OATP1B1.

Members of the solute carrier organic anion transporting polypeptide (OATPs) family function as transporters for a large variety of amphipathic organic anions including endogenous metabolites and clinical drugs, such as bile salts, steroids, thyroid hormones, statins, antibiotics, antivirals, and anticancer drugs. OATP1B1 plays a vital role in transporting such substances into the liver for hepatic clearance. FDA and EMA recommend conducting in vitro testing of drug-drug interactions (DDIs) involving OATP1B1. However, the structure and working mechanism of OATPs still remains elusive. In this study, we determined cryo-EM structures of human OATP1B1 bound with representative endogenous metabolites (bilirubin and estrone-3-sulfate), a clinical drug (simeprevir), and a fluorescent indicator (2',7'-dichlorofluorescein), in both outward- and inward-open states. These structures reveal major and minor substrate binding pockets and conformational changes during transport. In combination with mutagenesis studies and molecular dynamics simulations, our work comprehensively elucidates the transport mechanism of OATP1B1 and provides the structural basis for DDI predictions involving OATP1B1, which will greatly promote our understanding of OATPs.

Cryo-EM structure of the human sodium-chloride cotransporter NCC.

The sodium-chloride cotransporter NCC mediates the coupled import of sodium and chloride across the plasma membrane, playing vital roles in kidney extracellular fluid volume and blood pressure control. Here, we present the full-length structure of human NCC, with 2.9 Å for the transmembrane domain and 3.8 Å for the carboxyl-terminal domain. NCC adopts an inward-open conformation and a domain-swap dimeric assembly. Conserved ion binding sites among the cation-chloride cotransporters and the Na2 site are observed in our structure. A unique His residue in the substrate pocket in NCC potentially interacts with Na1 and Cl1 and might also mediate the coordination of Na2 through a Ser residue. Putative observed water molecules are indicated to participate in the coordination of ions and TM coupling. Together with transport activity assays, our structure provides the first glimpse of NCC and defines ion binding sites, promoting drug development for hypertension targeting on NCC.

Implementation of novel boolean logic gates for IMPLICATION and XOR functions using riboregulators.

To date, several different types of synthetic genetic switches, including riboregulators, riboswitches, and toehold switches, have been developed to construct AND, OR, NOT, NAND, NOR, and NOT IMPLICATION (NIMP) gates. The logic gate can integrate multiple input signals following a set of algorithms and generate a response only if strictly defined conditions are met. However, there are still some logic gates that have not been implemented but are necessary to build complex genetic circuits. Here, based on the toehold switches and three-way-junction (3WJ) repressors, we designed two novel biological Boolean logic gates of IMPLICATION (IMP) and XOR. Subsequently, the outputs of these two logic gates were characterized by fluorescence analysis, indicating that they can achieve the truth tables of logical gates. Furthermore, the fluorescence intensity under the logical TRUE condition was significantly higher than under the logical FALSE condition, suggesting the high dynamic range of the ON/OFF ratios. Because of the programmability of synthetic RNA switches, the constructed RNA logic gates could serve as elementary units to build a versatile and powerful platform for translational regulation and RNA-based biological computation.