Nucleoside Transport

Structural Basis of Cellular Nucleoside Transport

Being that nucleosides are hydrophilic molecules, they require specific membrane transporter proteins—known as nucleoside transporters (NTs)—to cross the cell membrane. Such a process is mediated by two distinct protein families: concentrative and equilibrative nucleoside transporters (CNTs and ENTs). Since nucleoside transport is associated with essential physiological processes, such as nucleic acid synthesis and the termination of adenosine signaling, we think it’s important to interrogate the transport mechanisms of NTs in order to gain a deeper understanding of nucleoside-related physiological and drug pharmacology. But the biggest hurdle in revealing these mechanisms had been the lack of atomic structures that reveal the design principles behind CNTs and ENTs.

Concentrative nucleoside transporters (CNTs)

Snapshots of how nucleoside-derived anticancer and antiviral drugs and nucleosides are transported into the cell by concentrative nucleoside transporters (CNTs). Movement of the transport (yellow globs) and scaffold (blue helices) domains of CNT transport drugs across the cell membrane with a large elevator-type motion.







Solving the Crystal Structures

In the Lee Lab, we solved the crystal structure of a concentrative nucleoside transporter from Vibrio cholerae (vcCNT), the first reported structure of any CNT family protein (Nature 2012). We also determined crystal structures of vcCNT in complex with nucleoside-derived anticancer and antiviral drugs and studied their interactions using functional assays employing ITC, fluorescence-based equilibrium binding, and a radioactive flux assay to understand the principles of substrate recognition by CNTs (eLife 2014). We recently solved the structures of inward-facing, intermediate, and outward-facing states of CNT from Neisseria wadsworthii (Nature 2017).

Our Discovery

Our research provides proof of principle for utilizing transporter structural and functional information to help design compounds so they can enter cells more efficiently and selectively. Taking what we learned in our structural studies, we rationally modified an existing anti-cancer drug, resulting in higher transport selectivity and efficiency through a single human CNT subtype (eLife 2014). Furthermore, CNT is known to operate an elevator type transport mechanism, but the structural features concerning the conformational path that leads to such a transition was not yet known. By solving structures of CNT from inward to intermediate and outward-facing states, we showed direct evidence for the trajectory of conformational transition in the elevator model for the first time (Nature 2017).

Equilibrative nucleoside transporters (ENTs)

Atomic structures of human ENT1 in complex with adenosine reuptake inhibitors. We found that the inhibitors dilazep and NBMPR exhibit distinct structural mechanisms of inhibition.







Solving the Crystal Structures

The Lee group also solved crystal structures of an equilibrative nucleoside transporter from H. sapiens (human ENT1), the first reported structure of any ENT family protein (Nat Struc Mol Biol 2019). These structures feature human ENT1 in complex with two different adenosine reuptake inhibitors, which are pharmacological agents used to stimulate the tissue-protective effects of adenosine receptor signaling by ENT block. 

Our Discovery

This research revealed the molecular architecture of the ENT family protein fold, design principles of human ENT1 inhibition by two different adenosine reuptake inhibitors, while providing initial insight into the nucleoside transport mechanism exhibited by ENTs. We also discovered the reuptake inhibitor NBMPR inhibits this transporter in a unique manner, which sets the stage for the rational design of improved small-molecule therapeutics targeting human ENTs.