BPS2026 – Investigating the structural dynamics of substrate and ion binding in NaCT

NaCT is a human citrate transporter that has been implicated as a potential drug target for treatment of obesity and type 2 diabetes. It cotransports four Na + with each citrate molecule, but the binding sites of two Na + and the binding pose of citrate are unresolved in all cryo-electron microscopy (cryo-EM) structures of NaCT. To characterize this unresolved structural information, we previously developed a molecular dynamics-based approach that enhances sampling of binding events by preventing four Na + and a citrate molecule from diffusing too far out of the binding pocket but otherwise leaves all dynamics unperturbed. With this approach, we have obtained 200 μs of sampling of ion and substrate binding to NaCT under a variety of different conditions, including with NaCT in its inward-facing (IF) and outward-facing (OF) states and with a key binding pocket residue (Glu461) in different protonation states. From these simulations, we have identified a preferred binding pose for citrate that is consistent with low-resolution cryo-EM density data and strong candidates for the Na3 and Na4 sites. The Na3 site is mainly formed by interactions with citrate, and we have found that the locations of the citrate and Na3 binding sites shift depending on whether NaCT is in an IF or OF state. The Na4 site is deeply buried in the protein, and access to this site is dependent on subtle binding pocket dynamics present only when Glu461 is deprotonated. From our simulations, we can now predict the likely order of binding to each site, and we have started to investigate how completed binding initiates an IF⇌OF transition. These results provide valuable insight into key portions of NaCT’s transport cycle, and they also enable compelling follow-up simulations to characterize NaCT’s bound IF⇌OF transition.